microsoft word editorial.docx thank you to reviewers of negative results in 2014 david alcántara all res. j. biol., 2014, 5, 23 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com editorial issue 4, vol 5, 2014, 23 thank you to reviewers of negative results in 2014 david alcántara editor in chief, the all results journals:biol the mission of the all results journals:biol is the communication of excellent research work obtaining negative results from all fields of biology and biomedicine. clearly, a scientific journal’s greatest responsibility toward the scientific community is to ensure that all contributions accepted for publication are rigorously but fairly reviewed, specially in a journal of negative results like this. for this reason, all manuscripts published in the journal are reviewed by expert referees. the final quality of papers accepted for publication depends to a considerable extent on the reviewers, who give valuable constructive criticism to the authors. the input and dedication of all our reviewers are highly appreciated by both the editors and the authors. at the end of 2014, we gratefully acknowledge the valuable support of the following scientists who have reviewed papers for the journal during the past 12 months: benjamin mudrak carlos juan ceacero ruiz charumathi sabanayagam emilio gonzalez enrique cobos girish mahajan jordi martinez quintanilla joseph sullivan karen sagredo laura plaza arregui luis diaz maria pilar ayuda durán narasimhulu korrapati nicolas navrot osvaldo ernesto antonio arce robert mandell ron van noorden ronald dorenbos sonia malik tomas salmeron parra victor quesada vijay nema william smith 23 microsoft word editorial.docx thank you to reviewers of negative results in 2015 david alcántara all res. j. biol., 2015, 6, 30 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 3, vol 6, 2015, 30 thank you to reviewers of negative results in 2015 david alcántara editor in chief, the all results journals:biol the mission of the all results journals:biol is the communication of excellent research work obtaining negative results from all fields of biology and biomedicine. clearly, a scientific journal’s greatest responsibility toward the scientific community is to ensure that all contributions accepted for publication are rigorously but fairly reviewed, specially in a journal of negative results like this. for this reason, all manuscripts published in the journal are reviewed by expert referees. to be fair with authors and reviewers we decided to use double blind peer-review process since our first issue. the final quality of papers accepted for publication depends to a considerable extent on the reviewers, who give valuable constructive criticism to the authors. the input and dedication of all our reviewers are highly appreciated by both the editors and the authors. at the end of 2015, we gratefully acknowledge the valuable support of the following scientists who have reviewed papers for the journal during the past 12 months: amit misra carlos juan ceacero ruiz chima ngumah d. nagasamy venkatesh girish mahajan hamideh ofoghi mehdi mohebodini maria pilar ayuda durán nicolas navrot saurabh ghoshroy valeria righi zemin wang microsoft word realeditorial docx -revisado.docx editorial: a closer look david alcántara all res. j. biol., 2012, 3, i the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com editorial                                                                                                                          issue 1, vol. 3, 2012, i editorial: a closer look david alcántara1,2 1) instituto de nanociencia de aragón,zaragoza university, mariano esquillor s/n, i+d+i building (rio ebro campus), 50018 zaragoza (spain); 2) andalusian center for nanomedicine and biotechnology (bionand), severo ochoa 35, 29590 campanillas (spain). as editor-in-chief of the all res. j. biol. i would like first to thanks all the authors who have submitted their manuscripts to our journal. this has been a positive and encouraging sign for us to continue working and publishing good quality work focused on negative results. during the first semester of 2012 we have received almost twice as many submissions as during the same period of 2011, while at the same time, increased the number of rejections. our policy of only publishing peer-reviewed work ensures a good quality control standard for the journal. for publication, experiments have to be rigorously conducted and repeatable. works that showed significant flaws in scientific methodology or inconsistent presentation of results were not even submitted for peer-review. manuscripts that carefully review the possible reasons for negative results in the discussion section have generally been positively evaluated and submitted to external peer-review. here, the work of our expert reviewers (volunteers) has been essential and, for that, i would also like to thanks them for their dedication and valuable insights they have brought to this journal and for the improvements to the articles that we publish. this issue contains two articles which take a closer look at the important areas of 1) medical device testing and 2) polymerase chain reaction (pcr). the first article focuses on biocompatibility testing for lipophilic leachates. the food and drug administration (fda) requires medical devices and materials be tested for safety. biocompatibility testing is necessary for all devices requesting fda clearance1,2. in their work, dr. lucas et al. developed a system of indirect exposure of cells to oil, a system that may be useful in looking at surface contamination of devices. by placing cells in dialysis tubing and then putting these cells directly in oil, it was expected that exposure to lipophilic compounds would be increased; however, they found this was not the case. polymerase chain reaction (pcr) has secured its place in biochemical history as a revolutionary method. in the second article, dr. nema reports on experiments where the initial amplification of the 16srrna gene from purified dna isolated from a stool sample generated spurious sequences when amplified utilizing a routine taq polymerase enzyme. as the author stated “care must be taken at every step to ensure the quality of data being generated and made available as standards for future analysis in the databases”. our immediate goal at the all res. j. biol is to provide scientists with responsible and balanced information in order to advance faster, improve experimental designs and clinical decisions. publishing negative results ensures that finite research resources are better used, avoiding replication of previous experiments and leading to a more efficient use of resources. we encourage new submissions from researchers from all biology disciplines and look forward to publishing good quality work within the scope of our journal. references 1. fda guidance document. use of international standard iso-10993, 'biological evaluation of medical devices part 1: evaluation and testing'. http://www.fda.gov/medicaldevices/deviceregulati onandguidance/guidancedocuments/ucm080735.ht m.. 2. astm f 748 – 98, 1998. standard practice for selecting generic biological test methods for materials and devices. in: the annual book of astm standards, astm west conshohocken, pa, usa. 1998 microsoft word layoutediting_chinikar.docx surveillance of rift valley fever in iran between 2001 and 2011 sadegh chinikar,* nariman shah-hosseini, ehsan mostafavi, maryam moradi, sahar khakifirouz, tahmineh jalali and anthony r. fooks . all res. j. biol., 2013, 4, 16-18 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                  issue 2, vol. 4, 2013, 16-18 surveillance of rift valley fever in iran between 2001 and 2011 sadegh chinikar,*a nariman shah-hosseini,a ehsan mostafavi,b maryam moradi,a sahar khakifirouz,a tahmineh jalali a and anthony r. fooks c,d a) arboviruses and viral haemorrhagic fevers laboratory (national ref. lab), pasteur institute of iran; b) department of epidemiology, pasteur institute of iran, tehran, iran; c) animal health and veterinary laboratories agency, wildlife zoonoses and vector-borne diseases research group, department of virology, veterinary laboratories agency, weybridge, new haw, addlestone, surrey, united kingdom; d) national consortium for zoonosis research, university of liverpool, leahurst, neston, south wirral ch64 7te, united kingdom. *corresponding author email: sadeghchinikar@yahoo.com abstract rift valley fever virus (rvfv) is an acute zoonotic viral disease that mostly affects ruminants with an occasional spill over as human infection. following the outbreak of rvf in saudi arabia in 2000, surveillance of both animal and human population in iran increased until 2011. during this period 1206 ovine, 405 caprine, 325 bovine and 28 camel samples were tested for rvfv in nine provinces in iran. none of these samples tested igg positive. moreover, amongst 37 clinically suspected human cases of patients with rvf symptoms, none of these samples tested positive for rvfv. despite the fact that no positive cases in human or animal populations were identified in iran, surveillance and monitoring of viral haemorrhagic fevers including rvfv will continue. keywords: rvf, caprine, ovine, bovine, camel, human 1) introduction rift valley fever (rvf) is a viral disease affecting livestock. rvf virus (rvfv) is a member of the bunyaviridae family, genus phlebovirus1. rainy seasons and flooding prepare the ground for the hatching of the primary vectors, multiple species of mosquitoes known as floodwater aedes, which feed on nearby mammals. high levels of viremia in these animals lead to infection of secondary arthropod vector species and to subsequent infection of other mammals and livestock, in which it causes abortions and death in susceptible animals. 2 the first human infection with rvfv was reported after the isolation of the virus in 1930. there were no extensive human disease outbreaks until 1951, when an estimated 20,000 persons were infected during an epizootic in cattle and sheep in south africa. 3 the route of transmission from animals to humans is from rvfv carrying arthropod vectors, aerosols of blood or amniotic fluid, or other direct contact with infected animals. 4 rvf in humans has a wide spectrum of clinical manifestations, from asymptomatic infection or a benign febrile illness, to severe illness in approximately 1%-3% of cases, which can include retinitis, encephalitis, hepatitis and haemorrhagic fever. 5 rvf is mainly reported in africa. in 2000, the first confirmed rvf outbreak outside africa was reported in two ‘rvf hotspot’ countries bordering iran; namely the kingdoms of saudi arabia and yemen. because of livestock and meat imports from saudi arabia to iran, enhanced monitoring of rvfv in susceptible species was undertaken.4, 6 2) materials and methods 2.1. study area a surveillance and control programme of viral hemorrhagic fevers in iran, established by three collaborating organisations in 1999, including the centre for disease control (cdc) at the ministry of health (moh), pasteur institute of iran (pii) (with the establishment of arboviruses 16 all res. j.biol, 2013, 4, 16-18     and viral haemorrhagicfevers laboratory known as a national reference lab) and the veterinary organisation, all organised at the national level. these three organisations instituted a national expert committee on viral haemorrhagic fevers (nec). in the framework of the nec, an active collaboration on rvf monitoring has been undertaken over a span of 10 years between 2001 and 2011 in the nine provinces of iran which have led to this report. geographical distribution of these provinces were as follows: khorasan province (northeast iran), tehran province (northern iran), isfahan province (central iran), fars province (southern iran), kerman province (southeast iran), sistan va baluchistan (southeast iran), kurdistan (western iran), hormozgan province (southern iran) and bushehr province (southwest iran). these provinces were selected randomly from among the 30 provinces of the country. 2.2. sampling animal and human sampling was conducted at various intervals between 2001 and 2011. during this period, a total number of 405 caprine samples were gathered from suspected goats in four provinces (155 from hormozgan, 157 from fars, 80 from bushehr and 13 from tehran). to monitor seroprevalence of rvf among suspected sheep, 1206 ovine samples were collected in five provinces (283 from bushehr, 102 from fars, 296 from isfahan, 118 kurdistan and 407 from hormozgan). three provinces were selected to collect a total number of 325 bovine samples from suspected cattle (51 from sistan va baluchistan, 147 from hormozgan and 127 from bushehr). in 2001, 28 suspected camel sera were collected from sistan va baluchistan province. all animal samples were collected by the iranian veterinary organisation. animal samples were collected mostly from regions with casual reports of abortion. at the same time, 37 suspected human cases with rvf symptoms from kerman (17), bushehr (16) and khorasan (4) provinces were sent to the arboviruses and viral haemorrhagic fever laboratory (national. ref. lab) at the pasteur institute of iran, to be examined for rvf (figure 1). figure 1. geographical distribution of human and animal sampling in iran 2.3. igg detection for human and animal sera for elisa, the wells were coated overnight at 4°c with ag rvf, and the ag negative control was diluted at 1:800 in 1x phosphate buffered saline (pbs). the plates were then washed 3 times with phosphate buffered saline tween-20 (pbst). diluted positive control and negative control and human or animal samples (caprine, ovine, bovine and camel) in phosphate buffered saline tween-20 milk (pbstm) at 1:100 were added to the plate and incubated for 1 h at 37°c. the plates were then washed 3 times with pbst. anti igg human or animal conjugated with horseradish peroxidase (hrp), diluted in pbstm at 1/350, was added to the plate and incubated for 1 hour at 37°c. after washing, 3,3′,5,5′tetramethylbenzidine (tmb) was added and after a while the reaction was stopped with sulphuric acid 4n. ultimately, the od was read at 450 nm by elisa reader. 7 positive and negative rvfv antigens and also positive and negative controls were obtained from pasteur institute of dakar as who collaborating center. 2.4. rt_pcr for human sera viral rna was extracted from 140 µl of serum using qiaamp viral rna kit according to the instructions of the manufacturer (qiagen gmbh, hilden, germany). the samples were subsequently analysed by rt-pcr using specific primers nsca (5'-ccttaacctctaatcaac-3') and ns2g (5'-tgatttgcagagtggtcgtc-3'), which amplified an 800 bp fragment. amplification cycles were as follows: denaturation at 95°c for 30 s annealing at 55°c for 30 sec and extension at 72°c for 1 min. the final extension step was at 72°c for 5 min. amplified dna fragments were visualised after electrophoresis in 1.5% agarose gel and visualised under ultraviolet light. 8 positive (band 780 bp) 17 all res. j.biol, 2013, 4, 16-18     and negative controls were obtained from pasteur institute of dakar. to ensure the accuracy of the assays and eradicate potential false results, both serological and molecular assays were performed three times per sample. 3) results and discussion the study took place between 2001 and 2011. no rvfv igg positive cases were detected in 405 caprine, 1206 ovine, 325 bovine and 28 camel samples. in addition, none of the 37 suspected human cases with rvf symptoms tested positive for rvfv using serological and molecular tests. rvf is a viral disease affecting livestock, especially sheep and goats, causing abortion in females and a high mortality rate in newborn animals. 9 humans can be infected directly by contact with blood or abortion products of infected animals, or indirectly by mosquito bites. 5 animal serum samples were nevertheless collected from regions with some casual reports of abortion; none were igg positive for rfv. an explanation for this is that other diseases could have caused the animal abortions. 4) conclusion although disease is usually introduced by natural means 10, but the role of efficient surveillance and regional monitoring should not be omitted as protective strategies. for instance, by monitoring the situation of disease in hot spot countries and having put the import ban on live cattle in some years of outbreak, the potential risk of disease introduction would be declined.11 thus, despite the fact that no positive cases in human or animal population in studied areas were detected, successive surveillance and monitoring of viral haemorrhagic fevers, including rvf would seem logical, and can be effective for the prevention of the potential introduction of such uninvited zoonosis, as iran lies in close proximity to the ‘hot spot countries’ saudi arabia and yemen, which have notable outbreaks. 3, 4 in this regard, because of the aerosol infectivity and risk of dissemination of the virus, a need exists for on-going surveillance and monitoring of outbreaks in the country and region. acknowledgments we thank all members of the arboviruses and viral haemorrhagic fevers laboratory (national ref. lab); pasteur institute of iran, for their technical contributions. references 1. huiskonen jt, overby ak, weber f, grunewald k. (2009). electron cryo-microscopy and single-particle averaging of rift valley fever virus: evidence for gn-gc glycoprotein heterodimers. journal of virology 83(8), 3762-9. 2. woods cw, karpati am, grein t, mccarthy n, gaturuku p, muchiri e, et al. (2002). an outbreak of rift valley fever in northeastern kenya, 1997-98. emerging infectious diseases. 8(2),138. 3. madani ta, al-mazrou yy, al-jeffri mh, mishkhas aa, al-rabeah am, turkistani am, et al. (2003). rift valley fever epidemic in saudi arabia: epidemiological, clinical, and laboratory characteristics. clinical infectious diseases. 37(8), 1084-92. 4. shoemaker t, boulianne c, vincent mj, pezzanite l, al-qahtani mm, al-mazrou y, et al. (2002). genetic analysis of viruses associated with emergence of rift valley fever in saudi arabia and yemen, 2000-01. emerging infectious diseases. 8(12). 1415. 5. marrama l, spiegel a, ndiaye k, sall aa, gomes e, diallo m, et al. (2005). domestic transmission of rift valley fever virus in diawara (senegal) in 1998. 36(6). 1487-95 6. clements aca, pfeiffer du, martin v, otte mj. (2007). a rift valley fever atlas for africa. preventive veterinary medicine. 82(1), 72-82. 7. paweska jt, burt fj, anthony f, smith sj, grobbelaar aa, croft je, et al. (2003). igg-sandwich and igm-capture enzyme-linked immunosorbent assay for the detection of antibody to rift valley fever virus in domestic ruminants. journal of virological methods.113(2),103-12. 8. sall a, macondo e, sene o, diagne m, sylla r, mondo m, et al. (2002). use of reverse transcriptase pcr in early diagnosis of rift valley fever. clinical and diagnostic laboratory immunology.9(3),713-5. 9. pepin m, bouloy m, bird bh, kemp a, paweska j. (2010). rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention. veterinary research. 41(6). 10. martin v, chevalier vr, ceccato pn, anyamba a, de simone l, lubroth j, et al. (2008). the impact of climate change on the epidemiology and control of rift valley fever. revue scientifique et technique, office international des epizooties. 27(2), 413-26. 11. chevalier v, pepin m, plee l, lancelot r. (2010). rift valley fever--a threat for europe? euro surveillance: bulletin europeen sur les maladies transmissibles= european communicable disease bulletin. 15(10), 19506. 18 microsoft word 55-316-2-galleys.docx rectification of artificial molecular recombination with the use of high fidelity enzyme in the amplification of 16s rdna sequences from stool sample. vijay nema all res. j. biol., 2012, 3, 6-9 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 1, vol. 3, 2012, 6-9 rectification of artificial molecular recombination with the use of high fidelity enzyme in the amplification of 16s rdna sequences from stool sample. vijay nema 1 1) national aids research institute, 73 g midc bhosari, pune, maharashtra india 411026. e-mail: vnema@nariindia.org graphical abstract : abstract: reliance on routinely used taq polymerases for amplification may generate spurious sequences, especially in metagenomic studies consuming complex mixtures of various dna templates. this study reports one such incident wherein initial amplification of 16srrna gene from purified dna isolated from a stool sample, generated spurious sequences when amplified utilizing the taq polymerase used in routine amplifications. this was rectified when the same gene was reamplified using high-fidelity taq polymerase. use of high fidelity enzymes and verification of the sequences using various software tools before submission to the databases ensures better quality and confidence. keywords: metagenomics; 16s rdna; sequencing; chimera; high fidelity taq polymerase; pintail. 6 all res. j. biol, 2012, 3, 6-9 introduction with the legendary work of woese and his co-workers,1 a new kingdom archaea was defined using 16s ribosomal dna sequences and since then thousands of sequences have been submitted to ribosomal databases defining new strains and species. amplification of the 16s rdna sequences from various environmental and biological niches has become a general practice now.2-4 these pcr-based methods, which are otherwise very robust, have some significant drawbacks. chimera formation is one such problem that becomes more likely with the homology that exists between the different dna templates present in the samples from complex habitats. all 16s rdna sequences share a degree of homology due to the highly conserved nature of the 16s rrna gene. a chimera is a spurious gene sequence derived from two microorganisms that forms during pcr amplification.5,6 the formation of chimeras can be attributed to several phenomena like dna damage,7 high numbers of pcr amplification cycles8 and errors by non proof-reading enzymes during amplification.9 dna in stool samples may suffer these snags during amplification as it is always exposed to various kinds of deleterious conditions. this work reports one such incident where the use of high fidelity enzymes could rectify the problem of chimera formation through the use of pintail software to determine chimera like sequences.   materials and methods two frozen stool samples were obtained from stocks of microbiology division with the available background information about their origin for an exploratory study. sample 1, was obtained from a hiv positive individual with a diarrheal episode. sample 2 was obtained from a non hiv infected person with diarrhea. dna was isolated from these samples using qiamp dna stool mini kit (qiagen). isolated dna was amplified using universal primers viz. 27f and 1492r (table 1). the reaction mixture contained taq polymerase (bangalore genei 105914) with 10x buffer containing 100 mm tris (ph 9.0), 500 mm kcl and 0.1% gelatin with final concentration of 200µm of dntps and 20pm of each primer in a solution of 50µl adjusted with dnase free water. the reaction was repeated for 30 cycles with annealing temperature of 54°c for 1.5 minutes. the amplicons were purified by cutting the band from 1.5% low melting agarose gel after electrophoresis and eluting using qiaquick gel extraction kit. purified amplicons were cloned into strataclone™ pcr cloning vector psc-a that uses ligation of pcr product through a-u base-pairing followed by topoisomerase i-mediated strand ligation. clones obtained after transformation were picked and analyzed for insertion by gel electrophoresis. positive clones were subjected to plasmid isolation using qiagen plasmid mini kit. purified plasmids were used for sequencing the inserted gene using m13 forward and m13 reverse primers and pd primer as an intermediate primer to cover the complete sequence (table 1). table 1. primers and their sequences used for amplification and sequencing. the sequencing of plasmid dna was carried out using big dye terminator v. 3.1 cycle sequencing kit and an abi 3730xl dna analyzer, according to the manufacturer’s protocol. nucleotide sequence analysis and curing were done using applied biosystem seqscape v.2.5. the cured sequences were blasted to analyze sequence similarities with the sequences present in nucleotide database using blastn 2.2.25 from ncbi. results and discussion different sequences showed similarities with different genera in the cloning products from both the samples. ribosomal rnas are generally highly conserved and thus are expected to obtain similarities to other rrna sequences over the entire length. keeping this in consideration, the sequences were checked with pintail software10 for chimera formation or any other sequence anomalies. all the clones from sample 2 were found to be non-anomalous. in sample 1 all (except one) sequences were found to be non-anomalous. a sequence (1442bp long) from one of the clones from sample 1 showed the highest sequence similarity of 94% with 28 gaps to lactobacillus gasseri jv-v03 sca04-contig11, whole genome shotgun sequence (accession no. acgo02000005.1). the analysis was done using pintail software with the sequence in question as query (1442 nt) and subject was the most identical sequence as obtained with blast. the in-built parameters of the software determined the sequence variability with a 300 base window, moving 25 bases at a time along the sequence length. the software depicted the probability of two non-anomalous sequences producing a de (deviation from expectation) of 3.51, when they differ by 6.01 % overall, to be 0.25 > p > 0.05. hence despite an acceptable de value, the lower confidence indicated a caution (figure 1). to locate the segment of variation the sequence was run again in pintail by removing 100bp at a time from beginning to end of the query sequence and vice versa. it was observed that region around 750 to 950 bp according to the base position of 16s rrna gene gave the highest anomaly and could have been the region of chimera formation. the clone was again sequenced for 16srrna gene using the purified plasmid and exactly the same sequences were obtained. this indicated the problem with the insert used for cloning and not with the clone itself. this primer name nucleotide sequences 5´ to 3´ reference 27 forward gag ttt gat cmt ggc tca g 11 1492 reverse ggy tac ctt gtt acg act t 11 m 13 forward gta aaa cga cgg cca g universal sequencing primer m 13 reverse cag gaa aca gct atg ac universal sequencing primer pd cag cag ccg cgg taa tac 12 7 all res. j. biol, 2012, 3, 6-9     insert was the amplicon obtained after the amplification of 16srrna gene from purified dna of sample 1. figure 1. variation in % difference between clone sequence (query) and acgo02000005.1 sequence (subject). to confirm if the sequence has an artificial event of recombination or is a naturally occurring chimera-like sequence, the experiment was performed from the beginning by repeating the pcr amplification using the original dna isolated from the sample 1. this time, a high fidelity taq polymerase (platinum® taq dna polymerase high fidelity from invitrogen) was used for the amplification. a compatible 10x buffer was used as provided by the enzyme manufacturer. other reagents like dntps and primers were all kept constant like previous experiments. also, other steps of gel elution, cloning, sequencing and blast analysis remained the same as before. apart from other sequences showing similarity with other organisms, a few clone sequences were found to be 99-100% identical with lactobacillus gasseri gene for 16s rrna, complete sequence (accession no. ab517146.1). all these sequences when compared with the gene sequence mentioned above using pintail, gave no anomaly and were also observed to be practically identical. no other clone gave any such indication for chimera formation using pintail, when compared with the closest match as per the blast analysis. this indicates that the use of high fidelity enzymes for amplification of 16s rdna sequences from complex dna samples can help in obtaining reliable sequences. a few reagents and reaction conditions used in both the experiments were different but this did not produce any variation in other clones when the results from both experiments were compared. also within one experiment, when different clones were analysed for anomalies, only the experiment with taq polymerase without proofreading activity produced a clone with spurious sequences. this observation substantiated the outcomes of an earlier study that discussed the use of processivity-enhanced polymerases for the reduction in pcr mediated recombination.9 also in this study, amplification with high fidelity enzyme has generated 40 different positive clones identified after sequencing as compared to 30 different positive clones been generated with the use of routinely used taq polymerase when all other procedures were kept constant. later analysis of the results showed that sample 1 contained a diverse set of genus whereas sample 2 was limited in genus diversity (data not shown). hence the complexity of sample 1 but not sample 2 might have made the phenomenon of chimera formation possible. as metagenomic approaches are taking the front seat in the analysis of environmental samples, practices involving amplification of various genes, sequencing and sequence submission to various databases are becoming a routine. with the advanced knowledge and tools, we now have sufficient evidence that whatever is being amplified may not show the real picture. hence care must be taken at every step to ensure the quality of data being generated and made available as standards for future analysis in the databases. acknowledgements: author is thankful to director nari for infrastructural and moral support. also the help of dr. jayanta bhattacharya, head, molecular virology division at nari and his team during experimentation and constructive suggestions of the genbank submissions staff for sequence analysis is highly acknowledged. competing interest: the author declare no competing interests references: 1. woese, c. r., and fox, g. e. (1977). phylogenetic structure of the prokaryotic domain: the primary kingdoms. proc natl acad sci u s a 74, 50885090. 2. giovannoni, s. j., britschgi, t. b., moyer, c. l., and field, k. g. (1990). genetic diversity in sargasso sea bacterioplankton. nature 345, 60-63. 3. ward, d. m., weller, r., and bateson, m. m. (1990). 16s rrna sequences reveal numerous uncultured microorganisms in a natural community. nature 345, 63-65. 4. weller, r., and ward, d. m. (1989). selective recovery of 16s rrna sequences from natural microbial communities in the form of cdna. appl environ microbiol 55, 1818-1822. 5. meyerhans, a., vartanian, j. p., and wain-hobson, s. (1990). dna recombination during pcr. nucleic acids res 18, 1687-1691. 6. shuldiner, a. r., nirula, a., and roth, j. (1989). hybrid dna artifact from pcr of closely related target sequences. nucleic acids res 17, 4409. 7. paabo, s., irwin, d. m., and wilson, a. c. (1990). dna damage promotes jumping between templates during enzymatic amplification. j biol chem 265, 4718-4721. 8. wang, g. c., and wang, y. (1996). the frequency of chimeric molecules as a consequence of pcr coamplification of 16s rrna genes from different bacterial species. microbiology 142 ( pt 5), 11071114. 9. lahr, d. j., and katz, l. a. (2009). reducing the impact of pcr-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity dna polymerase. biotechniques 47, 857-866. 8 all res. j. biol, 2012, 3, 6-9     10. ashelford, k. e., chuzhanova, n. a., fry, j. c., jones, a. j., and weightman, a. j. (2005). at least 1 in 20 16s rrna sequence records currently held in public repositories is estimated to contain substantial anomalies. appl environ microbiol 71, 7724-7736. 11. frank, j. a., reich, c. i., sharma, s., weisbaum, j. s., wilson, b. a., and olsen, g. j. (2008). critical evaluation of two primers commonly used for amplification of bacterial 16s rrna genes. appl environ microbiol 74, 2461-2470. 12. weisburg, w. g., barns, s. m., pelletier, d. a., and lane, d. j. (1991). 16s ribosomal dna amplification for phylogenetic study. j bacteriol 173, 697-703.     9 editorial editorial david alcantara, joe blois, carlos juan ceacero all res. j. biol, 2010, 1, 1-3 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com editorial issue 1, volumen 1, 2010, 1-3 1 editorial david alcantara 1 , joe blois 2 , carlos juan ceacero 3 1) center for nuclear medicine and molecular imaging, mgh-harvard medical school, 13th st. blg 149, charlestown ma-02129, usa 2) genzyme corporation, 49 new york avenue, framingham, ma-01701, usa 3) cell biology department, pablo de olavide university, ctra. de utrera, km. 1 41013, seville, spain. dear readers of the all results journals:biol, we are pleased to introduce to you the all results journals: biol (all res. j. biol), a very unique journal that publishes articles and reviews with negative results in the field of biology. this journal represents the first total open access source for research concerning negative results and will be a valuable resource for researchers all over the world; experts and those new to the field alike. our immediate goal is to provide scientists with responsible and balanced information in order to advance faster, improve experimental designs and clinical decisions. many journals skew towards only publishing “positive” data; that is, data that successfully proves a hypothesis. the all results journals: biol is the home for negative or “secondary” data: experimental documentation of hypotheses that turn out not to be true, or other experiments that do not lead to an advance of a specific hypothesis but are, nevertheless, a true rendering of that experiment. for example, if a researcher set up a cell-based experiment and the experiment did not work in a particular set of conditions, it would be very useful for other researchers to know this (to avoid time and money wasting and better planning). there is a huge untapped resource of experimental data locked up in laboratory notebooks that could be of great service to the scientific community at large. many experiments fail to produce results or expected discoveries. this high percentage of „failed‟ research can still generate high quality knowledge. the main objective of the all results journals: biol is to recover and publish these valuable pieces of scientific information. as we publish negative results, the newer generation of researchers will not waste their time and money repeating the same studies and finding the same results (negative in this case). we believe that negative results are high-level pieces of knowledge that deserves to be published. the all results journals: biol is a peer reviewed journal developed to publish original, innovative and novel research articles resulting in negative results. this peer-reviewed scientific journal publishes theoretical and empirical papers that report negative findings and research failures in biology and related fields. submissions should have a negative focus; experiments that yield negative results will be given more preference. all theoretical and methodological perspectives are welcomed. we also encourage the submission of short papers/communications presenting counter-examples to usually accepted conjectures or to published papers. negative results in biology biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. biology is a vast subject containing many subdivisions, topics, and disciplines. it seems with our ever increasing ability to dissect biological systems to finer detail, simplicity of explanation becomes more elusive. with this in mind, the all results journals serves as a platform and resource for your important negative observations that stand the test of rigorous scientific scrutiny and methodology in the complex fields of biology. data collected under exceptional experimental design that may not support a convention in a given area of research should and can be reported. negative results shape the development of effective therapeutic agents, help us understand what cell types are critical for autoimmune pathogenesis and redefine the molecular targets of a drug. these data serve to drive the scientific method forward by showing the path not to follow. as scientists we strive for remarkable observations within biological systems that will further expand our understanding of the human condition, aging, cancer, autoimmunity, etc. at the all results journal we know how science gets done; sometimes the pieces just don‟t add up. these negative results drive our next step at the bench but are rarely published. we are working to bring to light these types of observations to be published under peer review for the greater good. our goal is to make accessible a manuscript about what didn‟t work so you can build on the mistakes of others rather than simply repeat them. instead of three steps forward and two steps http://en.wikipedia.org/wiki/natural_science http://en.wikipedia.org/wiki/life http://en.wikipedia.org/wiki/life http://en.wikipedia.org/wiki/organism all res. j. biol, 1, 2010, 1-3 2 back, science could just move forward. we now have an unbiased forum to present a negative finding. in cancer research or chemotherapeutic development, for example, the trend is to publish data showing efficacy. we‟d offer that inefficacy could also be of great importance to the scientific community. what agents failed, in what types of cancer and why; the latter question albeit difficult to answer. one could imagine the same trends emerging from this type of work in terms of gene expression profiling, proteomics and biomarkers. agent x will not be effective in cancer y because of overexpression of biomarker z. a manuscript focused on the inefficacy of a particular chemotherapeutic agent could assist in moving the cancer biology field forward by offering a forum to share with the greater cancer research community the same negative findings that may have contributed to the development of a highly effective agent. breaking this cycle of publishing only positive results will undoubtedly improve our ability to make educated decisions at the bench in biology. furthermore, there are many research based scientific disciplines that would benefit from bringing this important work to the mainstream of scientific publication and peer review. this trend (resistance to publish negative results or unsuccessful experiments) has been recently defined as “publication bias” and has major ramifications for the health of citizens. publication bias is a growing problem and some authors are now extensively writing about it. 1 not only health but also ecology has shown this publication bias and have been widely discussed by different authors in recent years. 2-5 generally, results that either fail to reject a null hypothesis or do not accord with the current consensus are often not published, which may lead to a biased representation of natural processes. 6 although it is believed that publication and dissemination bias is less pronounced in ecology than medicine, 3, 4 there is the same resistance among the authors of several fields to submit their negative results. this problem does exist in ecology and others sub-fields of biology (botany, biochemistry, genetics, etc.) and is probably accentuated by the lack of a venue for publishing negative results like the all results journals: biol. contradictions of current expectations can also suffer bias. this trend might be perpetuated by the attitude of researchers who have deliberately hidden negative results or by the ones who neglected or forgot about results entirely (mainly due to lack of time). large research groups might continue with other experiments without stopping, analysing or reporting negative results. in these cases, the authors contribute to a growing problem because they consider those results to be less interesting and important than they actually are. 3 it may contribute to biases in meta-analytical studies due to negative results being less accessible to the wider scientific community. another important type of publication bias in ecology (as in other sub-fields) arises from replication. biological systems are difficult and costly to replicate under natural conditions (i.e. natural variables are very heterogeneous in space and time) and replicate studies often reveal nothing new and/or produce negative results. 5 the all results journals: biol can help to fight the publication bias problem in biology (and its sub-fields) first, providing an excellent way for negative results (non-significant, contradict current expectations, lack of replication, etc.) publication and second, contributing to increase the negative results‟ knowledge for scientific progress. additionally, researchers must overcome their selfimposed barriers to the publication of negative results and give them the attention they deserve. in this issue in this first issue we feature an updated review on malaria, highlighting some negative results obtained in treatments. the paper highlights the plasmodium genes of interest playing a role in resistance to first line therapies such as chloroquine and sulfadoxine-pyrimethanine. the respective mode of action of these and other second generation compounds are discussed and presented as the next line of combination therapies that will hopefully overwhelm resistance genes. the authors provide the scope and history of antimalarial drug development as well as the problems facing implementation of drug regimens, diagnosis and follow-up statistics of patients. at the heart of this review is the failure and limitations of some of the most recently developed anti-malarial agents at various stages of clinical development. therein the authors review shortcomings in study design germane to current nonhuman primate models available. they go on to discuss the biochemical rationale of the various agents and offset this with potential side effects of the drugs. the subject matter at hand, namely the difficulties with development of an effective antimalarial agent and achieving clinical success are in the spirit of the all results journal. the second article describes the negative results obtained when testing a new protecting ischemic stroke drug. thromboembolic occlusion of intracerebral vessels is responsible for the majority of ischemic strokes. the intrinsic pathway for thrombus formation is initiated when coagulation factor xii (fxii) becomes activated on a negatively charged surface followed by successive activation of factor xi (fxi) and factor ix (fix). it has been shown that fxii-deficient mice were protected from pathological thrombus formation so the use of fxii inhibitors would be associated with relatively low rates of therapy-related hemorrhages, the major clinical complication associated with current anticoagulant therapies. the authors tested the new chemical cou254, a 3carboxamide-coumarine that selectively inhibits fxiia, in a rodent model. the authors found no differences between controls and mice treated with cou254 when they induced cerebral ischemia. in addition, they didn‟t find any significant all res. j. biol, 1, 2010, 1-3 3 differences in infarct volumes in both groups. furthermore, analysis of the neurological status in both groups did not reveal any beneficial effects of cou254 in acute ischemic stroke or any differences in thrombus formation. the authors pointed out some reasons why these negative results were obtained like optimum dosage or correct timing of drug administration after ischemic stroke induction. we agree with the authors that further preclinical evaluation is needed. this negative result opens the door to new antithrombotic drug improvements. epilogue we strongly believe that the total open access format of the new journal has clear benefits for science, medicine and the general public: first, all articles are freely and universally accessible online, and so an author's work can be read by anyone at no cost. the easy and widespread availability of articles significantly enhances reading and citation of the results. second, all accepted articles are immediately published with no delay and therefore, allow particularly rapid dissemination of new results. third, the all results journals: biol. allows interactive discussion and annotation of articles providing an online tool for open discussion of data. fourth, there is no size restriction for articles and no publication charges to authors. authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited. there is an ethical imperative and a significant challenge to ensure that finite research resources are better used, avoiding replication of previous experiments leading to an optimization on the use of resources. the all results journals: biol is tackling that challenge, providing to scientists a new tool for publishing their negative results. we invite you to dig into your file drawer or hard drive for the negative results and submit them to the all results journals: biol. all results are good results. references 1. mcgauran, n.; wieseler, b.; kreis, j.; schuler, y. b.; kolsch, h.; kaiser, t. (2010). reporting bias in medical research a narrative review. trials, 11, 37. 2. kotiaho, j. s.; tomkins, j. l. (2002). meta-analysis, can it ever fail? oikos, 96, (3), 551-553. 3. koricheva, j. (2003). non-significant results in ecology: a burden or a blessing in disguise? oikos, 102, (2), 397-401. 4. leimu, r.; koricheva, j. (2004). cumulative meta-analysis: a new tool for detection of temporal trends and publication bias in ecology. proc biol sci, 271, (1551), 1961-6. 5. kotze, d. j., johnson, c.a., o'hara, r.b., vepsäläinen k. & fowler, m.s. (2004). editorial. the journal of negative results ecology and evolutionary biology, 1, 1-5. 6. knight, j. (2003). negative results: null and void. nature, 422, (6932), 554-5. microsoft word 124-725-4-layout.doc evaluation of the antibacterial activity of adansonia digitata l. seed oil obtained from zaria, kaduna state, nigeria on some strains of bacteria p.r.o. edogbanya, e. dangana, y. apeji all res. j. biol., 2015, 6, 37-41 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 3, vol 6, 2015, 37-41 evaluation of the antibacterial activity of adansonia digitata l. seed oil obtained from zaria, kaduna state, nigeria on some strains of bacteria p.r.o. edogbanya*1, e. dangana2, y. apeji2 1 department of biological sciences, faculty of science, ahmadu bello university, zaria, nigeria. 2 department of pharmaceutics, faculty of pharmaceutical science, ahmadu bello university, zaria, nigeria. *corresponding author: +23408138381212, ocholiedogbanya@gmail.com graphical abstract abstract the use of essential oils as antibacterial agents have become popular over the years, this is in a bid to search for alternative ways of dealing with strains of bacteria that have become resistant to conventional antibiotics. this study was carried out to evaluate the antibacterial potentials of adansonia digitata seed oil obtained from zaria, kaduna state, nigeria, on the clinical isolates of some bacteria (bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa and escherichia coli). the oil was extracted from the seeds using the soxhlet extraction method with n-hexane as the solvent. the well diffusion method was used to test the susceptibility of the strains of bacteria to the oil, using gentamycin and streptomycin as standard positive controls. experiments were carried out in duplicates. data obtained from the experiment was analysed using one-way analysis of variance (anova) and duncan multiple range test (dmrt), with p < 0.05 considered significant. the results revealed that a. digitata oil was unable to create any inhibition zones in the bacteria cultures. from this research, it can be concluded that a. digitata oil had no antibacterial activity. keywords: adansonia digitata; antibacterial; bacteria; essential oils; seed 37 all res. j.biol, 2015, 6, 37-41     introduction the world health organisation has reported that diseases caused by pathogenic bacteria are one of the main cause of morbidity and mortality worldwide1. drug resistance has become a mitigating factor in the effective treatment of many bacterial diseases, therefore, there is an intense search for alternative methods of treating these diseases2, 3. many plants are known to contain essential oils (also known as volatile oils) which are aromatic in nature. these oils are by-products of metabolism secreted by special glands and stored up in various parts (such as leaves, flowers, roots, buds, twigs, rhizomes, bark, seeds and fruits etc.), and are aromatic in nature. from time immemorial the antimicrobial properties of essential oils have been known. the ‘first-aid' kit containing myrrh essential oil was used by few greek soldiers in the battlefield to treat wounds and was introduced by physician galen4. many of them have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases5. adansonia digitata l. – baobab (commonly known as “kuka” in northern nigeria) is a deciduous tree belonging to the malvaceae family and is indigenous to arid central africa6, 7. it is a large imposing tree which reaches heights of about 18-25 m and produces a rounded crown showing a stiff branching habit. it has a peculiarly swollen trunk of up to 10 m in diameter, usually tapering or cylindrical and abruptly bottleshaped; often buttressed. giant individuals can reach a girth of up to 28 m. it is widely distributed and can be found in most of sub-saharan africa’s semi-arid and sub-humid regions as well as in western madagascar 8. different parts of the tree are used as foods and medicines including the back fibres. no part of the tree is a waste9, 10, 11, 12. the seeds have a characteristic bean shape and have been reported to contain essential oil11, 12. this research attempts to evaluate the antibacterial efficacy of the seed oil of adansonia digitata. materials and methods collection of plant material dried fruits of adansonia digitata were collected from the department of biological sciences, ahmadu bello university, zaria, kaduna state, nigeria, and were verified in the herbarium of the department, with a voucher number of 2512. the fruits were split open and seeds were mechanically removed, properly washed, dried, pulverized into powder using mortar and pestle, sieved through a pore size of about 1mm, and stored in airtight containers. extraction of essential oils the extraction of essential oil was done using n-hexane in electro-thermal soxhlet extractor (gallenkamp, england). 30 g of powdered seeds was weighed and put into the thimbles of the soxhlet extractor, the apparatus was mounted and allowed to run for 1 hour, after which the mixture of essential oil and n-hexane was collected in a beaker and evaporated over a hot water bath to collect the oil as residue. bacterial strains clinical isolates of bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa and escherichia coli were obtained from the department of pharmaceutics and pharmaceutical microbiology, faculty of pharmaceutical sciences, ahmadu bello university, zaria. overnight cultures obtained using 10 ml nutrient agar were used for the study. well diffusion assays mueller hinton's agar was prepared according to manufacturer's instructions, poured into petri dishes, and allowed to set. the surface of the agar was flooded with 2 ml of isolate and the excess was drained into a disinfectant container. a cork borer was flamed and used to bore three wells in each agar. 100 µml each of undiluted adansonia digitata oil, 1 mg/ml of gentamycin and 0.1 mg/ml of streptomycin (which served as standard positive controls), were put separately in the three wells and allowed to stand for 1 h to diffuse properly. the plates were incubated for 24 h at 37°c and inhibition zones were measured13. the experiments were done in duplicates. statistical analysis one way analysis of variance (anova) was used to compare the mean inhibition zones of the different treatment groups and duncan multiple range test (dmrt) was used to separate means where significant. p < 0.05 was considered significant. results at the end of the experiment, it was observed that a. digitata oil had no inhibitory effect on all the bacteria isolates, this was confirmed by its failure to cause any inhibition zone around the wells containing the oil, 38 all res. j.biol, 2015, 6, 37-41     unlike the standard antibiotics used (table 1 and figure 1 – 4). table 1: mean inhibition zones of the different treatments on bacterial isolates mean ± s.e.m; n = 2; means in the same row with different superscripts are significantly different ( p < 0.05). figure 1: inhibitory effect of a. digitata oil (a), gentamycin (g) and streptomycin (s) on b. subtilis. figure 2: inhibitory effect of a. digitata oil (a), gentamycin (g) and streptomycin (s) on s. aureus figure 3: inhibitory effect of a. digitata oil (a), gentamycin (g) and streptomycin (s) on p. aeruginosa mean inhibition zones (mm) bacteria gentamycin (1 mg/ml) streptomycin (0.1 mg/ml) a.digitata oil b. subtilis 37.00 ± 1.00a 39.00 ± 1.00a 0.00 ± 0.00b s. aureus 36.00 ± 0.05b 37.00 ± 0.05a 0.00 ± 0.00c p. aeruginosa 34.50 ± 2.50a 33.50 ± 1.50a 0.00 ± 0.00b e. coli 34.50 ± 0.50a 29.50 ± 0.50b 0.00 ± 0.00c 39 all res. j.biol, 2015, 6, 37-41     figure 4: inhibitory effect of a. digitata oil (a), gentamycin (g) and streptomycin (s) on e. coli discussion the findings of this research were not in agreement with a similar work of samie et al. (2012)14 which reported that a. digitata oil was able to weakly inhibit the growth of a number of bacterial organisms, of which p. aeruginosa and s. aureus were amongst them. although samie et al. (2012) 14 also acknowledged that even though the oil showed inhibitory effect, it, however, had little bactericidal activity. the failure of a. digitata oil to cause an inhibitory effect on the bacterial organisms used in this study may be due to the fact that it is a weak antibacterial agent, and the strains of bacteria were resistant to it. djouahri et al. (2013) 15 reported that methods used in the extraction of essential oils causes variation in their chemical composition of the active compounds, and this may also be responsible for the discrepancy in results. samie et al. (2012)14 used the hydrodistillation method while the soxhlet extraction method was used in this research. ozcan and chalchat (2005) 16 reported that location causes a variation in the chemical composition of essential oils and this may be another possible factor that affected the results. samie et al. (2010) 14 used baobab seeds obtained from the south african region, while the seeds used for this research were obtained from the central african region (zaria, kaduna state, nigeria). it has also been reported that the hydrocarbon monoterpenes compounds found in essential oils show low antibacterial activity while oxygenated compounds possess high potential, especially phenol type compounds as thymol and carvacrol. oxygenated monoterpenes, exhibit strong antimicrobial activity, especially pronounced on whole cells, while hydrocarbon derivatives possess lower antimicrobial properties, as their low water solubility limits their diffusion through the medium 17, 18. the limitation of a. digitata oil’s antibacterial activity may be due to the fact that it contains more hydrocarbon monoterpenes than it does oxygenated compounds, although this has not been verified. however other parts of a. digitata have been reported to possess high antibacterial activity such as the root and stem barks 19, fruit pulp 20, and leaves 21. conclusion from this research, it can be concluded that a. digitata oil had no antibacterial activity against bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa and escherichia coli. further research could be done using pure strains of these bacteria and a different method of extraction of oil. references   1. world health organization (who) (1998). the world health report. life in the 21st century: a vision for all 2. measuring health. (world health organization, geneva, switzerland), pp. 39-60. 2. reynolds, j.e.f. (1998). martindale: the extra pharmacopeia (31st ed.). (london: royal pharmaceutical society of great britain), pp. 592-601. 3. prabuseenivasan, s., manickkam, j. and savarimuthu, i. (2006). in vitro antibacterial activity of some plant essential oils. bmc compl. alter. med., 6:39 doi:10.1186/1472-6882-6-39. 4. soni, s. and soni, u.n. (2014). in-vitro antibacterial and anti-fungal activity of some selected essential oils. int. j. pharm. sci., 6 (6): 586-591. 5. tepe, b., daferera, d., sokmen, m., polissiou, m. and sokmen, a. (2004) in vitro antimicrobial and antioxidant activities of the essential oils and various extracts of thymus eigii m. zohary et p.h. davis. j. agric. food chem., 52:1132-1137. 6. yazzie, d., vanderjagt, d.j., pastuszyn, a., okolo, a. and glew, h. (1994). the amino acid and mineral content of baobab (adansonia digitata l.) leaves. j. food comp. anal., 7, 189-193. 7. bremer, b., bremer, k., chase, m.w., reveal, j.l., soltis, d.e., soltis, p.s. zmarzty, s. (2003). an update of the angiosperm phylogeny group classification for the orders and families of flowering plants: apg ii. bot. j. linn. soc., 141, 399-436. 40 all res. j.biol, 2015, 6, 37-41     8. diop, a.g., sakho, m., dornier., m., cisse, m. and reynes, m. (2005). le baobab africain (adansonia digitata l.): principals caractéristques et utilisations. fruits, 61, 55-69. 9. igboeli, l.c., addy, e.o.h. and salami, l.i. (1997). effects of some processing techniques on the antinutrient contents of baobab seeds (adansonia digitata). bioresour. technol., 59, 29-31. 10. gebauer, j., el-siddig, k. and ebert, g. (2002).baobab (adansonia digitata l.): a review on a multipurpose tree with promising future in the sudan. gartenbauwissenschaft, 67, 155-160. 11. sidibe, m. and williams, j. t. (2002). baobab. adansonia digitata. (southampton, united kingdom: international centre for underutilised crops), pp. 14 – 16. 12. de caluwé, e., halamová, k. and van damme, p. (2010). adansonia digitata l. – a review of traditional uses, phytochemistry and pharmacology. afrik. foc., 23 (1): 11-51. 13. das, k., tiwari, r.k.s. and shrivastava, d.k. (2010). techniques for evaluation of medicinal plant products as antimicrobial agents: current methods and future trends. j. med. plants res., 4(2):104-111. 14. samie, a., nefefe, t., gundidza, m., mmbengwa, v., magwa, m. and mtshali m. s. (2012). antimicrobial activities and time kill profiles of five essential oils from southern africa against selected bacterial and fungal organisms. afr. j. pharm. pharmacol. 6(44):3086-3095. 15. djouahria, a., boudarenea, l. and meklatib, b.y.(2013) effect of extraction method on chemical composition, antioxidant and anti-inflammatory activities of essential oil from the leaves of algerian tetraclinis articulata (vahl) masters. ind. crops prod., 44 (2013): 32– 36. 16. ozcan, m. and chalchat, j.c. (2005). effect of different locations on the chemical composition of essential oils of laurel (laurus nobilis l.) leaves growing wild in turkey. j. med. food, 8(3) 8(3):408411. 17. knobloch, k., weigand, h., weis, n., schwarm, h.m. and vigenschow, h. (1986) action of terpenoids on energy metabolism. in progress in essential oil research; brunke, e.j., (ed.) (berlin, germany: walter de gruyter), pp. 429-445. 18. soković, m., glamočlija, j., marin, p.d., brkić, d. and van griensven, l.j. l. d.(2010) antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model. mol., 15, 7532-7546; doi: 10.3390/molecules15117532. 19. masola, s., mosha, r.d., wambura, p.n. (2009). assessment of antimicrobial activity of crude extracts of stem and root barks from adansonia digitata (bombacaceae) (african baobab). afr. j. biotechnol. 8(19):5076-5083. 20. afolabi, o.r. and popoola, t.o.s. (2005). the effects of baobab pulp powder on the microflora involved in tempe fermentation. eur. food res. technol., 220, 187-190. 21. ananil, k., hudson, j.b., de souzal, c., akpaganal, k., tower, g.h.n., amason, j.t. and gbeassor, m. (2000). investigation of medicinal plants of togo for antiviral and antimicrobial activities. j. pharm. biol. 38(1): 40-45. 41 microsoft word 116-717-2-le.docx an attempt to use immunohistochemical methods for semi-quantitative determination of surfactant in bronchial secretion after hyperbaric exposures piotr siermontowski, agnieszka pedrycz, janusz kopczyński, dorota kaczerska, katarzyna van damme-ostapowicz, romuald olszański all res. j. biol., 2015, 6, 31-36 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 3, vol 6, 2015, 31-36 an attempt to use immunohistochemical methods for semi-quantitative determination of surfactant in bronchial secretion after hyperbaric exposures piotr siermontowski1*, agnieszka pedrycz 2, janusz kopczyński3, dorota kaczerska4, katarzyna ostapowicz 5, romuald olszański1 1 maritime & hyperbaric medicine department, military institute of medicine in gdynia 3; 2 department of histology and embryology, medical university of lublin; 3 pathology department, holycross cancer center in kielce; 4 department of clinical nutrition, medical university of gdańsk; 5 department of integrated medical care, medical university of bialystok. *correspondence to: piotr siermontowski, maritime & hyperbaric medicine department, military institute of medicine, grudzinskiego 4, 81-103 gdynia, poland, e-mail: nurdok@tlen.pl abstract: background the most significant index of pulmonary oxygen toxicity is a decrease in vital capacity (vc) dependent on the duration of exposure and partial pressure of oxygen. the only method to measure this decrease is spirometry performed directly after exposure. objective the aim of the study was to check whether the extent of lung damage could be assessed by quantitative determination of pulmonary surfactant in bronchial secretion. design sputum samples were collected before, during and after hyperbaric air or oxygen exposures; histological preparations were prepared and stained immunohistochemically to visualize surfactant. amongst 781 samples collected, 209 contained sputum and 126 were included in the study. in this group, only 64 preparations could be paired for comparison. results the semi-quantitative method used and statistical findings have not demonstrated any significance. conclusions the method suggested for assessing the extent of lung damage has been found unsuitable for practical use due to difficulties in obtaining the proper sample; moreover, the study findings do not allow to draw conclusions concerning its effectiveness. keywords: oxygen toxicity, lung surfactant, immunochemical methods, detection 31 all res. j. biol, 2015, 6, 31-36     introduction toxic effects of oxygen are physiological and pathological reactions of organisms resulting from exposure to partial pressure of oxygen higher than that in the atmospheric air. the problem is becoming increasingly common due to more frequent oxygen use during diving, hyperbaric therapy1-3 and long-term oxygen therapy in patients with respiratory failure. the effect of oxygen during hyperbaric exposure is of particular relevance. although the exact reactions have not been fully elucidated, oxygen toxicity is assumed to be associated with the generation of its reactive forms4 and interactions with the adjacent cell structures, which lead to functional and morphological deficits5-8. the effector organs that are affected by oxygen are the central nervous system (cns) and lungs. the cns symptoms arise due to the partial pressure of oxygen9 whereas the lung symptoms depend on the partial pressure and duration of exposure10. when the partial pressure of oxygen in the pulmonary alveoli exceeds the arterial pao2, the lungs are considered to be exposed to a high oxygen pressure. at oxygen pressures of 0.05 – 0.2 mpa, maximum time of exposure is limited by the development of symptoms of pulmonary oxygen toxicity1,11,12. this limits the time of exposure of a diver under such conditions. in addition to clinical symptoms such as dyspnoea, pain and burning sensation in the throat and/or thorax, cough, retrosternal pain, or easy fatigue13-15, the basic measurable clinical index of pulmonary oxygen toxicity is a decrease in vital capacity (vc)13-15. when the partial pressure exceeds 50 kpa, the “oxygen clock” kicks in. the following units were introduced to determine quantitatively the toxic effects of oxygen on pulmonary parenchyma: unit of pulmonary toxicity dose (uptd), cumulative pulmonary toxicity dose (cptd), and oxygen tolerance unit (otu), which corresponds to damage resulting from one-minute exposure to 100% oxygen at a pressure of 0.1 mpa (1 ata)13 the out plays an essential role in estimation of a decrease in vc related to oxygen toxicity. a decrease in vc is associated with: • damage to surfactant structure • desquamation of surfactant to the alveolar lumen • toxic damage to type ii pneumocytes, i.e. inhibition of surfactant production. all the mechanisms mentioned above can be associated with the function of pulmonary surfactant, which maintains surface tension and alveolar size. the only reliable test determining the extent of lung damage (atelectasis) caused by hyperbaric oxygen is spirometry. its limitation is the necessity to perform the test immediately after diving. the use of histopathological or cytological methods would enable simultaneous collection and storage of samples from a large group of divers and performing the tests at any time after diving. aim the aim of the study was to design a method for indirect determination of lung damage under conditions of oxygen hyperbarism by examining the amount of surfactant in the bronchial secretion. materials and methods the sample, i.e. bronchial secretion, can be obtained from: • brochoaspirate • bronchial lavage • sputum the first two methods are invasive and complex. therefore, in our study the sputum was examined. the samples were collected between 2000-2011 during hyperbaric exposures in a pressure chamber using air or oxygen. air exposures were administered in a series of two 30-minute exposures with the first one to 0.4 mpa and the second one to 0.7 mpa with a one-day interval. the exposure profiles are presented in fig.1 and fig. 2. 32 all res. j. biol, 2015, 6, 31-36     figure 1. profile of 30m/30min. exposure16 exposure parameters: time taken to reach maximum depth: 3 min (v=10m/min), time between leaving surface and initiation of ascent (within any one immersion): 30 minutes, time taken to ascend from maximum depth to the first decompression stop: 3 min (v=8m/min), decompression:   6m/5min,  3m/15min. figure 2 .   profile   of   60m/30min. exposure16 parameters: time taken to reach maximum depth: 6 min (v=10m/min), time between leaving surface and initiation of ascent (within any one immersion): 30 minutes, time taken to ascend from maximum depth to the first decompression stop: 5 min (v=7.8m/min), decompression:   21m/12min, 18m/15min, 15m/16min, 12m/19min, 9m/28min, 6m/40min, 3m/52min. the samples from oxygen exposures were collected during oxygen tolerance test. this group was administered a single exposure. the exposure profile is presented in fig.3. figure 3. oxygen exposure profile17 the study encompassed 270 individuals undergoing training or experimental exposures in the hyperbaric complex dgkn 120, department of diving and underwater work technology, naval academy. in total, 781 samples were collected, including 84 from oxygen exposures. all participants were instructed on how to expectorate to limit the number of non-diagnostic samples such as the saliva. the sputum was expectorated into a polyethylene container filled three quarters with 80% ethyl alcohol after – rinsing mouth. the samples were marked in the following way: • “a” – before exposure • “x” – during exposure (only the exposures in the hyperbaric chamber) • “b” – after exposure • “c” – before the second exposure • “y” – during the second exposure (only the exposures in the hyperbaric chamber) • “d” – after the second exposure. the sputum was fixed in 80% ethyl alcohol for about 7 days and subsequently in 10% neutralized formalin directly before embedding in paraffin blocks. histopathological specimens were prepared using the paraffin method and sliced into 4-micron sections with a sledge microtome; 586 paraffin blocks were obtained from 33 all res. j. biol, 2015, 6, 31-36     781 samples. the remaining samples (195) formed a suspension in the fixer, from which the samples were not recovered despite the use of methods for cytological material recovery. similarly, some samples were lost during embedding in paraffin. histological specimens from paraffin blocks were stained with haematoxylin-eosin and selected for further tests. two pathologists assessed the same preparations independently. pulmonary macrophages were used to indicate the origin of sample from not only the oral cavity but also the bronchial tree. during the second stage of selection, the surface occupied by the sample identified as sputum was evaluated. when ten nonoverlapping fields were observed under 100 x magnification, the material qualified for further tests. two hundred and nine preparations were identified as “sputum”, including 126 selected for further tests based on the surface occupied by the bronchial tree material. the selected paraffin blocks were re-sliced and the assay for the presence of human surfactant using surfact protein a, rb x (millipore) was performed. a red colour denoted the presence of the reaction in the preparation. the microscopic picture of an example reaction in the preparation is shown in fig.4. figure 4. human surfactant immunohistochemical reaction preparations were assessed semi-quantitatively as follows: 0 – no reaction 1 – single colour fields in the preparation 2 – reaction involving over 25% of surface 3 – reaction involving over 75% of surface. the results were compiled in tables; whenever possible, they were paired and statistically analysed. results thirty-two cases (64 preparations) were found in which comparisons were possible as they formed pairs from the same individual and from the same exposure. the following pairs were used for comparisons: • “a” and “b” • “a” and “x” • “c” and “d” • “c” and “y” first, the normality of variable distribution was tested. all the variables showed the normal distribution. subsequently, descriptive statistics were used. high values of standard deviations were of interest, which is presented in fig.5. wykres ramka-wąsy średnia średnia±błąd std średnia±odch.std a x b c y d -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 figure 5. high values of standard deviations in all groups. ▫ – average, □ – average± se (standard error), ┬ ┴ average ± sd.(standard deviation) statistical significance was tested using the t test for dependent samples. based on the results indicated in the table below, we concluded that there were no statistically significant intergroup differences. the findings and significance level are depicted in tables 1 and 2.   34 all res. j. biol, 2015, 6, 31-36     table 1. the student’s ttest for dependent samples; p > 0.05 for each pair group a x b a 1.000000 0.476179 0.087693 x 0.476179 1.000000 0.194171 b 0.087693 0.194171 1.000000 table 2. the student’s ttest for dependent samples. the differences in bold are significant at p < 0.05 group c y d c 1.000000 0.234745 0.047892 y 0.234745 1.000000 0.284931 d 0.047892 0.284931 1.000000 the findings demonstrated that there were no statistically significant differences between the pairs of groups, except for groups c-d. however, the level of significance p is worth noticing, which is close to p=0.05. assessment of the efficiency of the method revealed that only 589 paraffin blocks were prepared from 781 samples . according to the first selection, sputum – non-sputum, 209 preparations were qualified; 126 preparations were suitable for semi-quantitative examinations, including 64 preparations that formed pairs enabling comparisons. percentage distribution is presented in table 3. table 3. percentage distribution number material percentage 781 collections 100 586 blocks 75 209 sputum 27 126 for analysis 16 64 preparations forming pairs 8 32 pairs discussion our study showed that pulmonary surfactant is desquamated, is present in the sputum and can be detected using immunohistochemical methods. moreover, the presence of surfactant in the preparation can be semi-quantitatively analysed. mouth rinsing was found essential for material collection. after careless mouth rinsing, food residues constituted a substantial part of the preparation; hard parts, when present (e.g. parts of nuts), hindered or prevented cutting of the paraffin block. the designed method for material collection fulfilled the conditions of simplicity and longer storage of material for further analysis. however, it did not fulfil the basic condition of probability of obtaining the diagnostic sample. although the procedures were followed strictly, only 27% of the sample was usable sputum. for comparisons, pairs of preparations were required and only 8% of preparations formed pairs. obtaining only 8% of preparations suitable for use disqualifies the method in question. furthermore, low efficiency of the method prevents its routine use for assessment of lung parenchyma damage after hyperbaric exposures. the authors plan to undertake further studies on the group of patients, who have been receiving a long-term oxygen treatment in intensive care units. the results obtained from their sputum will be compared with the results obtained from the bal. in spite of low efficiency of the method, semi-quantitative determinations and statistical analysis were carried out, which confirmed that the method was not useful. comparisons of pairs did not demonstrate statistical significance; extremely high scattering of results and high standard deviations in all groups hindered interpretation of results. 35 all res. j. biol, 2015, 6, 31-36     in our opinion, the only reliable method of determining lung damage caused by breathing oxygen, is the current method of spirometry. conclusions 1. there are no significant differences in the amount of surfactant in sputum before, during and after hyperbaric exposure. 2. the immunohistochemical method for determination of surfactant in sputum is not suitable for assessment of pulmonary oxygen toxicity due to difficulties in obtaining the diagnostic sample. acknowledgement special thanks to cmdr maciej konarski m.d. ph.d. for his help in study preparations. conflict of interest the authors declare that they have no conflict of interest in the research. the study as financed by kbn/ncn (state committee for scientific research/national science centre) research grant no. nn 404 109 739 references 1. clark j.m.: oxygen toxicity. in: bennett pb, elliott dh (eds.) the physiology and medicine of diving. w.b. saunders company ltd, london 1993, 121-169. 2. konarski m, klos r, nitsch-osuch a, korzeniewski k, prokop e.: lung function in divers. adv exp med biol. 2013, 788: 221-7 3. hamilton r.w.: tolerating exposure to high oxygen levels: repex and other methods. mar tech soc j 1989, 23, 19-25. 4. fisher a.b., bassett d.j.p., forman h.j.: oxygen toxicity of the lung: biochemical aspects. in: fishman a.p., 5. freeman b.a., crapo j.d.: biology of disease: free radicals and tissue injury. lab invest 1982, 47, 412-426. 6. donald k.w.: oxygen and the diver. the spa ltd, harley swan 1992 7. dweik r.a., laskowski d., abu – soud a.m., kaneko t., hutte r., stuehr d.j., erzurum s.c.: nitric oxide synthesis in the lung. regulation by oxygen through a kinetic mechanism. j. clin. invest. 1998, 101, 3, 660–666 8. gerschman r.: biological effects of oxygen. in: dickens f., neil e., (eds.) oxygen in the animal organism. macmillan, new york 1964, 475-494. 9. harabin a.l., survanshi s.s., homer l.d. 1994. a model for predicting central nervous system toxicity from hyperbaric oxygen exposure in man: efects of immersion, exercise, and old and new data. bethesda : naval medical research institute, 1994. nmri 94-0003; ad-a278 348 10. clark j.m., thom s.r. 2003. oxygen under pressure. [aut. książki] neuman t.s. brubakk a.o. bennett and elliott's physiology and medicine of diving. edinburgh : saunders, 2003, rozdział 9.4 11. clark j.m.: the toxicity of oxygen. am rev resp dis 1974, 110, 40-50. 12. jamieson d.: oxygen toxicity and reactive oxygen metabolites in mammals. free radical biol med 1989, 7, 87108. 13. konarski m., kłos r., siermontowski p.: preliminary evaluation of oxygen pulmonary toxicity risk during the oxygen tolerance test w: olszański r., (red.) wybrane problemy medycyny morskiej i nurkowej. wyd. ptmith, gdynia 2012, 136-179. (in polish) 14. clark j.m., lambertsen c.j.: pulmonary oxygen toxicity: a review. pharm rev 1971, 23, 37-133 15. clark j.m., lambertsen c.j.: rate of development of pulmonary oxygen toxicity in man during oxygen breathing at 2.0 ata. j appl physiol 1971, 30, 739-752. 16. tab. nr 3 mw rp syg. mar.woj. 860/81 17. kłos r. inherent unsaturation. the risk of central nervous system oxygen toxicity part 1. polhypres 2014, 1(46) in press. 36 microsoft word 70-436-1-galley.docx genetic analysis of congenital hemimelia in buffaloes from southern italy simona tafuri, luigi m. pavone*, dionea santoro, sara albarella, silviana rea, valeria de pasquale, rossella della morte, vincenzo peretti . all res. j. biol., 2013, 4, 2-6 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 1, vol 4, 2013, 2-6 genetic analysis of congenital hemimelia in buffaloes from southern italy simona tafuria, luigi m. pavoneb,*, dionea santorob, sara albarellaa, silviana reab, valeria de pasqualeb, rossella della mortea, vincenzo perettia adepartment of veterinary medicine and animal productions, university of naples federico ii, via f. delpino 1, 80137 naples, italy; bdepartment of molecular medicine and medical biotechnology, university of naples federico ii, via s. pansini 5, 80131 naples, italy; *corresponding author: luigimichele.pavone@unina.it abstract: hemimelia is a common congenital limb abnormality found in water buffaloes from southern italy. in humans, such a defect has been associated with mutations in wnt7a and esco2 genes. these two candidate genes were analyzed by polymerase chain reaction in the genomic dna extracted from the blood of buffaloes, and cows for control. no differences in wnt7a and esco2 sequences between affected and healthy buffaloes were identified. however, comparing sequences of control cows and buffaloes, wnt7a showed simple species polymorphisms, and esco2 showed seven base-pair substitutions. these results demonstrate that limb malformations in buffaloes are not related to congenital defects in wnt7a gene. interestingly, our findings highlight for the first time differences in the sequences of wnt7a and esco2 genes between buffaloes and cows. keywords: buffaloes, cows, hemimelia, esco2, wnt7a. hemimelia is a congenital malformation characterized by the absence of a limb portion. it is anatomically classified into two types: transversal hemimelia, characterized by complete absence of distal portion of the limb, and paraxial hemimelia characterized by aplasia of either radius or ulna, or tibia and fibula. in humans, these malformations are sporadic or very rare, with an incidence of approximately 1 per 1 million of live births. some genetic mutations that cause limb deficiencies are associated with an autosomal dominant inheritance; other genetic causes include an autosomal recessive inheritance and chromosomal aberrations. however, different teratogenic agents and drugs have been also related to hemimelia1. many case reports of hemimelia in cattle, sheep, dog, cat and goat have been reported2-4. recent studies highlighted the major molecular components that coordinate limb outgrowth along three axes: fibroblast growth factors (fgfs) control the proximodistal axis, the sonic hedgehog (shh) the anteroposterior axis, while the dorsoventral axis is regulated by bone morphogenetic proteins (bmps), engrailed-1 (en1), and wingless-type member 7a protein (wnt7a)5. the activities of these genes are mutually dependent. the wnt7a gene encodes a secreted signalling molecule that plays several roles in vertebrate development, and it is expressed in limbs, central nervous system, and urogenital tract. functional studies have demonstrated that wnt7a is needed for normal patterning of the limb buds6. homozyguos missense mutations in wnt7a cause two distinct limb-malformation disorders in humans: the fuhrmann syndrome and the al-awadi/raasrothschild phocomelia syndrome7. however, the wnt7a g204s mutation results to be associated with both al-awadiraas rothschild syndrome and fuhrmann syndrome phenotypes8. a phenotype similar to fuhrmann syndrome was detected in wnt7a knockout mice9. furthermore, the autosomal recessive human disorder roberts syndrome, characterized by craniofacial anomalies, tetraphocomelia, and loss of cohesion at heterochromatic regions of centromeres and y chromosome, has been associated to mutations in esco2 gene10. this gene encodes a protein belonging to the highly conserved eco1/ctf7 family of acetyl-transferases, and it is involved in the regulation of sister chromatid cohesion11. in humans, most esco2 mutations cause premature stop codons that may result in truncated proteins or mrna instability due to nonsensemediated mrna decay12. 2 all res. j.biol, 2013, 4, 2-6     in livestock, several congenital malformations such as amelia, polymelia, ectromelia and hemimelia have been associated with genomic instability4,13. in this study, we screened wnt7a and esco2 genes in water buffaloes affected by limb defects in order to check whether mutations in these genes could be associated with their hemimelia phenotype. bovine and human wnt7a genes differ for one additional exon present in the human genome, and bovine esco2 includes 10 exons whereas human esco2 comprises 11 exons spanning 30.3 kb, with the start codon in exon 2 and the stop codon in exon 11. we analyzed twenty-six mediterranean italian buffaloes from one day to six month old, 13 of which were affected by hemimelia (figure 1), and 13 were healthy. thirteen healthy cows were also studied controls. figure 1. italian mediterranean buffalo calf with left hind limb amputated off proximal epiphysis metatarsus. the clinical and radiological patterns observed in the malformed animals are reported in table 1. in their malformed limbs, all the animals showed more or less developed outlines of claws. materials and methods genomic dna was extracted from peripheral blood samples (1 ml) of all the animals using a purelink genomic dna mini kit (invitrogen, milan, italy) according to manufacturer’s instructions. using genomic dna templates, polymerase chain reaction (pcr) was performed to amplify the three exons of the bovine wnt7a gene and exon 2 of bovine esco2 gene. this esco2 exon was selected because of its high susceptibility to mutations in humans. primers were selected from bovine wnt7a and esco2 gene sequences (ensembl genome browser) using the primer3 input 0.4.0 program, because buffalo genome has not been sequenced yet. pcr mix contained in a final volume of 25 µl: 60 ng of genomic dna, 1 µm primer, 1.5 mm mgcl2, 1 u taq polymerase (eppendorf, milan, italy), and 0.2 mm dntps. pcr to amplify wnt7a exons was performed using a gene amp mj mini (biorad laboratories, rome, italy) as it follows: 1x (94°c for 4 min) and 38x (94°c for 45 s, 58.2°c (exon 3) and 63°c (exon 1 and 2) for 30 s, 72°c for 1 min). pcr to amplify esco2 exon 2 from genomic dna was performed as it follows: after an initial 5 min denaturation step at 95°c, 35 amplification cycles (94°c for 40 s, 60°c for 30 s, 72°c for 1 min) were carried out followed by a 10 min incubation at 72°c. the oligonucleotide primer sequences, and pcr product size for each exon are reported in table 2. table 1. clinical and radiological patterns observed in malformed animals. 1 female hind limbs amputated, the right amputated off the second tarsus bones and the left amputated off the proximal epiphysis metatarsus, and the right thoracic limb hypoplasic 2 females 1 male left hind limb amputated off the proximal epiphysis metatarsus 1 female left hind limb amputated off the third tarsus bones 1 female 1 male left hind limb amputated off the tibia 1 female left hind limb amputated off the distal epiphysis metatarsus 1 male left hind limb amputated off the first phalanx 1 male right hind limb amputated off the proximal epiphysis metatarsus 1 female left hind limb amputated off the proximal epiphysis tibia 2 males right hind limb amputated off the proximal epiphysis tibia 3 all res. j.biol, 2013, 4, 2-6     table 2. primers for amplification and sequencing of wnt7a exons and esco2 exon 2. forward primer reverse primer bp wnt7a exon 1 5’gtctgcaggct gtgccccgc-3’ 5’ccactttgagc tccttgccg-3’ 298 wnt7a exon 2 5’ggagccggga ggccgccttc3’ 5’cttccggcctg cctcattat-3’ 272 wnt7a exon 3 5’atcctggagg aaaacatgaa3’ 5’tcacttgcacg tgtagacct-3 480 esco2 exon2part1 5’atcaatggac tgtttccttt3’ 5’ggcttagaac tcgaggagca3 579 esco2 exon2part2 5’tgcaaggaaa accagtctgc3’ 5’ttagaagctat gaatttcca-3 504 results and discussion the pcr products were separated by electrophoresis to verify the expected length of amplified fragments. figure 2 shows the pcr products of the three wnt7a exons and esco2 exon 2 from cows, healthy and malformed buffaloes. no length differences were observed between the amplified products from cows, healthy and malformed buffaloes. pcr products were purified and sequenced, and the sequence of each exon was analyzed using codoncode aligner software. pcr amplifications and sequencing were performed in triplicate. table 3 summarizes the nucleotide differences between wnt7a sequences of cows, healthy and malformed buffaloes. the base-pair substitutions between cows and healthy buffaloes encode for the same amino acid, thus suggesting the occurrence of polymorphisms. no mutations were observed in wnt7a coding sequences of malformed buffaloes compared to healthy animals table 4 reports the specie specific nucleotide differences observed between esco2 exon 2 sequences of cows and healthy buffaloes. in three cases, the base-pair substitutions encode for the same amino acid, whereas, in five cases, the base-pair substitutions encode for amino acids with similar chemical properties, and, in two cases, for amino acids with different chemical properties. figure 2. pcr products of wnt7a exons and esco2 exon 2. a. pcr product of wnt7a exons from extracted dna of control cow (lane 1), healthy buffaloes (lane 2) and malformed buffaloes (lane 3). lane 4: negative control, sm: dna ladder. b. pcr product of esco2 exon 2 part 1 and exon 2 part 2 from extracted dna of control cows (lanes 1 and 4), healthy buffaloes (lanes 2 and 5) and malformed buffaloes (lanes 3 and 6). lanes 7 and 8: negative controls, sm: dna ladder. arrows indicate the size of pcr products. figure 3 shows the sequences of esco2 exon 2 that give rise to different amino acids between cows and healthy buffaloes. no differences were observed in the esco2 exon 2 sequences between healthy and malformed buffaloes (table 4). 4 all res. j.biol, 2013, 4, 2-6     table 3. wnt7a bp differences between control cows, healthy and malformed buffaloes. control cow healthy buffaloes malformed buffaloes bp aa bp aa bp aa 48 g l 16 48 a l 16 48 a l 16 273 t t 91 273 c/t t 91 273 c/t t 91 372 c t 124 372 g/c t 124 372 g t 124 393 c c 131 393 t c 131 393 t c 131 474 c y 158 474 t/c y 158 474 t y 158 519 a k 173 519 g k 173 519 g k 173 609 c h 203 609 t h 203 609 t h 203 633 g t 211 633 c t 211 633 c t 211 684 c l 228 684 t l 228 684 t l 228 798 t t 266 798 c t 266 798 c t 266 969 g q 323 969 a q 323 969 a q 323 the bp numbers are referred to bos taurus wnt7a cdna (ensbtat00000002188) table 4. esco2 bp and aa differences between control cows, healthy and malformed buffaloes. control cow healthy buffaloes malformed buffaloes bp aa bp aa bp aa 209 g r 70 209 a k 70 209 a k 70 262 g a 88 262 t s 88 262 t s 88 272 t v 91 272 c a 91 272 c a 91 313 t l 105 313 c l 105 313 c l 105 498 g v 166 498 a v 166 498 a v 166 566 a y 189 566 c s 189 566 c s 189 571 g a 191 571 a t 191 571 a t 191 626 t v 209 626 c a 209 626 c a 209 673 t s 225 673 a t 225 673 a t 225 831 c n 277 831 t n 277 831 t n 277 the bp numbers are referred to bos taurus esco2 exon 2+exon 1 cdna (ensbtat00000008606). in red highlighted species polymorphisms, in black highlighted conservative mutations and in green highlighted mutations for amino acids with different properties. figure 3. sequence analysis of the esco 2 exon 2 shows differences in the coding sequence between control cow and healthy buffaloes. in recent years, an increasing number of calves born in southern italy shows limb defects, and in particular, transversal hemimelia4. genomic instability has been demonstrated in these animals as proved by the high rates of structural chromosomal aberrations and increased sister chromatid exchanges detected in affected calves4,13-14. due to the economic and social impact of such a problem, molecular genetic studies, which allow identifying the genes responsible for these congenital defects, will help to find adequate strategies for the prevention of the disease. here, we investigated for the first time the wnt7a and esco 2 genes that are the main candidate genes involved in human severe limb pathologies such as fuhrmann syndrome and roberts syndrome. our results do not show genetic alterations in the wnt7a exons and esco2 exon 2 coding sequences of malformed buffaloes, although further studies on esco2 gene are needed to rule out its involvement in the pathogenesis of these congenital malformations. these findings suggest that the pathogenesis of hemimelia in buffaloes from southern italy could be probably related to genetic alterations in other genes involved in embryonic limb development. interestingly, our findings highlight for the first time differences in the sequences of wnt7a and esco2 genes between buffaloes and cows. 5 all res. j.biol, 2013, 4, 2-6     acknowledgments this work was supported by a grant “analisi genetica dell'emimelia congenita nel bufalo” from regione campania, italy. we thank dr. e. cirillo for administrative help. references 1. sanders, d.d. and stephens, t.d. (1991) review of drug-induced limb defects in mammals. teratology 44, 335-354. 2. baum, k.h., hull, b.l., weisbrode, s.e. (1985). radial agenesis and ulnar hypoplasia in two caprine kids. j. am. vet. med. association 186, 170-171. 3. lapointe, j.m., lachance, s., steffen, d.j. (2000). tibial hemimelia, meningocele, and abdominal hernia in shorthorn cattle. vet. pathol. 37, 508-511. 4. peretti, v., ciotola, f., albarella, s., restucci, b., meomartino, l., ferretti, l., barbieri, v., iannuzzi, l. (2008). increased sce levels in mediterranean italian buffaloes affected by limb malformation (transversal hemimelia). cytogenet. genome res. 120, 183-187. 5. niswander, l. (2003). pattern formation: old models out on a limb. nat. rev. genet. 4, 138-143. 6. yang, y., niswander, l. (1995) interaction between the signaling molecules wnt7a and shh during vertebrate limb development: dorsal signals regulate anteroposterior patterning. cell 80, 939-947. 7. woods, c.g., stricker, s., seemann, p., stern, r., cox, j., sherridan, e., roberts, e., springell, k., scott, s., karbani, g., sharif, s.m., toomes, c., bond, j., kumar, d., al-gazali, l., mundlos, s. (2006). mutations in wnt7a cause a range of limb malformations, including fuhrmann syndrome and the al-awadi/raas-rothschild phocomelia syndrome. am. j. hum. genet. 79, 402-408. 8. al-qattan mm, shamseldin he, alkuraya fs. (2013) the wnt7a g204s mutation is associated with both al-awadi-raas rothschild syndrome and fuhrmann syndrome phenotypes. gene 516, 168170. 9. parr, b.a., avery, e.j., cygan, j.a., mcmahon, a.p. (1998). the classical mouse mutant postaxial hemimelia results from a mutation in the wnt7a gene. dev. biol. 202, 228-234. 10. vega, h., waisfisz, q., gordillo, m., sakai, n., yanagihara, i, yamada, m., van gosliga, d., kayserili, h., xu, c., ozono, k., jabs, e.w., inui, k., joenje, h. (2005). roberts syndrome is caused by mutations in esco2, a human homolog of yeast eco1 that is essential for the establishment of sister chromatid cohesion. nat. genet. 37, 468-470. 11. gordillo, m., vega, h., trainer, a.h., hou, f., sakai, n., luque, r., kayserili, h., basaran, s., skovby, f., hennekam, r.c., uzielli, m.l., schnur, r.e., manouvrier, s., chang, s., blair, e., hurst, j.a., forzano, f., meins, m., simola, k.o., raasrothschild, a., schultz, r.a., mcdaniel, l.d., ozono, k., inui, k., zou, h., jabs. e.w. (2008). the molecular mechanism underlying roberts syndrome involves loss of esco2 acetyltransferase activity. hum. mol. genet. 17, 2172-2180. 12. vega h, trainer ah, gordillo m, crosier m, kayserili h, skovby f, uzielli ml, schnur re, manouvrier s, blair e, hurst ja, forzano f, meins m, simola ko, raas-rothschild a, hennekam rc, jabs ew. (2010). phenotypic variability in 49 cases of esco2 mutations, including novel missense and codon deletion in the acetyltransferase domain, correlates with esco2 expression and establishes the clinical criteria for roberts syndrome. j. med. genet. 47, 30-37. 13. albarella, s., ciotola, f., dario, c., iannuzzi, l., barbieri, v., peretti, v. (2009). chromosome instability in mediterranean italian buffaloes affected by limb malformation (transversal hemimelia). mutagenesis 24, 471-474. 14. di berardino, d., iannuzzi, l., fregola, a., matassino, d. (1983). chromosome instability in a calf affected by congenital malformation. vet. rec. 112, 429-432. 6 microsoft word 156-845-1-layoutediting.docx activity on non-methylated dna limits the use of endonuclease mspji for epigenetic analyses maría belén jerez* and maximiliano juri ayub* all res. j. biol., 2018, 9, 1-6 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol 9, 2018, 1-6 activity on non-methylated dna limits the use of endonuclease mspji for epigenetic analyses maría belén jerez* and maximiliano juri ayub* instituto multidisciplinario de investigaciones biológicas de san luis, imibio-sl-conicet and facultad de química, bioquímica y farmacia, universidad nacional de san luis, san luis argentina. * corresponding authors. e-mail: jerezbel@gmail.com; mjuriayub@hotmail.com graphical abstract: abstract: cytosine methylation of dna in mammals has been associated with both physiological and pathological changes in gene-expression. dna treatment with methylation sensitive and/or dependent restriction enzymes, followed by pcr amplification is a widely used approach to test cpg methylation. recently, restriction endonuclease mspji has been proposed as a promising tool for epigenetic analyses. in this paper, we have tested mspji as a tool for detecting cpg methylation on mammalian genomic dna. for this experiment mouse genomic sequences harboring or lacking cpg sites were selected. the extent of degradation was evaluated by pcr using primers flanking the chosen genomic regions. digestion of mouse genomic dna, in combination with end-point and real-time pcr reactions, revealed that mspji treatment reduced the amplification of genomic regions either containing or lacking of cpg motifs. in addition, treatment of bona fide non-methylated (in vitro amplified) dna samples definitely demonstrated that mspji shows significant activity against non-methylated dna. these results show that star activity can be an important concern when using mspji, even under standard conditions. therefore, we conclude that (in contrast to classical restriction enzymes), careful case by case evaluation of reaction conditions is mandatory for optimizing the usefulness of mspji in epigenetic studies. keywords: dna methylation; restriction enzyme; star activity; epigenetic; cytosine; mspji; real time pcr 1 all res. j.biol, 2018, 9, 1-6 introduction methylation of dna is associated with transcriptional repression, as well as other highly specialized processes such as genomic imprinting and x chromosome inactivation [1]. there is also growing evidence linking dna methylation to pathological processes such as cancer development [2]. these findings have raised interest about methodologies addressing dna methylation states. in particular, cytosine methylation at cpg sites on gene promoters of animal genomes is a widely explored feature, since 5-methylcytosine (5mc) has been described as a major regulator of gene expression [3]. basically, two general approaches are currently used for detecting 5mc, namely bisulfite conversion and restriction with methylation sensitive and/or dependent enzymes. a comprehensive study comparing the relative merits of these strategies has been recently performed [4]. bisulfite treatment followed by sequencing is the ‘gold standard’ for methylation quantification. however, this method is often time-consuming and demands lots of resources, impairing its wide usage in many non-specialized laboratories, in particular for exploratory or preliminary analyses. therefore, restriction-based approaches remain as a valuable alternative to analyze cpg methylation at the single-locus level. one of these strategies involves the treatment of genomic dna with methylation-dependent endonucleases. then, the methylation level of the selected genomic region is evaluated by pcr (preferably qpcr), using primers flanking the dna region under study (figure 1) [5]. one of the drawbacks of the restriction-based methods is that they are able to detect a subset of mcpg sites; namely those where the surrounding bases yield an adequate restriction site. therefore, the discovery of new methylation-dependent enzymes may allow expansion of the toolkit and the coverage of different methylation sites. recently, several new promising methyl-dependent enzymes have been discovered: mspji, fspei, lpnpi, aspbhi, rlai, and sgrti [6]. in particular, mspji has been characterized and described as specifically recognizing the mcnnr sequence and cleaving both strands by a few bases on the 3´ side of methylated cytosine, being inactive on non-methylated dna. for this reason, it represents a powerful tool for epigenetic studies, as it is able to detect around 50% of methylated cpg sites: mcgna and mcgng [6]. digestion of arabidopsis thaliana genomic dna with mspji followed by size fractionation of digested product has been reported to be useful for generating a methylated cytosine enriched sequencing library [7]. more recently, mspji has been used in combination with 5-methyldctp to achieve dna fragmentation prior to running samples in short-read dna sequencing platforms [8]. figure 1. schematic representation of the experimental design. genomic dna is incubated in the presence or in the absence of methylation-dependent endonucleases. if dna molecules are nonmethylated (left), both samples remain intact and can be amplified by pcr. if dna is methylated (right), endonuclease treated dna is (partially or completely) degraded leading to weaker or no amplification, as compared to non-enzyme control. partial methylation can be better detected by qpcr as a shift in cq values. therefore, we decided to evaluate this enzyme combined with pcr, as a tool to unveil the cpg methylation state of a selected locus. as proof of concept, the mouse interleukin 12b promoter gene was used. as control, a 95 bp region lacking cpg was selected. the relevant features of these selected genomic regions are depicted in figure 2. in this work, we provide evidence revealing that although mspji selectively degrades methylated dna, its activity on nonmethylated dna is not negligible. therefore, cautions should be taken when using this enzyme as an epigenetic tool. materials and methods all experiments shown in this paper have been performed at least three times. dna isolation dna was purified from different sources using the wizard® genomic dna purification kit (promega), following manufacturer’s instructions. integrity was confirmed by agarose electrophoresis and quantification was spectrophotometrically performed by measuring absorbance at 260 nm. 2 all res. j.biol, 2018, 9, 1-6 digestion with mspji genomic dna from different sources (1 µg) was digested according to the manufacturer’s instructions, using 4 units of mspji (neb) in the presence of 1 µl of double-stranded dna activator, in a final volume of 30 ul. all reactions were incubated at 37°c for 4 or 8 h. control and mspji treated samples were incubated at 65 °c for 20 min in order to inactivate the enzyme. after that, samples were always kept on ice or stored at -20 °c. to exclude any artifacts, control reactions included all the components except mspji, and were processed in parallel with mspji treated samples. digestion products were visualized by gel electrophoresis. it should be noted that in order to avoid artifacts, dna and enzyme concentrations, temperature and incubation times were kept in the range suggested by the manufacturer (0.5-1 µg dna; 2.5-5 u mspji, incubation at 37ºc for 4-16 h). digestion products were analyzed by agarose electrophoresis and/or used directly as templates for end-point or real-time pcr reactions. end-point pcr pcr reactions were performed in 20 µl volume using taquba dna polymerase (gifted by dr. mauro morgenfeld) essentially as previously described [9]. reaction buffer included tris-hcl (ph 9.0) 10 mm, kcl 50 mm, triton x-100 0.1 %, mgcl2 1.5 mm). we used 50 ng of genomic dna as a template, which was previously incubated at 37ºc in the presence or in the absence of mspji, as described above. the following oligonucleotides were designed to amplify a 1,799 bp fragment from the il-12b promoter gene: il-12bfw_1: 5’tcggccccatattgctttgt-3’ and il-12brev_1: 5’acagcctctagatgcaggga-3’ (figure 2). cycling conditions were as follows: 1 step at 94°c for 30 s; 35 cycles of 94 °c for 30 s, 63 °c for 45 s, 72 °c for 2 m; followed by a final elongation step of 72 °c for 10 m. amplicons were visualized by electrophoresis on 1.5 % agarose gel. real-time pcr pcr amplification mixtures contained 50 ng of genomic dna, faststart universal sybr green master (rox) (lifesciences, roche), 500 nm primers and ultrapure™ dnase/rnase-free distilled water (life technologies). reactions were run on an abi prism 7500 (applied biosystems) instrument. cycling conditions comprised 2 m at 50 °c, 10 m polymerase activation at 95 °c and 40 cycles of 95°c for 15 s and 60 °c for 60 s. the following primers were used to amplify a 333 bp sub-fragment from the il-12b promoter: il-12bfw_2: 5’-aagtgtgtggctgggaag-3’ and il-12brev_2: 5’-gttgatgttacctcccttcctc-3’. in addition, as negative control, a 95 bp cpg-lacking fragment of the beta-actin gene was amplified using the following oligonucleotides: actin-fw: 5’cttgatcttcatggtgctaggag-3’ and actin-rev: 5’cagtgctgtctggtggtac-3’ (figure 2). figure 2. scheme depicting the relevant features of target amplified sequences. left: a 1,799 bp fragment belonging to the il-12b promoter (including 40 cpg sites) was used for end-point pcr. a sub-fragment of 333 bp was used for qpcr amplification. right: a 95 bp fragment belonging to the beta actin gene, lacking cpg sites was used as a negative control in qpcr. green and yellow circles are cpg sites which can be or not interrogated by the mspji specificity, respectively. these sites are represented schematically on the figure and their number does not match with the actual number on these genome regions. results and discussion the mouse genome harbours a high percentage of cytosine methylation (7.6 %) [10]. as expected, a smeared pattern was observed after digestion of mouse dna with mspji under standard conditions (figure 3, left panel). as a negative control, genomic dna from the parasite giardia lamblia, which was available at our lab, was used. it is known that cytosine methylation is very low or even null in all the studied eukaryotic microorganisms [11,12,13,14,15]. in addition, it has been reported that the genome of g. lamblia is completely devoid of cytosine methyl transferase encoding genes [16]. we have further confirmed this by searching on the g. lamblia genomic databases for genes encoding the corresponding protein domain (interpro: ipr001525/pfam: pf00145). as expected, smearing of g. lamblia dna treated with mspji was negligible (figure 3, right panel). these results are consistent with a specific digestion of methylated dna by mspji, according to previous reports [6,7,8,17]. 3 all res. j.biol, 2018, 9, 1-6 however, careful observation of figure 3 right panel shows that the band size of the digested sample (lane 7) is slightly lower compared to the non-treated dna (lane 6). this fact is compatible with both a low proportion of methylated dna and/or non-specific activity of mspji on unmethylated dna. next, we used mspji-digested mouse dna as template to amplify a 1,799 bp fragment from the il-12b gene (gene id: ah004859) (nt -1693 to +106, according to the transcription initiation site), containing 40 cpg sites (see figure 2). out of these 40 cpg sites, 27 are overlapped with mspji recognition sites on one or both strands, and their methylation could be addressed (through digestion) with this enzyme. notably, mspji treatment completely prevented the amplification of the il-12b promoter, suggesting that this region was densely methylated at cpg sites (figure 4). this result prompted us to set up a quantitative amplification analysis by using qpcr instead of end-point pcr. we selected two different pcr targets (see figure 2): a 333 bp sub-region of the il-12b promoter (nt -1136 to -801, according to the transcription initiation site) including 19 cpg sites (13 overlapping with mspji restriction sites), and a 95 bp fragment belonging to the beta-actin gene (gene id: nc_000071.6, nt +999 to +1093 according to the transcription initiation site) lacking cpg sites. surprisingly, in both cases mspji treatment produced a 2-3 cycle shift of the cq value, when amplifying both regions containing or lacking of cpg sites (figure 5a). the identity of amplified fragments was confirmed by thermal dissociation (figure 5b). figure 3. electrophoresis of genomic dna from m. musculus and g. lamblia digested with mspji. lane 1 and 5: 1 kb dna ladder; lanes 2 and 6: undigested dna; lanes 3 and 7: dna incubated with mspji for 4 h; lane 4: dna incubated with mspji for 8 h. figure 4. amplification of il-12b promoter gene by end-point pcr. lane 1: 1 kb dna ladder. two replicates of genomic dna (1 and 2) were incubated in the absence (nd) or in the presence (d) of mspji for 4 h, and subsequently used as templates for pcr. the cq value shift for the 95 bp fragment lacking cpg sites strongly suggests that unmethylated dna is digested by mspji. however, as an alternative hypothesis it can be postulated that methylated cytosines at sequences other than cpg were present in the genomic samples, recognized and cleaved by mspji. in fact, non-cpg methylation has been reported in stem cells and, more recently, in differentiated mammalian cells [18]. therefore, in order to definitively evaluate the specificity of the assay using a bona fide nonmethylated sample, we replaced genomic dna by an aliquot of the 1,799 bp pcr product. interestingly, mspji degraded this unmethylated dna (figure 6a) and, consequently, prevented further pcr amplification (figure 6b). this experiment definitely confirmed that mspji degraded nonmethylated dna at a significant extent. these results demonstrate that although mspji selectively degrades methylated dna (figure 3) [6,7,8,17], residual activity on unmethylated dna should be considered and case by case evaluation and optimization is needed to improve its usefulness for epigenetic analyses based on methylationdependent restriction coupled to qpcr strategies. 4 all res. j.biol, 2018, 9, 1-6 figure 5. effect of mspji treatment on the real time amplification of genomic templates. top panel: real-time amplification of the 333 bp il-12b promoter fragment harboring 13 mspji-detectable cpg sites. bottom panel: real-time amplification of the 95 bp beta-actin fragment lacking cpg sites. a. amplification plots for genomic samples treated with (d) or without (nd) mspji. curves are representative of 12 independent experiments performed in duplicates. fluorescence values are shown on a logarithmic scale in order to better detect cq differences among samples. the identity of amplified fragments was confirmed by thermal dissociation (b). figure 6. degradation of bona fide non-methylated dna by mspji. a. an aliquot of the 1799bp pcr product was incubated in the absence (lane 2) or in the presence (lane 3) of mspji and reactions were analyzed by agarose electrophoresis. b. digestion products (2 ng) of (a) were used as templates for pcr amplification and products were analyzed by agarose electrophoresis. a negative pcr control was run in parallel (lane 4) with controls. in summary, we have demonstrated that mspji displays significant activity against non-methylated dna. therefore, cautions should be taken when using this enzyme for epigenetic studies. our results indicate that further characterization experiments on mspji would be helpful in order to improve the performance of this enzyme as an epigenetic tool. it would be relevant to address whether nonspecific activity is sequence-independent, or unmethylated target sequences (cnnr) are preferentially cleaved. providing a better understanding of these issues would allow developing genetic engineered variants of higher selectivity mspji. this would, in turn, yield high fidelity variants for this enzyme, a strategy that has shown to be successful with classical restriction enzymes. acknowledgments we are grateful to dr. maria l. mascotti for her helpful comments and contributions about the manuscript. we are also in debt with dr. walter j. lapadula for his help in searching for protein domains in the g. lamblia database. we also thank the two anonymous reviewers for their comments and suggestions about the manuscript. 5 all res. j.biol, 2018, 9, 1-6 references 1. goll mg, bestor th (2005) eukaryotic cytosine methyltransferases. annu rev biochem 74: 481-514. 2. ting ah, mcgarvey km, baylin sb (2006) the cancer epigenome--components and functional correlates. genes dev 20: 3215-3231. 3. deaton am, bird a (2011) cpg islands and the regulation of transcription. genes dev 25: 1010-1022. 4. redshaw n, huggett jf, taylor ms, foy ca, devonshire as (2014) quantification of epigenetic biomarkers: an evaluation of established and emerging methods for dna methylation analysis. bmc genomics 15: 1174. 5. hashimoto k, kokubun s, itoi e, roach hi (2007) improved quantification of dna methylation using methylation-sensitive restriction enzymes and real-time pcr. epigenetics 2: 86-91. 6. cohen-karni d, xu d, apone l, fomenkov a, sun z, et al. (2011) the mspji family of modification-dependent restriction endonucleases for epigenetic studies. proc natl acad sci u s a 108: 11040-11045. 7. huang x, lu h, wang jw, xu l, liu s, et al. (2013) high-throughput sequencing of methylated cytosine enriched by modification-dependent restriction endonuclease mspji. bmc genet 14: 56. 8. shinozuka h, cogan no, shinozuka m, marshall a, kay p, et al. (2015) a simple method for semi-random dna amplicon fragmentation using the methylation-dependent restriction enzyme mspji. bmc biotechnol 15: 25. 9. mascotti ml, lapadula wj, juri ayub m (2015) the origin and evolution of baeyer-villiger monooxygenases (bvmos): an ancestral family of flavin monooxygenases plos one (in press). 10. capuano f, mulleder m, kok r, blom hj, ralser m (2014) cytosine dna methylation is found in drosophila melanogaster but absent in saccharomyces cerevisiae, schizosaccharomyces pombe, and other yeast species. anal chem 86: 3697-3702. 11. gorovsky ma, hattman s, pleger gl (1973) ( 6 n)methyl adenine in the nuclear dna of a eukaryote, tetrahymena pyriformis. j cell biol 56: 697-701. 12. cummings dj, tait a, goddard jm (1974) methylated bases in dna from paramecium aurelia. biochim biophys acta 374: 1-11. 13. ammermann d, steinbruck g, baur r, wohlert h (1981) methylated bases in the dna of the ciliate stylonychia mytilus. eur j cell biol 24: 154-156. 14. rae pm, spear bb (1978) macronuclear dna of the hypotrichous ciliate oxytricha fallax. proc natl acad sci u s a 75: 4992-4996. 15. bracht jr, perlman dh, landweber lf (2012) cytosine methylation and hydroxymethylation mark dna for elimination in oxytricha trifallax. genome biol 13: r99. 16. ponger l, li w-h (2005) evolutionary diversification of dna methyltransferases in eukaryotic genomes. molecular biology and evolution 22: 1119-1128. 17. zheng y, cohen-karni d, xu d, chin hg, wilson g, et al. (2010) a unique family of mrr-like modificationdependent restriction endonucleases. nucleic acids res 38: 5527-5534. 18. pinney se (2014) mammalian non-cpg methylation: stem cells and beyond. biology (basel) 3: 739-751. 19. capuano f, mülleder m, kok r, blom hj, ralser m (2014) cytosine dna methylation is found in drosophila melanogaster but absent in saccharomyces cerevisiae, schizosaccharomyces pombe, and other yeast species. analytical chemistry 86: 3697-3702. 6 microsoft word 165-888-1-layoutediting.doc phytochemical screening and antimicrobial activity of terminalia catappa (l.) seed oil obtained from anyigba, kogi state, nigeria, on some selected clinical isolates p.r.o. edogbanya, m.o. suleiman, j.b. olorunmola, r.f. aminu and i.j. oijagbe all res. j. biol., 2020, 11, 1-5 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol 11, 2020, 1-5 phytochemical screening and antimicrobial activity of terminalia catappa (l.) seed oil obtained from anyigba, kogi state, nigeria, on some selected clinical isolates p.r.o. edogbanya *1, m.o. suleiman1, j.b. olorunmola1, r.f. aminu2 and i.j. oijagbe 3 1department of plant science and biotechnology, kogi state university, anyigba, nigeria. 2department of microbiology, kogi state university, anyigba, nigeria. 3department of biology, ahmadu bello university, zaria, nigeria. *corresponding author: ocholiedogbanya@gmail.com +2348138381212 grafical abstract abstract: this research was carried out in a bid to seek for alternative ways of dealing with strains of microorganisms that have become resistant to conventional antibiotics. the study was carried out to evaluate the antimicrobial efficacy of terminalia catappa seed oil obtained from anyigba, kogi state nigeria, on some selected clinical isolates of selected microorganisms (aspergillus niger, staphylocuccos aureus, escherichia coli). the oil was extracted from the seeds using the soxhlet extraction method with n-hexane as the solvent. the mueller hinton (well diffusion method) was used to test the susceptibility of the strains of the microorganisms to the oil, using ciproflaxin as standard positive controls, alongside tween and dmso as negative control. experiments were carried out in duplicates. the results obtained revealed that terminalia catappa oil was unable to create any inhibition zones on the clinical isolates. from this research, it can be concluded that terminalia catappa oil had no antibacterial activity. keywords: antimicrobial, terminalia catappa, essential oils, seed introduction although many different antibacterial agents are available in the field of medicine, many of these agents are increasingly being incapacitated by the microorganisms through the evolution of different mechanisms that amount to resistance to these drugs.1 presently in the developing countries, synthetic drugs are not only expensive and inadequate for the treatment of diseases but also are often with adulterations and side effects.2 there is therefore a continuous and urgent need to discover new antimicrobial components with diverse chemical structures and novel mechanisms of actions because of the increase in the incidence of new and re-emerging infectious diseases3 to replace those that have lost their 1 all res. j. biol, 2020, 11, 1-5 efficacy. research has, however, shown that many herbs possess varying degree of antimicrobial activities. kaufman et al. (1999)4 had reported that more than 25% of the prescribed drugs contained at least one active ingredient of plant origin and about 80% of world population relies on traditional medicine for significant part of their primary health care needs. belonging to the family combretaceae, terminalia catappa linn. is naturally occurring and widespread in the subtropical and tropical zones of the indian and pacific oceans, and is planted extensively in many countries as an ornamental tree.5 the type of oil that is obtained from terminalia catappa seeds is safe for consumption, in addition oliveria et al., (2000)6 indicated that the oil content of terminalia catappa (583.0 g/kg dry matter) is comparable to that of other oil seeds such as peanut, rapeseed, and sunflower. termianalia catappa is used primarily as an ornamental shade, and salt-tolerant street tree, but the leaves provide food for the tasar silkworm, and the seeds are edible like almonds with similar oils. on the malay peninsula and through the canary islands this tree is known as the tropical almond. termianalia catappa has been claimed to have therapeutic effects for liver related diseases.7 it is attributed with cholagogue action. in india, it is used as cardiac stimulant. its leaves are widely used as a folk medicine in southeast asia for the treatment of dermatosis and hepatitis8. more and more pharmacological studies have reported that the extract of termianalia catappa leaves and fruits have anticancer by (fan et al., 2004)9, antioxidant by (zhai et al., 2001)10, anti-hiv reverse transcriptase, anti-inflammatory, and anti-diabetic effects as stated by (xu et al., 2000)11 and hepato-protective activities as stated by (nagappa et al., 2003)12 but the effective components and related mechanisms remain unknown. numerous studies have been conducted with the extracts of various plants, screening antimicrobial activity as well as for the discovery of new antimicrobial compounds13,14,15,16. the efforts of scientists in establishing plants with promising antimicrobial property is yielding fruitful results as a number of plants with high antimicrobial property have been elucidated. this work was carried out to determine the phytochemical components as well as evaluate the antimicrobial activity of terminalia catappa seed oil. methodology the essential oil used in this study was extracted from nuts of indian almond. ripe fresh seeds were collected directly from the tree plant and dried in an oven for 5 days to reduce moisture content and enable easy breaking to get reasonable amount of the nuts. the seeds were cracked open with a nut cracker and the nuts are pounded using mortar and pestle into a smooth powder to increase surface area so that the solvent can permeate properly into it to extract the oil. extraction of essential oil by soxhlet method the extraction of essential oil was done using n-hexane in electro-thermal soxhlet extractor (gallenkamp, england). 30g of powdered seeds was weighed and put into the thimbles of the soxhlet extractor, the apparatus was mounted and allowed to run for 6 hours, after which the mixture of essential oil and n-hexane was collected in a beaker and evaporated over a hot water bath to collect the oil as residue. the method was repeated thrice to get sufficient amount of oil.17 determination of relative density of oil two density bottles were washed and dried in an oven and allowed to cool in a desiccator. each of the bottles was then weighed empty and the weights recorded. one was filled with water and the other with terminalia catappa seed oil. both was weighed again and the weight of the water and oil were determined by the difference in weight.18 where: weight of empty bottle = w1 weight of empty bottle + oil = w2 weight of empty bottle + water = w3 determination of the phytochemical constituents of oil chemical tests is carried out on the seeds oil of terminalia catappa using standard procedures to identify the constituents as described by (sofowora, 1982)19, (trease and evans, 1989)20 and (harborne,1973)21. alkaloids: about 0.5ml of the extracts is warmed with 2% of the h2so4 for two minutes. it was filtered and a few drops of dragondoff reagent is added. orange red precipitate indicates the presence of alkaloids. tannins: small quantity of extracts is mixed using water and heated on water bath and filtered. few drops of ferric chloride is added to filtrate. dark green solutions indicates the presence of tannins. glycosides: extracts is hydrolyzed using hcl and neutralized with naoh solution. few drops of fehling’s solution is added. red precipitate indicates the presence of glycosides. saponins: about 0.5ml of the extract is shaken with 5ml of distilled water and then heated to boil. frothing (appearance of creamy miss of small bubbles) shows the presence of saponins. flavonoids: extract of about 0.5ml is dissolved in diluted naoh and hcl added. a yellow solution that turns colourless indicates the presence of flavonoids. total phenol: extract of about 0.5ml is dissolved in dennis solution. a red colour precipitate indicates the presence of phenol. culture of clinical isolates clinical isolates (aspergillus niger, staphylocuccos aureus, escherichia coli) were obtained from the department of microbiology, faculty of natural sciences, kogi state 2 all res. j. biol, 2020, 11, 1-5 university, anyigba. overnight cultures were obtained using 10ml nutrient agar and used for the study. well diffusion assays antimicrobial activity testing was carried out by using agar diffusion method. three dilutions (30, 60 and 90%v/v) of terminalia catappa seed oil were made using 0.005%v/v tween 20 with 0.005%v/v dimethyl sulfoxide (dmso) in the ratio 1:1 as a co-diluent. mueller-hinton sterile agar plates were flooded with 2ml indicator microbial stains (aspergillus niger, staphylocuccos aureus, escherichia coli), and the excess drained. a cork borer was flamed and used to bore five wells which contained 0.005% tween and 0.005% dmso (1:1) (negative control); 30%, 60%, and 90% v/v of oil (treatments), and 0.5mg/l ciprofloxacin (positive control) and allowed to stay at 37ᵒc for 3 hours. the zones of growth inhibition around the disks were measured using venier caliper and a meter rule after 18 to 24 hours of the incubation at 37ᵒc for bacteria and 48 to 96 hours for fungi at 28ᵒc. the experiments were done in duplicates. statistical analysis one way analysis of variance (anova) was used to compare the mean inhibition zones of the different treatment groups and duncan multiple range test (dmrt) was used to separate means where significant. p < 0.05 was considered significant. results results revealed that essential oil of termianalia catappa seed contained tannins, flavonoids and glycosides (table 1) table 1: phytochemical components of oil extract parameter test/reagent observation inference alkaloids dragondorff no colour change (-) tannins ferric chloride dark green colour (+) saponins frothing test no frothing (-) flavonoids naoh-hcl test slight yellow colour (+) glycosides fehlings a and b test slightly red colour (+) phenols dennis test no colour change (-) key: + present absent relative density of the oil the relative density of terminalia catappa oil obtained was 0.875 (table 2) table 2: relative density of terminalia catappa oil sample weight of empty density bottle weight of empty bottle × sample mass of substance density g/ml oil 9.55 18.35 8.80 0.875 water 9.55 19.61 10.06 effect of the extracts on isolates results showed that the extract (termianalia catappa seed oil), at 30% (a) , 60% (b) , and 90% (c) v/v had no significant effect on the test organisms, the three organisms being escherichia coli, staphylococcus aureus , and aspergillus niger respectively. the negative control (nc), like the extract, had no effect as well on all the test organisms. the positive control (pc) however had effects on e.coli, s. aureus, a. niger with inbition zones of 12mm, 17mm and 22.5mm respectively (table 3; fig 1 -3). table 3: antimicrobial activity of terminalia catappa seed oil extract on a few selected pathogenic organisms pictorial presentation of the agar plates figure 1: effect of terminalia catappa seed oil on escherichia coli : nc = negative control (0.5% dmso and 0.5% tween 20) ; a = 30% oil extract ; b = 60% oil extract ; c = 90% oilextract; pc = positive control (0.5mg/l ciprofloxacin) test organism inhibition zone(mm) nc a b c pc e. coli 12.0 ± 2.0 s. aureus 17.0 ± 3.0 a. niger 22.5 ± 2.5 3 all res. j. biol, 2020, 11, 1-5 figure 2: effect of terminalia catappa seed oil on staphylococcus aureus: nc = negative control (0.5% dmso and 0.5% tween 20) ; a = 30% oil extract ; b = 60% oil extract; c = 90% oil extract; pc = positive control (0.5mg/l ciprofloxacin) figure 3: effect of terminalia catappa seed oil on aspergillus niger: nc = negative control (0.5% dmso and 0.5% tween 20) ; a = 30% oil extract ; b = 60% oil extract ; c = 90% oil extract; pc = positive control (0.5mg/l ciprofloxacin) discussion the seed oil extract of terminalia catappa was found to have tannins, flavonoids and glycosides; but had no inhibitory or antimicrobial activity against the micro-organism isolates (escherichia coli, staphylococcus aureus, and apergillus niger) this was confirmed by its failure to exert any form of inhibition zone around the wells containing the oil. this could be due to the fact that the concentration of the phytochemicals present in the oil was not enough to exert antimicrobial activity against the organisms22 and the low concentration of the phytochemicals could be due to the soxhlet method used in extracting the oil, which has been reported to be less effective than methods like steam distillation.23 miksusanti et al (2019)24 reported that 0.5% terminalia catappa seed oil showed no antibacterial effect on streptococcus mutans (with inhibition zone of 0 mm2), but on ethanolysis of the oil the resultant product had antimicrobial effect on the same isolate (with inhibition zone of 183 mm2).the lack of antimicrobial activity of the oil may also be as a result of its inability to diffuse through the medium properly due to its poor solubility.22,23 other parts of terminalia catappa have however shown antimicrobial activities. studies by babayi et al. (2004)25 using leaf extracts of terminalia catappa containing saponins, tannins and steroids had antimicrobial activity against escherichia coli and staphylococcus aureus which are well known to be involve in cutaneous diseases, wounds, burns and other different ailments. the antimicrobial activity of the leaf extracts of terminalia catappa may be due to the presences of saponins and steroids present which are absent in the seed oil. studies by pawar and pal (2002)26 on the chloroform extracts of terminalia catappa root also show good antimicrobial activity against gram-positive and gramnegative microorganisms. conclusion the phytochemical analysis carried out on the oil extract revealed the presence of tannins, flavonoids and glycosides and the absence of saponins, alkaloids and phenols. the antimicrobial investigations carried out on terminalia catappa seed oil base on this study prove had no antimicrobial activity against escherichia coli, aspergillus niger, and staphylococcus aureus. recommendations though terminalia catappa oil had no significant effect on the organisms tested in this research, it may be further tested on other microorganisms at different concentrations. adoption of more suitable and effective method of extraction and isolation of active compounds such as stem distillation and column chromatography is suggested. other uses of the oil should be investigated; such as the possibility of its use as a cooking oil and in the manufacture of cosmetics. references 1. walsh, c. (2000). molecular mechanisms that confer bacteria drug resistance. nature, 406(84):235-289. 2. shariff, z.u. (2001). modern herbal therapy for common ailments. nature pharmacy series (vol. 1). united kingdom: spectrum books limited, ibadan, nigeria in association with safari books (export) limited. pp. 9-84. 3. nair, r. and sumitra, c. (2008). antimicrobial activity of terminalia catappa, manikara zapota and piper betel leaf extract. indian journal of pharmaceutical science, 70 (3):390-393. 4. kaufmann, b. p., calson, f. t., dayanandan, p. e., fisher, b. j., parks, c and wells, r. j. (1999).plants: their biology and importance. new york : harper and row publishers. 681-700. 5. world health organization statistics (2002). are herbs safe. herb quarterly. pp.37. 6. oliveria, j.t.a., vasconcelos, i.m., bezerra l.c.n.m., silveira, s.b., monteiro, aco. and moreira, r.a. (2000). composition and nutritional properties of 4 all res. j. biol, 2020, 11, 1-5 seeds from pachira aquatica aubl, sterculia striata st hil et naud and terminalia catappa linn. food chemistry, 70:185-91. 7. chiu n. y. and chang, k. h. (1986). the illustrated medicinal plants of taiwan (vol.1). taipei: smc publishing, inc. p. 129. 8. lin, c.c., chen y.l, lin, j.m. and ujiie, t. (1997). evaluation of the antioxidant and hepatoprotective activity of terminalia catappa. american journal of chinese medicine, 25:153‑61. 9. fan, y.m., xu, l.z., gao .j., wang, y., tang, s.h., zhoa x.n. and zhang z.x. (2004) phytochemical and anti‑inflammatory studies on terminalia catappa. fitoterapia, 75:253‑60. 10. zhai, y. f, yao, j., fan, y. m., xu, l. z., gao, j. and zhao, x. n. (2001). inhibitory effects of lr-9 on proliferation of hepatocarcinoma cells. journal of nanjing university of natural science, 37 (2001): 213-217. 11. xu, l. z., gao, j., zhu, l., xu, m., lu, s. y., zhao, x. n. and zhang, z. x. protective effects of lr-98 on hepato-toxicity induced by carbon tetrachloride and d-galac-tosamine in mice. journal of nanjing university of natural science, 36 (2000):197-201. 12. nagappa, a.n, thakurdesai p.a., venkat rao .n., and singh .j. (2003); anti‑diabetic activity of terminalia catappa fruits. journal on ethno pharmacological studies. 88:45‑50. 13. parekh, j., karathia, n. and chanda, s. (2006). screening of some traditionally used medicinal plants for potential antibacterial activity. indian journal of pharmaceutical science, 68 (2006):832834. 14. parekh, j. and chanda, s. (2007). antibacterial and phytochemical studies on twelve species of indian medicinal plants. african journal of biomedical research, 10 (2007):175-181. 15. hediat, m. h. s. and marraiki, n. (2010). antimicrobial activity and phytochemical analyses of polygonum aviculare l. (polygonaceae), naturally growing in egypt. saudi journal of biological sciences,17 (1): 57-63. 16. pearson, d. (1991). the chemical analysis of foods (7th ed.). church living stone: longman group limited. pp. 493-494 17. meyer, e.w. (1966). soya bean protein concentrate and isolate. proceedings of international conference on soy bean protein foods. usda/ars-71:36-38 18. sofowora, a. (1982). medicinal plants and traditional medicine in africa. chichester, new york john: wiley and sons. p. 256. 19. trease, g.e. and evans, w.c. (1989). pharmacognosy (13th ed). bailliere tindal, london: english language book society. pp. 378-480. 20. harborne, j.b. (1973). phytochemical methods: a guide to modern techniques of plant analysis. london: chapman and hall limited. pp. 279. 21. knobloch, k., weigand, h., weis, n., schwarm, h.m. and vigenschow, h. (1986) action of terpenoids on energy metabolism. in progress in essential oil research; brunke, e.j., (ed.) (berlin, germany: walter de gruyter). pp. 429-445. 22. samie, a., nefefe, t., gundidza, m., mmbengwa, v., magwa, m. and mtshali m. s.(2012). antimicrobial activities and time kill profiles of five essential oils from southern africa against selected bacterial and fungal organisms. african journal of pharmacy and pharmacology. 6(44):3086-3095. 23. soković, m., glamočlija, j., marin, p.d., brkić, d. and van griensven, l.j. l. d.(2010) antibacterial effects of the essential oils of commonly consumed. medicinal herbs using an in vitro model. mol., 15, 7532-754 24. miksusanti, herlina, fithri a.n., ferlinahayati (2019). properties of ethanolysis product from ketapang seed oil ( terminalia catappa linn.) incorporated in mucoadhesive path film. iop conference series: earth and environmental science 347 (012034): 1 – 9 25. babayi, h., kolo, i. okogun, j. i., ijah, u.j. j. (2004).the antimicrobial activities of methanolic extracts of eucalyptus camaldulensis and terminalia catappa. against some pathogenic microorganisms. biokemistri. 16 (2):106-111. 26. pawar, s.p and pal s.c. antimicrobial activity of extracts of terminalia catappa root. indian journal of medicinal science. 2002; 56(6): 276-278. 5 microsoft word 61-362-1_galleys.docx some limits of biocompatibility testing for lipophilic leachates anne d. lucas* and edward a. gordon all res. j. biol., 2012, 3, 1-5 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                          issue 1, vol. 3, 2012, 1-5 some limits of biocompatibility testing for lipophilic leachates anne d. lucas* and edward a. gordon u.s. food and drug administration, center for devices and radiological health, office of science and engineering laboratories (division of biology); 10903 new hampshire ave, bldg 64 room 4010, silver spring md 20903. *corresponding author: anne.lucas@fda.hhs.gov abstract:  medical device standards recommend using both a polar and non-polar solvent to extract materials prior to in vitro testing. testing lipophilic extracts in cell culture systems is limited by the toxicity of the lipophilic solvents used in extraction. use of agar overlay and direct contact methods do not directly address the problem of testing for highly lipophilic leachates from device or material extracts. this particular problem was approached by 1) use of hydrotropes, and 2) by sealing the suspended cells in dialysis tubing and placing it directly in oil or media. the use of hydrotropes to eliminate micelle formation and increase the solubility of lipophilic compounds was not useful as the hydrotropes themselves were toxic to the cells at concentrations that significantly increased analyte solubility. diffusion of hydrophobic compounds from either peanut oil or cell culture media into the dialysis tubing, where the test cells resided in media, was significantly higher for the cell culture media than the peanut oil. there were significant differences in toxicity for cells in dialysis tubing from devices extracted between peanut oil and media. this study illustrates the importance of examining if cell toxicity is due to micelle formation or to the soluble chemicals from lipophilic extracts. keywords: lipophilic, biocompatibility, medical device extracts introduction the food and drug administration requires medical devices and materials be tested for safety. biocompatibility testing is necessary for all devices requesting fda clearance1,2. the standards used by the agency for biocompatibility testing recommend using at least 2 different extraction solutions of different polarity3,4. lipophilic solvents, such as dmso and vegetable oil, are recommended extraction solvents for medical devices and materials prior to cell culture assay5,6. however, mammalian cells used in biocompatibility testing are affected by low concentrations (approximately 1% or less) of lipophilic solvents7. the ability to perform in vitro tests on lipophilic analytes is limited by the solubility of the analyte in aqueous solutions. to attempt to address this problem, a system of indirect exposure of cells to oil was developed. by placing cells in dialysis tubing, then putting these cells directly in oil, it was expected that exposure to lipophilic compounds would increase. in addition, diffusion and solubility of the lipophilic analytes limits the amount of the analyte reaching the test cells. to attempt to increase exposure of the cells to hydrophobic leachates, 2 detergents were examined for their ability to increase the diffusion of lipophilic leachates into the cell culture test system. the use of hydrotropes to increase solubility has previously been applied to drugs8 and its application to device leachate testing was a logical step. materials and methods cell cultures and conditions jurkat cells, a human lymphoma cell, were obtained from atcc (atcc crl 8163). the jurkat cell line was chosen because it is a suspended cell line, not requiring surface attachment in order to grow. although not commonly used for biocompatibility testing, this cell line is appropriate to use for toxicity testing because it is derived from human blood cells and blood cells have great potential to be exposed to and transport most drugs and leachates of devices. cells were grown and maintained in co2 independent medium (gibco laboratories) supplemented with l-glutamine and 10% heatinactivated fetal bovine serum (fbs, sigma chemical co.). stock cells were maintained at 37οc. 1 all res. j.biol, 2012, 3, 1-5     chemicals peanut oil and virgin olive oil were purchased from a local supermarket. cottonseed oil, dulbecco’s phosphate buffed saline (dpbs), atrazine and tween-20 were purchased from sigma-aldrich. polyethylene glycol 4000 was obtained from mallinckrodt chemicals, diethylhexyl-phthtalate (dehp) from fluka, and actonitrile from fisher scientific. a commercially available enzymatic cleaner for reprocessing medical devices was used to deliberately contaminate gi biopsy forceps. dialysis tubing was obtained from spectra/por (3500, 60008000, and 1200-14000 dalton mwco, semipermiable regenerated cellulose); nominal flat width of both 32 mm (‘large’) and 10 mm (‘small’) were used. tubing was rinsed, then boiled in distilled water to remove any glycerol residues and any other contaminates prior to use; the dialysis tubing could then be autoclaved. cell exposure exposure of the cells to various oils was accomplished by placing usually 5 ml of cells in media directly into the dialysis tubing with the ends of the tubing tied tightly and any excess material was trimmed off. cells were then put directly into a 50 ml polypropylene test tube with the appropriate extraction solution (figure 1). initially, 25 ml of the extraction oils (cottonseed, olive, or peanut oil) were tested to select the best oil with cell culture media as a point of comparison. after selecting the oil best tolerated by the cells, chemicals and extracts of medical devices were then assessed for cytotoxic response. the test device or materials was placed directly in the extraction solution, then 5 to 10 ml of cells enclosed in the large (32 mm) dialysis tubing was added. the potential effect of the dialysis tubing on the cells themselves was explored by placing a 16 cm long piece of tubing in a 50 ml polypropylene test tube with 10 ml of media. to this 10 ml of cells in media were added; the control for this experiment was 10 ml of media and 10 ml of cells in a 50 ml polypropylene test tube. different pore sizes and nominal flat widths of various dialysis membranes were also evaluated. figure 1: picture of the test system: cells enclosed in dialysis tubing, placed in peanut oil with a device. analysis of cells cytotoxic affects were analyzed using a flow cytometer (facscan, becton dickenson) as previously described9, 10. cells were analyzed according to the side-scattering profile (proportional to cellular granularity) versus the forwardscattering profile (proportional to the cellular cross sectional area). changes in cell populations were evaluated by gating on the normal cell population and comparing to the test groups over time. the use of propidium iodide dye (propidium iodide is actively excluded from viable, healthy cells) to determine viability was not possible because of the fluorescent background observed when 10 ml of media was placed in with oil and analyzed. there was no background observed in the gated region for dead cells. experiments were run in triplicate with the exception of range finding experiments. data are expressed as an average of the percentage of dead cells, and the variability of the assay is expressed as the standard error of the mean11. student t-test was used to evaluate significance. devices devices used for extraction were latex gloves and gi biopsy forceps. for the latex, 3 different natural rubber latex (nrl) standard examination gloves from 3 different manufacturers were cut into pieces and placed in a polypropylene test tube with oil or media (1 g/10 ml). 5 ml of cells in dialysis tubing were placed directly in the oil or media with the glove pieces. the test tubes were incubated overnight at 37oc. in the preparation of the biopsy forceps, 6 cm pieces of a gi biopsy forceps were placed in either water or a commercially available cleaning agent for devices and diluted as per manufacturer’s instructions. 5 pieces of the forceps soaked in 2 all res. j.biol, 2012, 3, 1-5     water were placed in a 50 ml polypropylene test tube with peanut oil; 2 pieces were placed in culture media. 5 ml of cells in dialysis tubing were directly added to the polypropylene test tube with the pieces of forceps in oil or media. the identical procedure was followed with pieces of the biopsy forceps soaked in the cleaning agent; the cleaning agent soaked forceps were not rinsed before use. evaluation of hydrotropes and hydrophilic compound diffusion tween-20 and peg were serially diluted in dpbs with 1% acetonitrile to determine any increase in solubility of dehp. dehp was initially examined as the test lipophilic analyte due to its low solubility in aqueous solutions and its prevalence in medical device materials. however, because peanut oil came in a plastic container and many plasticizers are ubiquitous low level contaminates, the herbicide atrazine was also used as a model compound to evaluate the movement of a lipophilic analyte from oil into the test cell culture system. the use of acetonitrile with the detergent was essential as stock solutions of dehp and atrazine needed to be made in organic solvents. the tolerance of the test cells for the various concentration of tween-20 or peg in 1% acetonitrile was also examined. to assess the actual concentration of small analytes to pass from either the peanut oil or the media into the dialysis tubing with cells, dehp and atrazine were spiked into the oil or media and measured using hplc12, 13. results and discussion initially, the ability of cells to tolerate various oils was examined. 10 ml of jurkat cells were put inside the dialysis tubing, then the cells were placed in a 50 ml test tube containing 25 ml of cottonseed, olive, or peanut oil (figure 1). media was used as the negative control. in addition, cottonseed, olive, or peanut oil was partitioned against an equal volume of dpbs (‘washed’) to examine if any aqueous components were responsible for any negative effects on the cells. both the cottonseed and the olive oil were toxic to the cells. “washing” the oil with dpbs did not acceptably reduce the toxicity of the cottonseed or olive oil to the cells (data not shown). the peanut oil showed minimal toxicity to the cells at 24 hours, but toxicity insignificantly increased after that, p<0.02 at 48 hrs and p<0.002 at 72 hrs (figure 2). there was a small, but significant, increase in the percentage of dead cells in the ppe control when compared to that in the ps flasks (data not shown). for the media samples, the increase in dead cells was significant at 72 hrs (p<0.02). when comparing the different size dialysis tubing to the ppe control, there was not a significant difference in cell viability. figure 2: the effect of extraction media on cells over time in the test system. student t-test indicated statistical significance between the media and oil at 48 hrs (p<0.01) and 72 hrs (p<0.001).     to evaluate any background fluorescence or micelle formation that would interfere with the flow cytometer, cottonseed, olive, peanut oil, or media were placed in 50 ml test tubes; however, 10 ml of media only was placed in the dialysis tubing. analysis of these background samples indicated a large fluorescent background in the area where live cells are gated. nominal background (less than 0.25%) was seen in the area where dead cells were gated, therefore, the percentage of dead cells was used as an indication of toxicity or the forward vs. side scattering profile. addition of dialysis tubing alone to cells did not exert a toxic affect on the cells (data not shown). application of this interface of cells and oil in order to evaluate the cytotoxicity of natural rubber latex gloves is depicted in figure 3. the gloves extracting in the media showed higher toxicity than the same gloves extracting in peanut oil. cutting the gloves in pieces probably had no effect on the cell toxicity, and was done to provide a uniform size sample and to prevent air pockets. this system may be useful in looking at surface contamination of devices. occasionally, reused devices contain residues of the cleaning solution if the devices are not rinsed thoroughly. gi biopsy forceps deliberately contaminated with a cleaning agent then extracted in media showed significantly higher toxicity than those extracted with peanut oil (figure 4). gi biopsy forceps soaked in water showed no toxicity using either peanut oil or media to extract the device. however, when acetonitrile was added to either media or peanut oil and the toxicity of the cells in dialysis tubing was examined the next day, the cells in peanut oil showed a much larger increase in the percentage of dead cells (figure 5). 0 2 4 6 8 10 12 14 2 24 48 72 time, hours p er ce nt ag e d ea d c el ls media peanut oil 3 all res. j.biol, 2012, 3, 1-5     figure 3: the toxicity of 3 different gloves on the cells in dialysis tubing using either oil or media in extraction. significant difference was noted between extraction solutions for glove 1 (p<0.004) and glove 2 (p<0.007). the use of detergents to aid in the diffusion of lipophilic analytes into the cell culture system was not successful; the solubility of dehp can be estimated by using the flow cytometer to visualize micelle formation14. toxicity due to a chemical in solution is different than toxicity due to micelles in solution. cell membrane damage from micelles is due to the solubilization of cell membrane components, which is a separate issue than a soluble chemical toxicity15. using 0.025% tween-20 with 1% acn in pbs increased the solubility of dehp to 25 µg/ml from 5 µg/ml in pbs alone. however, at the 0.025% tween-20 concentration, the cells died. at the tolerable tween-20 level of 0.00625%, there was no significant increase in dehp solubility.   figure 4: the toxicity of a commercially available enzymatic cleaning agent on cells in test system.   the hplc analysis of dehp from peanut oil into dialysis tubing with media alone showed that no detectable dehp had diffused into the dialysis tubing with media. atrazine is slightly more water soluble than dehp and was also examined in diffusing from peanut oil or media into the dialysis tubing with media alone. 15.5 µg/ml of atrazine was present in the dialysis tubing that was placed in media with an initial concentration of 25 µg/ml of atrazine; 0.8 µg/ml of atrazine was present in the dialysis tubing that was placed in peanut oil. the hydrophobic/hydrophilic interaction appears to prevent diffusion across the dialysis from the oil across the dialysis tubing to the cells. the higher concentration of atrazine in the media and the greater toxicity of the cleaning agent in media (figure 4), would support this hypothesis. figure 5: the toxicity of acetonitrile on cells in test system. significant increase in the toxicity of oil compared to media was seen at and above the 0.05% v/v level (p<0.005). conclusion medical device standards recommend using both a polar and non-polar solvent to extract materials prior to in vitro testing. to attempt to address this problem, a system of indirect exposure of cells to oil was developed. by placing cells in dialysis tubing then putting these cells directly in oil, it was expected that exposure to lipophilic compounds would be increased; however, this was not the case. atrazine, a lipophilic pesticide with limited solubility in water, actually diffused 5 times more from the test system with media than the test system with peanut oil. it is possible other semipermeable membranes may yield different results. attempts to increase exposure of the cells to hydrophobic leachates using 2 detergents were examined for their ability to increase the diffusion of lipophilic leachates into the cell culture test system. the use of hydrotropes to increase solubility has been applied to drugs8 and its application to device leachate testing using this in vitro system did not work at concentrations that were non-toxic to cells. this study does illustrate the importance of identifying toxicity due to micelle formation versus that of a soluble chemical(s) acknowledgments the authors wish to thank gail matson for her editorial expertise, ann koustenis for laboratory support, and dr. megan shoff for the technical review. fda chief scientist challenge grant partially supported this work. 0 10 20 30 40 50 60 70 glove 1 glove 2 glove 3 no glove glove sample p e rc e n ta g e d e a d c e ll s media oil 0 10 20 30 40 50 60 water cleaner water cleaner media peanut oil p e rc e n ta g e d e a d c e ll s 0 10 20 30 40 50 60 70 0 0.5 1 1.5 2 2.5 % acn v/v p er ce n ta g e d ea d c el ls media peanut oil 4 all res. j.biol, 2012, 3, 1-5     abreviations dpbs dulbecco’s phosphate buffed saline dehp diethylhexyl-phthtalate nrl natural rubber latex peg polyethylene glycol hplc high performance liquid chromatography ppe polypropylene ps polystyrene gi gastrointestinal acn acetonitrile references 1. fda guidance document. use of international standard iso-10993, 'biological evaluation of medical devices part 1: evaluation and testing'. http://www.fda.gov/medicaldevices/deviceregulati onandguidance/guidancedocuments/ucm080735.ht m. accessed january 27 2012. 2. astm f 748 – 98, 1998. standard practice for selecting generic biological test methods for materials and devices. in: the annual book of astm standards, astm west conshohocken, pa, usa. 1998 3. astm f 619 03, 2003. standard practice for evaluation of medical plastics. in: the annual book of astm standards, astm west conshohocken pa (usa). 2003 4. international standards organization. biological evaluation of medical devices part 12: sample preparation and reference materials. in aami standards and recommended practices biological evaluation of medical devices arlington, va, usa. 2007 5. international standards organization. biological evaluation of medical devices part 5: testing for cytotoxicity: in vitro methods. in aami standards and recommended practices biological evaluation of medical devices arlington, va, usa. 1999 6. astm f 619 79 (97), 1997. standard practice for evaluation of medical plastics. in: the annual book of astm standards, astm west conshohocken, pa, usa. 1997 7. lucas, a.d., lappalainen, s.k., wray-cahn, d. (2009). hyperthermia can increase the cytotoxicity of exogenous compounds. biomedical instrumentation and technology. 43, 73-79. 8. sharma. a,, jain, c.p. (2009). techniques to enhance solubility of poorly soluble drugs: a review. j of global pharma tech. 2(2), 18-28. 9. godar, d.e., miller, s.a. thomas dp. (1994). immediate and delayed apoptotic cell death mechanisms: uva verses uvb and uvc radiation. cell death and differentiation. 1, 59-69. 10. godar, d.e., lucas, a.d. (1995). spectral dependence of uv-induced immediate and delayed apoptosis: the role of membrane and dna damage. photochemistry and photobiology. 62,108-113. 11. nagele, p. (2001). misuse of standard error of the mean (sem) when reporting variability of a sample. a critical evaluation of four anaesthesia journals. british journal of anesthesiology. 90(4), 514-516. 12. steinheimer, t.r. (1993). hplc determination fo atrazine and principal degradates in agricultural soils and associated surface and ground water. j agric and food chem. 41(4), 588-595. 13. kambia, k., dine, t., gressier, b., gerne, a-f., luyckx, m., brunet, c., michaud, l., gottrand, f. (2001). high-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma. j. chromatog. b. 755, 297-303. 14. mcclelland. r,, pinder. a. (1994). detection of salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies. applied and environmental microbiology. 60(12), 4255-4262. 15. warisnoicharoen, w., lansley, a.b., lawrence, m.j. (2003). toxicological evaluation of mixtures of nonionic surfactants, alone and in combination with oil. j of pharmaceutical sciences. 92(4), 859868. 5 microsoft word ngumah_proofreaded.docx                                                                                                                    issue 2, vol 6, 2015, 24-29 antifungal  potencies  of  ethanol  leaf  extracts  of  four  plants  found  around   traditional  yam  barns  in  south  eastern  nigeria  on  five  yam  pathogens  implicated  in   postharvest  yam  rot chima    ngumah1*,  sarah  umeh1,  etienne  chinakwe1,  jane  ngumah2   1department  of  microbiology,  federal  university  of  technology  owerri,  p.m.b  1526  owerri,  nigeria.   2department  of  biology,  federal  university  of  technology  owerri,  p.m.b  1526  owerri,  nigeria.   *e-­‐mail:    ccngumah@yahoo.com   abstract the  susceptibilities  of  five  fungi   implicated  in  postharvest  rot  of  yams,  namely,  aspergillus  niger,   penicillium   oxalicum,   mucor   circinelloides,   fusarium   oxysporum,   and   rhizopus   nigricans   were   evaluated  using  ethanol  leaf  extracts  from  four  plants  commonly  found  around  traditional  yam  barns   in   south   eastern   nigeria.     the   plants   screened   were   fagara   rubescens,   neubouldia   laevis,   pterocarpus   soyauxii,   and   vernonia   amygdalina.   antimicrobial   susceptibility   was   measured   as   an   index  of  turbidity  of  broth  cultures  after  an  incubation  period  of  72  hours  at  ambient  temperature   (29  –  310c).  the  optical  densities  (od650)  were  measured  by  spectrophotometry.  none  of  the  tests   showed   susceptibility   to   any   of   the   plant   extracts   screened   irrespective   of   the   concentration   employed.     this   study   also   revealed   that   although   all   the   plants   contained   bioactive   secondary   metabolites,  no  antifungal  potency  was  detected  for  any  of  the  plant  extracts  on  the  test  organisms.   keywords: yam  pathogens,  plant  extracts,  yam  rot,  susceptibility,  phytochemicals introduction                    yams  are  monocotyledonous  plants  belonging  to   the   genus   dioscorea   in   the   family   dioscoreaceae.     yams  serve  as  a  staple  food  for  millions  of  inhabitants   of  the  tropics  and  sub-­‐tropics.    dioscorea  species  are   important   food   crops   in   west   africa,   the   caribbean,   and   some   parts   of   asia   (including   china,   japan,   malaysia  and  oceania).1                      shrines   and   traditional   yam   barns   in   south   eastern   nigeria   serve   as   home   for   various   plants,   some  of  which  have  been  proven  to  be  very  valuable.     some   of   these   plants   are   used   for   therapeutic   purposes.2   this   study   investigates   the   antifungal   properties  of  the  leaves  of  some  of  these  plants  found   around   traditional   yam   barns   on   common   fungi   implicated  in  the  postharvest  rot  of  yam.  the  plants   screened   in   this   study   were:   fagara   rubescens   (planch),   neubouldia   laevis   (seem),   pterocarpus   24 all res. j.biol, 2015, 6, 24-29     soyauxii   (taub),   and   vernonia   amygdalina   (linn).     these   plants   were   chosen   because   they   are   commonly  used  in  constructing  traditional  yam  barns   in  south  eastern  nigeria,  and  are  unofficially  believed   to  play  a  role  in  the  preservation  of  stored  yams.   material  and  methods   sample  collection                      rotten   yam   tubes   were   collected   from   yams   stored   on   horizontal   bamboo   platforms   (improvised   yam   barns)   at   okwuaba   village   in   okpofe,   ezinihitte   mbaise  local  government  area  of  imo  state  (nigeria).     fresh  leaf  samples  were  plucked  at  about  midday  at   okwuaba  okpofe.    the  leaves  were  authenticated  by   the   department   of   forestry,   imo   state   ministry   of   agriculture,   owerri.   the   leaves   were   air   dried   at   ambient   room   temperature   (280c   –   310c)   until   constant  weight  was  achieved.    the  dried  leaves  were   then  ground  to  powder  using  a  mechanical  grinder.     extraction  of  plant  materials                        70%  ethanol  was  used  for  the  extraction  of  the   plant  materials.    for  the  cold  ethanol  extraction,  100g   of   each   powdered   plant   material   was   steeped   in   500ml   of   70%   ethanol   for   48   hours,   while   the   hot   ethanol  extraction  was  carried  out  by  seeping  100g  of   each   powdered   plant   material   in   500ml   of   70%   hot   ethanol  which  was  maintained  at  600c  for  1  hour  in  a   water  bath.2  the  slurries  were  filtered  through  folds   of  sterile  cheese  cloth.    the  filtrates  were  evaporated   to   dryness   by   forced   air   pressure   using   a   rotary   evaporator   to   a   yield   of   about   12.5%   w/w   (with   respect  to  the  powdered  plant  material).3     preparation  of  plant  extract  diluent                      1000mg  quantity  of  each  ethanolic  extract  (hot   and   cold)   was   reconstituted   with   5   ml   of   10%   dimethyl  sulfoxide  (dmso)  to  obtain  a  concentration   of  200  mg/ml.4      a  two  fold  serial  dilution  was  used  to   obtain  the  following  concentrations  in  sterile  distilled   water:   100   mg/ml,   50   mg/ml,   25   mg/ml,   and   12.5   mg/ml.    these  were  stored  by  refrigeration  at  40c  in   sterile  amber  coloured  bottles  until  required.     isolation  of  fungal  pathogens  from  rotten  yam                      the   rotten   yam   tubers   were   rinsed   in   sterile   distilled   water   and   surface   sterilized   with   95%   ethanol.    the  rotten  yam  tubers  were  then  cut  open   with  a  sterile  knife  (flamed).    about  3  pieces  (3  mm   diameter)   of   each   infected   tissue   were   picked   with   flamed  sterilized  forceps  and  inoculated  on  separate   sterile  solid  sabouraud’s  dextrose  agar  (sda)  plates.5     the   plates   were   incubated   at   ambient   room   temperature   (29   –   310c)   for   up   to   5   –   7   days   and   examined   daily   for   growth   of   moulds.     the   isolates   were  then  sub-­‐cultured  to  obtain  pure  cultures  of  the   organisms,  which  were  eventually  transferred  to  sda   slants  and  stored  at  40c  until  required.         characterization  of  fungal  isolates                      the   fungal   isolates   were   identified   using   their   growth  (colonial)  morphology  on  sda  and  microscopic   morphology.     the   colonial   morphology   on   sda   was   determined   by   observing   the   surface   and   reverse   views  of  the  fungi  growing  on  each  agar  plate.    the   25 all res. j.biol, 2015, 6, 24-29     colour,  shape,  elevation,  and  spore  head  colouration   were   noted.     to   view   the   microscopic   morphology,   two  drops  of  cotton  blue  lactophenol  were  placed  on   a  clean  grease-­‐free  glass  slide.    then,  sterile  (flamed)   inoculating   needles   were   used   to   transfer   a   small   portion   of   mycelial   growth   to   the   cotton   blue   lactophenol.    the  fungal  growth  was  then  teased  out   in   the   cotton   blue   lactophenol   and   covered   with   a   clean   grease-­‐free   coverslip   and   then   examined   microscopically  using  x10  and  x40  objectives.6     preparation  of  inoculum                        all   fungal   isolates   were   aseptically   inoculated   onto  sterile  sda  slants  prepared  in  mccartney  bottles   and   incubated   at   ambient   room   temperature   (29   -­‐   310c)   for   4   days   to   obtain   young   actively   growing   cultures   consisting   of   mycelia   and   conidia   /   arthrospores   /   blastospores.   the   fungal   growth   on   each  agar  slant  was  aseptically  scraped  off  and  placed   in   10   ml   sterile   saline   (0.9%   w/v)   and   shaken   vigorously   using   a   vortex   mixer   until   the   fungal   filaments  were  broken  into  small  colony  forming  units   (cfu).   each   suspension   was   standardized   using   a   haemocytometer  to  obtain  104  -­‐  106  cfu/ml  and  this   was  used  as  the  inoculum.       testing  for  anti-­‐fungal  potency  of  the  plant  extracts                      the   susceptibility   of   the   isolated   fungal   pathogens   to   different   concentrations   of     the   plant   extracts   were   examined   using   a   spectrophotometer     by   measuring   their   optical   density   (absorbance)   at   620nm.3    0.5  ml  of  the  standardized  fungal  suspension   was  inoculated  into    a  separate  test  tube  containing   9.0   ml   of   sterile   sabouraud’s   dextrose   broth   (sdb).     then,   0.5   ml   of   the   reconstituted   plant   extract   was   finally  inoculated  into  each  test  tube.    this  was  carried   out   for   the   following   concentrations   of   each   plant   extract:   100   mg/ml,   50   mg/ml,   25   mg/ml,   and   12.5   mg/ml.   the   test   tubes   were   incubated   at   ambient   room  temperature  (29  –  310c)  for  72  hours  and  then   transferred   to   a   spectrophotometer,   where   their   optical   densities   were   measured.   for   each   set   of   experiments  there  was  a  control  test  tube  containing   sdb   and   plant   extract   without   fungal   isolate.     the   control  was  used  to  blank  the  spectrophotometer.               phytochemical  analysis                      powdered  leaf  plant  samples  were  used  to  carry   out  phytochemical  tests  using  standard  procedures.7                   26 all res. j.biol, 2015, 6, 24-29       results  and  discussion          table  1.    optical  densities  of  72  hours  sabouraud’s  dextrose  broth  cultures  of  five  yam  (postharvest)  pathogens   treated  with  four  leaf  extracts.     each   leaf   extract   recorded   the   same   optical   density   for   all   the   concentrations   screened   per   test   isolate.     the   presence   of   fungal   growth   was   confirmed   using   microscopy   by   viewing   samples   from   the   broth   cultures   using   x10   and   x40   objectives,   and   then   plating  0.1ml  on  solid  sda.    none  of  the  leaf  extract   concentrations   screened   in   this   work   showed   antifungal   potency   on   any   test   organism,   this   is   at   variance   with   the   reports   of   researchers   who   demonstrated   that   ethanol   leaf   extracts   of   carica   papaya,  glyphaea  brevis,  and  spondias  mombin  were   potent   against   the   same   test   organisms   used   in   this   work  (ngumah,  2012;  and  ngumah  et  al.,  2013).8,9     comparison   using   analysis   of   variance   (anova)   showed  no  significant  difference  in  the  od650  (p>0.05):     within   different   concentrations   of   the   same   leaf   extract  for  each  test  isolate,  and  among  the  different   plant  extracts  screened  on  each  test  isolate.     although   the   plant   leaves   screened   in   this   work   contained   bioactive   secondary   metabolites   (namely:   alkaloids,   tannins,   and   flavonoids)   reported   by   workers   to   confer   antimicrobial   activity   to   plant   extracts,   no   antifungal   activity   was   recorded   in   this   work   by   these   leaf   extracts.10,11,12     this   lack   of   antifungal  activity  may  be  attributed  to:  probable  low   concentration  levels  of  phytochemicals,13,14  resistance   levels  among  strains,15  age  of  leaves,16  state  of  leaf  at   point   of   extraction-­‐dry   or   fresh,17,18   and   method/extraction  solvent.19         plant  extracts                                                                                                                                      optical  density  at  620nm  (od620)  ±  sd                                                                                        a.  niger                        p.  oxalicum                      m.  circinelloides                  f.  oxysporum                        r.  nigricans   fagara  rubescens                      0.78±0.023              0.8±0.03                                  0.8±0.042                                        0.8  ±0.042                                  0.77±0.035     neuboldia  laevis                        0.82  ±0.025              0.8±0.042                              0.79±0.041                                  0.77±0.035                                0.8±0.042   pterocarpus  soyauxii        0.77±0.031                0.78±0.046                          0.8±0.042                                        0.82±0.012                              0.8±0.042   vernonia  amygdalina      0.81±0.023                0.81±0.017                        0.77±0.038                                    0.8±0.02                                        0.82±0.006   control                                                                              0.56                                          0.56                                              0.56                                                          0.56                                                    0.56   27 all res. j.biol, 2015, 6, 24-29     conclusion   although   data   obtained   in   this   work   revealed   the   presence  of  bioactive  substances,  none  of  the  plants   screened  showed  antifungal  activity  on  any  of  the  test   isolates.     this   suggests   that   the   mere   presence   of   bioactive  secondary  metabolites  does  not  necessarily   guarantee  the  antimicrobial  potency  of  plant  extracts.     secondly,  though  there  was  no  antimicrobial  activity   recorded   for   the   test   organisms,   the   same   plant   extracts  may  exhibit  antimicrobial  properties  on  other   organisms.     in   addition,   quantitative   analysis   should   be   done   to   ascertain   the   phytochemical   levels   in   these  extracts  and  compare  them  with  levels  found  in   other   plant   extracts   that   have   been   documented   to   exhibit  antifungal  activity  on  the  same  test  organisms   used  in  this  work.     references   1. okigbo,  r.,  and  ikediugwu,  f.  (2000).  studies   on   biological   control   of   postharvest   rot   of   yams  with  trichoderma  viride.  j.  phytopathol.   148(6),  35-­‐355.     2. osadebe,   p.   and   ukwue,   s.   (2004).   a   comparative  study  of  the  phytochemical  and   antimicrobial   properties   of   the   eastern   nigerian   specie   of   african   mistletoe   (loranthus   micranthus)   sourced   from   different  host  trees.  j.  biol.  res.  biotech.  2(1),   18  –  23.   3. esimone,   c.,   adikwu,   m.,   and   okonta,   j.   (1998).  preliminary  antimicrobial  screening  of   the   ethanolic   extract   from   the   lichen   usnea   subfloridans  (l.).  j.  pharm.  res.  dev.  3(2),  99-­‐ 102.   4. nweze,   e.,   okafor,   j.,   and   njoku,   o.   (2004).   antimicrobial  activities  of  methanolic  extracts   of  trema  guineensis  and  morinda  lucida  used   in  nigerian  herbal  medicinal  practice.   j.  biol.   res.  biotech.,  2(1),  39  –  46.   5. okigbo,   r.   and   nmeka,   i.   (2005).   control   of   yam   tuber   rot   with   leaf   extracts   of   xylopia   officinale.  african  j.  biotechnol.  4(8),  804-­‐807.     6. ogbulie,   j.n.,   uwaezuoke,   j.c,   and   ogiebor,   s.i.   (2001).   introductory   microbiology   practicals.   second   ed.   (owerri,   nigeria:   concave  publishers).   7. agomuo,  e.n.,  amadi,  b.a.,  chikezie,  p.c.,  and   ibegbulem,   c.o.   (2002).   basic   analytical   and   research   methods.   maiden   ed.   (owerri,   nigeria:  supreme  publishers).   8. ngumah,   c.   (2012).   antifungal   potencies   of   leaf   extracts   of   carica   papaya   on   fungi   implicated  in  soft  rot  of  yam.  annals.  food  sci.   technol.  13(2),  202-­‐209.   9. ngumah,   c.,   ogbulie,   j.,   orji,   j.   (2013).   antifungal   potencies   of   leaf   extracts   of   glyphaea   brevis   and   spondias   mombin   on   fungi   implicated   in   dry   rot   of   postharvest   yam.   annals.   food   sci.   technol.   14(2),   330-­‐ 335.   10. dathak,   p.   and   iwu,   s.   (1991).   inhibition   of   xanthine  oxidase  activity  by  some  flavonoids.   filoterapia  63:  385.   11. draughon,  f.  (2004).  use  of  botanicals  as  bio-­‐ preservatives   in   foods.   food   technology   58(2),  20  -­‐28.   12. hassan,  s.,  umar,  r.,  ladan,  m.,  nyemike,  p.,   wasagu,   r.,   lawal,   m.,   and   ebbo,   a.   (2007).   nutritive  value,  phytochemical  and  antifungal 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 content  of   the   leaves   of   four   nigerian   pterocarpus   species.  int.  j.  mol.  med.  adv.  sci.  3(1),  6  -­‐11.   17. doughari,   j.   sunday,   d.   (2008).   antibacterial   activity   of   phyllanthus   muellerianus.   pharmaceut.  biol.  46(6),  400  –  405.   18. osuagwu,   g.,   and   akomas,   c.   (2013).   antimicrobial   activity   of   the   leaves   of   three   species  of  nigerian  pterocarpus  (jacq.).  int.  j.   med.  and  arom.  plants,  3(2),  178  -­‐183.   19. khan,   j.,   yadav,   j.,   srivastava,   y.,   and   pal,   p.   (2012).   in   vitro   evaluation   of   antimicrobial   properties  carica  papaya.   int.  j.  biol.  pharm.   allied   sci.   (ijbpas)   1(7),   933   –   945.     29 microsoft word 106-592-1-le-2.docx reusable areas of clinically used ventilators carry low numbers of aerobic bacteria elizabeth gonzalez, julie kase, fassil getachew, mark cowan, pamela scott, iacovos kyprianou, barre jones, victoria hitchins all res. j. biol., 2014, 5, 24-29 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 4, vol 5, 2014, 24-29 reusable areas of clinically used ventilators carry low numbers of aerobic bacteria elizabeth gonzalez1, julie kase2, fassil getachew3, mark cowan3, pamela scott4, iacovos kyprianou4, barre jones3, victoria hitchins1 1. division of biology, chemistry, and material sciences, office of science and engineering laboratories, center for devices and radiological health, fda, silver spring, md, usa. 2. division of microbiology, office of regulatory science, center for food safety and applied nutrition, fda, college park, md, usa. 3. baltimore va medical center, baltimore, md, usa. 4. office of the center director, center for devices and radiological health, fda, silver spring, md, usa. abstract: ventilator associated pneumonia (vap) remains a serious problem for critically ill patients. we swabbed nine reusable areas on 20 clinically-used maquet servo ventilators from a va hospital; shortly after they had been removed from patients and identified bacterial isolates. no bacteria were isolated from most of the samples and of the samples that did grow bacteria, the majority of those had fewer than 10 colonies. the bacteria that were isolated were primarily non-pathogenic gram-positive skin flora. of the 20 ventilators swabbed, only one of the cultured bacteria was associated with nosocomial infections: methicillin-resistant s. aureus. the most commonly contaminated areas were those most likely to be touched by healthcare professionals: the power button and the screen. the areas in closest proximity to the patients, the inspiratory and expiratory ports were the least often contaminated areas. overall, very few bacteria were transferred to the reusable areas of the ventilators following clinical use. keywords: ventilators, reusable, cleaning, disinfecting, bioburden, ventilator associated pneumonia introduction while mechanical ventilation can be a life-saving and necessary procedure, it often presents complications for patients such as ventilator associated pneumonia (vap).1 vaps result in an increase in hospital stay time and cost for patients.2,3 additionally, patients that acquire vap while in the icu have a higher mortality rate, although the exact risk of death associated with vap is still unclear due to confounding variables.1 while vap rates have decreased in recent years, at least partly due to changes in clinical practices such as elevating patients at an angle rather than lying horizontally,4 they still remain a problem. many modern ventilators have features that limit crosscontamination from one patient to another,   such as frequent changes of disposable tubing and heat and moisture exchangers.5 however, it is unclear how many bacteria and number of strains are transferred via the surface of the ventilators, often touched by healthcare caregivers directly after touching patients. due to the danger of nosocomial infections, it is imperative to understand the risk of spreading infection via the ventilator surface. the objective of this study was to swab ventilators after patient use to determine the number and strains of bacteria present on areas of the ventilators that are not single use,   and therefore will come into contact with subsequent patients. methods sampling twenty clinically used maquet servo ventilators at the va hospital in baltimore, md were swabbed approximately 2448 hours after being removed from a patient. the machines were covered by an equipment bag and isolated after being removed from the patient until they were swabbed. the patients had been admitted to the hospital in either the surgical intensive care unit or the medical intensive care unit. they were ventilated for an average of 2.49 days due to one of the following issues: to protect the airway following surgery, chronic obstructive pulmonary disease (copd), cardiopulmonary arrest, respiratory failure, or respiratory insufficiency. nine components of the ventilator were chosen to swab based on either their proximity of contact with patients or the likelihood of them being touched by healthcare providers (figure 1.) these components were: 1. 24 all res.j.biol, 2014, 5, 24-29     expiratory inlet, 2. inspiratory outlet, 3. exhaust port, 4. interface buttons, 5. power button, 6. knob, 7. screen,   figure 1:nine reusable areas of the maquet servo ventilator were swabbed to recover aerobic bacteria. the areas swabbed were: 1. expiratory inlet, 2. inspiratory outlet, 3. exhaust port, 4. interface buttons, 5. power button, 6. knob, 7. screen, 8. diaphragm, 9. exhalation pressure port. 8. diaphragm, 9. exhalation pressure port. the expiratory inlet and inspiratory outlet are where the ventilator is connected to disposable tubing, which then directly connects to patients. the exhaust port returns used air to the environment. the interface buttons and knob are present around the screen and allow healthcare providers to interact with the ventilator. the power button turns the unit on and off and is found on the back of the machine. the screen is a touch screen and is often pushed to turn off the alarm. the diaphragm and exhalation pressure port regulate the air pressure within the ventilator. to collect samples, the ampule within an individually packaged sterile bactiswab (remel, lenexa, ks) was broken to moisten each swab immediately before swabbing one area of the ventilator. the swab was then vigorously swished approximately 10 times in a microcentrifuge tube containing 1 ml of sterile phosphate buffered saline. in order to maximize the amount of sample collected, the swab was then pressed against the internal side of the tube to squeeze out fluid. samples were plated in triplicate (250 µl/plate) on trypticase soy agar (tsa) plates, incubated aerobically at 37°c, and examined the following day for microbial growth. each morphologically unique colony type from each plate was restreaked onto a tsa plate and incubated as above. identification isolates were identified as gram-positive or -negative using the gram staining set (bd, franklin lakes, nj.) based on these results, the isolates were then run through the vitek 2 system (biomerieux, durham, nc) on either the grampositive or -negative id card according to manufacturer’s instructions. two samples were identified as s.aureus. as methicillin-resistant s. aureus (mrsa) is known to cause serious nosocomial infections, we used oxacillin selective media (fisher scientific, pittsburgh, pa) to determine the cultures’ resistance to beta-lactam antibiotics. five isolates were unidentifiable by the vitek 2 system and the sixth had confounding results. these samples were subsequently identified using 16s rrna sequencing technology provided by ge healthcare (houston, tx). results samples were taken from 9 areas on 20 ventilators making 180 samples in total. the majority of the samples (129/180) had no bacteria (figure 2a); but 19 of the 20 ventilators cultured positive for bacteria on at least one swabbed area (figure 2b.) table 1 presents a full list of all the bacteria identified. the areas that most commonly contained bacteria were the power button and the screen with 12 (60%) and 13 (65%) ventilators culturing positive, respectively (figure 2b.) these two areas are likely the most frequently contaminated because they are often touched by the gloved hand of healthcare providers. the two areas that have the closest proximity to the patients, the expiratory inlet and inspiratory outlet, were amongst the least frequently contaminated having only one (5%) ventilator testing positive for each of the two outlets (figure 2b.) in general, the organism number of instances bacillus licheniforms 1 bacillus megaterium 1 bacillus pumilusor 1 bacillus simplex 1 cronobacter sp. 1 enterococcus casseliflavus 4 enterococcus columbae 1 enterococcus faecium 1 kocuria rosea 1 kocuria varians 1 micrococcus luteus/lylae 4 pantoea vagans 1 rhizobium radiobacter 2 rothia nasimurium 1 sphingobacterium spiritivorum 1 sphingomonas paucimobilis 2 staphylococcus aureus† 2 staphylococcus capitis 2 staphylococcus epidermidis 23 staphylococcus haemolyticus 2 staphylococcus hominis 11* staphylococcus intermedius 2 staphylococcus lentus 7 staphylococcus warneri 2* table 1: a complete list of the bacteria identified from the 9 areas swabbed from the 20 clinically used ventilators. † 25 all res.j.biol, 2014, 5, 24-29     resistant to β-lactam antibiotics (mrsa), * one isolate could not be differentiated between s. hominis and s. warneri and therefore is represented twice. ventilators had relatively low contamination levels present shortly after patient use,  as the majority of the areas that were figure 2: (a) the number of samples out of a total of 180 that had 0, 1-10, 10-20, 20-50, 50-100, or more than 100 colonies isolated. (b) the number of ventilators that had bacteria isolated from 9 different areas swabbed: expiratory inlet, inspiratory outlet, exhaust port, interface buttons, power button, knob, screen, diaphragm, and exhalation pressure port. (c) the proportion of bacteria isolated that were gram-positive and -negative. (d) the proportion of the gram-positive bacteria isolated in each of the genera represented. (e) the proportion of the gram-negative bacteria isolated in each of the genera represented. 26 all res.j.biol, 2014, 5, 24-29     contaminated with bacteria (43/55) contained fewer than 10 colonies (figure 2a). of the bacteria that were present, the majority (89.3%) was gram-positive (figure 2c) and 74.6% of these belonged to the staphylococcus genus (figure 2d.) the rest of the gram-positive isolates were divided amongst five genera: micrococcus, enterococcus, kocuria, bacillus, and rothia (figure 2d.) while the gram-positive bacteria were clearly skewed towards one genus, the gram-negative bacteria were more evenly split amongst five genera: sphingomonas, sphingobacterium, rhizobium, cronobacter, and pantoea (figure 2e.) all of the bacteria isolated are characterized as either biosafety level (bsl)-1 or -2 and the majority of the bacteria are associated with common skin flora that do not typically cause infections unless the patient is immunocompromised. one important exception to this observation is that methicillin-resistant s. aureus (mrsa) was isolated from the interface and power buttons of one of the ventilators. discussion the ventilators included in this study were used to treat chronic obstructive pulmonary disease (copd), cardiopulmonary arrest, respiratory failure, respiratory insufficiency, and surgery in order to protect the airway of the patient. cardiopulmonary arrest and respiratory failure are considered acute respiratory failures, which along with copd make up approximately 79% of indications for ventilation, indicating that our sample was representative of some of the most common reasons for ventilation.6,7 overall, the ventilators were relatively free of bacterial contamination. the gram-positive bacteria isolated are primarily commensal skin flora and were principally found on the power button and screen, indicating that the primary mode of contamination is likely the healthcare provider touching the ventilator’s interface after touching the patient. after combining the microbiological data with knowledge of normal ventilation protocols, the reason behind the pattern of contamination becomes clear. when the healthcare professional begins ventilation on a patient they start by using a gloved hand to turn on the power button and then adjust the settings using the interface buttons, the knob, and the screen. they then connect the inspiratory and expiratory outlets to single use tubing, which is then connected to the patient’s face. the hospital reports that the tubing is replaced between patients and also when visibly soiled. once the initial setup is complete, healthcare professionals will enter the room of a patient and interact with the ventilator approximately 50 times a day; the exact frequency at which they enter the room will depend on the patient’s individual needs. during each of these interactions, the healthcare professional will put on new gloves when her/she enters the room. they will then touch the patient in order to perform a variety of tasks such as suctioning, listening to breathing sounds, performing chest therapy, and checking vitals. none of these activities require taking the patient off the ventilator as it is a closed system, however they can all cause the machine to register a change in oxygen flow and will therefore cause it to alarm. the healthcare professional will then touch the screen in order to silence the alarm before continuing to work on the patient. this practice of frequently touching the screen directly after patient contact explains why the screen is one of the most contaminated areas of the ventilator and why the contamination is mostly skin flora. the power button is the second most frequently contaminated and only seems to be touched when a patient is connected or disconnected from the machine. this may be because it is behind a retractable panel, making it more of a challenge to clean. while the retractable panel protects the ventilator from accidentally being turned off after being bumped, it makes the power button more difficult to clean and disinfect. the outlets connecting tubing are touched when a patient is initially connected to the machine or disconnected as well as once a day in order to change the filters. the hospital reports that when the healthcare professional enters the room to change the filter, he/she will do so with newly gloved hands before touching the patient, indicating that the majority of the interactions with these areas are not preceded by direct contact with the patient. this would be consistent with the fact that most of the outlets connecting tubing to and from patients were found to have no bacteria present. however there was one outlet that connected the machine to the patient which tested positive for enterococcus casseliflavus while another outlet that connected to the machine from the patient tested positive for sphingobacterium spiritivorum. these two instances of contamination are important to note because, while infrequent, they are associated with ventilator components that have a high proximity to the patient. both e. casseliflavus and s. spiritovorum are bsl-2 organisms that can cause opportunistic infections.8,9 bacteria that are commonly associated with soil, such as rhizobium radiobacter and bacillus pumilus, were also isolated, indicating that outside soil is also likely a contributing source of contamination. it is important to note that bacillus sp. are difficult to kill because they form spores. while none of these soil bacteria cause infections in healthy individuals, they can do so in immunocompromised patients.10,11 none of the patients included in this study were immunocompromised, however immunocompromised patients are at an increased risk to develop both infectious and non-infectious pulmonary issues.12,13 once immunocompromised patients are ventilated, they have an increased rate of vap and mortality.14,15 the most concerning bacteria isolated from the ventilators were s. aureus resistant to beta-lactam antibiotics, or mrsa. mrsa has become an increasingly problematic organism for hospitals to control due to its resistance to multiple antibiotics.16 while we swabbed 3 ventilators used on patients positively diagnosed with a mrsa infection, only one ventilator tested positive for the bacteria. as we swabbed directly after use on a patient and before the ventilator was subjected to disinfection or sterilization, we can see that the transmission of the bacteria from the patient to the ventilator is low. additionally, the cleaning/disinfecting/sterilization processes in place are likely working to prevent crossover mrsa contamination given that we did not see any mrsa present on ventilators that did not come directly from isolated 27 all res.j.biol, 2014, 5, 24-29     patients. furthermore, another ventilator isolated due to an infection with a bacteria found in human fluids and cosmetics,17,18 enterobacter gergoviae, did not test positive for the bacteria in question. after the ventilator is removed from a patient, the single use components, such as the tubing, are replaced and the reusable areas are cleaned and disinfected. the respiratory therapists wipe down the surfaces of the ventilator (inspiratory and expiratory ports, pressure port, screen, heater, and stand) with caviwipes; a commercially available cleaning and disinfecting wipe. the expiratory filter is replaced every 24 hours or sooner if soiled. the hospital has also instituted respiratory isolation for patients that have been diagnosed with infections such as mrsa or clostridium difficile. c. difficile has the potential to cause devastating gut infections in patients that have recently been treated with antibiotics.19 healthcare personnel and visitors must wear clean gloves and scrubs when entering an isolated patient’s room and dispose of them when exiting. if a ventilator was used on a respiratory isolated patient, the expiratory cassette is removed and sent to sterile processing for steam sterilization. bleach wipes are additionally used to clean all external ventilator surfaces, if the ventilator was used on a patient in isolation because of a c. difficile infection. it should be noted that one of the limitations of our study is that we are not able to comment on the absence of c. difficile in our samples, because we only grew our samples aerobically keeping in mind that all of the surfaces that we sampled are exposed to air and therefore are in aerobic conditions. additionally, it is possible that there are more bacteria present on the ventilators immediately after use that may die off before we are able to swab them. however, these bacteria would be unlikely to be transmitted to the subsequent patient as they would be dead before the next patient was in contact with the machine. overall, we have found that there is a low transmission rate of bacteria onto the maquet servo ventilators at the baltimore va hospital. acknowledgments the authors would like to thank mr. ed gordon for his photography expertise and support. this project was supported in part by an appointment to the research participation program at the authors’ center administered by the oak ridge institute for science and education through an interagency agreement between the author’s agency. the authors also thank the author’s agency’s critical path program for providing funding. disclaimer: the mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the u.s. department of health and human services. references 1. chastre, j., fagon, j-y. (2002). ventilator-associated pneumonia. am. j. resp. crit. care. med 165, 867-903. 2. warren, d., shukla, s., olsen, m., kollef, m., hollenbeak, c., cox, m., cohen, m., fraser, v. (2003). outcome and attributable cost of ventilator-associated pneumonia among intensive care unit patients in a suburban medical center. crit. care. med31, 1312-1317. 3. kappstein, i., schulgen, g., beyer, u., geiger, k., schumacher, m., daschner, f. (1992).prolongation of hospital stay and extra costs due to ventilator associated pneumonia in an intensive care unit. eur. j.clin.microbiol. infect.dis11,504-508. 4. drakulovic, m., torres, a., bauer, t., nicolas, j., nogue, s., ferrer, m. (1999). supine body position as a risk factor for nosocomial pneumonia in mechanicallyventilatedpatients: a randomised trial. lancet 354, 1851-1858. 5. cadwallader, h., bradley, c., ayliffe, g. (1990). bacterial contamination and frequency of changing ventilator circuitry. j. hosp.infect15, 65-72. 6. tobin, m. (2001). advances in mechanical ventilation. n. engl. j. med 344, 1986-1996. 7. esteban, a., anzueto, a., alia, i., gordo, f., apezteguia, c., palizas, f., cide, d., goldwaser, r., soto, l., bugedo, g., rodrigo, c., pimentel, j., raimondi, g., tobin, m. (2000). how is mechanical ventilation employed in the intensive care unit? am. j. resp. crit. care med 161, 1450-1458. 8. reid, k., cockerill, f., patel, r. (2001). clinical and epidemiological features of enterococcus casseliflavus/flavescens and enterococcus gallinarum bacteremia: a report of 20 cases. clin. infect. dis32, 15401546. 9. lambiase, a., rossano, f., del pezzo, m., raia, v., sepe, a., de gregorio, f., catania, m. (2009).sphingobacterium respiratory tract infection in patients with cystic fibrosis.bmc res. notes 2, 262. 10. kimouli, m., vrioni, g., papadopoulou, m., koumaki, v., petropoulou, d., gounaris, a., friedrich, a., tsakris, a. (2012). two cases of severe sepsis caused by bacillus pumilis in neonatal infants. j. med.microbiol61, 596-599. 11. lai, c., teng, l., hsueh, p., yuan, a., tsai, k., tang, j., tien, h. (2004).clinical and microbiological characteristics of rhizobium radiobacter infections.clin. infect. dis 38, 149-153. 12. crawford, s. (1999). noninfectious lung disease in the immunocompromised host. respiration 66, 385-395. 28 all res.j.biol, 2014, 5, 24-29     13. baughman, r. (1999). the lung in the immunocompromised patient: infectious complications part 1. respiration 66, 95-109. 14. combes, a., costa, m., trouillet j., baudot, j., mokhtari, m., gibert, c., chastre, j. morbidity, mortality, and qualityof-life outcomes of patients requiring> or = 14 days of mechanical ventilation. crit. care med 31, 1373-1381. 15. rano, a., agusti, c., benito, n., rovira, m., angrill, j., pumarola, t., torres, a. (2002). prognostic factors of nonhiv immunocompromised patients with pulmonary infiltrates. chest122, 253-261. 16. grgurich, p., hudcova, j., lei, y., sarwar, a., craven, d. (2012). management and prevention of ventilator-associated pneumonia caused by multidrug-resistant pathogens. expert rev. respir. med 6, 533-555. 17. davin-regli, a., chollet, r., bredin, j., chevalier, j., lepine, f., page, j. (2006). enterobacter gergoviae and the prevalence of efflux in parabens resistance. j. antimicrob. chemother 57, 757-760. 18. brenner, d., richard, c., steigerwalt, a., asbury, m., mandel, m. (1980) enterobacter gergoviae sp. nov.: a new species of enterobacteriaceae found in clinical specimens and the environment. int. j. syst.bacteriol 30, 1-6. 19. theriot, c., young, v. (2013). microbial and metabolic interactions between the gastrointestinal tract and clostridium difficile infection. gut microbes 5, 86-95. 29 microsoft word 171-878-1-ce_layoutediting.docx women’s preferences for male facial masculinity are not condition-dependent in a large online study stefan van dongen, femke danckaers, jessie beerten & toon huysmans all res. j. biol., 2019, 10, 1-6 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol 10, 2019, 1-6 women’s preferences for male facial masculinity are not condition-dependent in a large online study stefan van dongen1,*, femke danckaers2, jessie beerten1 & toon huysmans2 1: evolutionary ecology group, university of antwerp, belgium 2: mec-vision lab, department of physics, university of antwerp, belgium *corresponding author: evolutionary ecology group, university of antwerp, universiteitsplein 1, b-2610 wilrijk, belgium. email: stefan.vandongen@uantwerpen.be abstract: while several studies have found evidence for conditional-dependent effect on women’s preferences for male masculinity, others have questioned the relative importance of these effects. in this study, we evaluated variation in women’s preference for male facial masculinity in a forced-choice experiment. nearly 1200 participants scored preference for manipulated photos and surface scans. between-individual variation in preferences were relatively small, especially for the evaluation of the surface scans. nevertheless, preferences from the evaluations of photos and scans correlated positively, indicating that both stimuli provide similar biological information. only few condition-dependent variables correlated significantly with preference for masculinity, and not all in predicted directions. stronger preference for masculine male faces – albeit only significant for the photos – with higher own women attractiveness was observed as expected. yet, for perceived infectability, consistently across the photos and scans, a negative association with preference for masculine faces was observed, which is opposite to theoretical predictions. in addition, no effects of pathogen exposure, germ aversion (a correlate of disgust), relational status, preference for short term relationships and sociosexuality were detected. thus, overall, our study is in line with recent large studies that also find only very weak conditiondependent effects, if any. keywords: woman preferences; masculinity; forced-choice experiment, context-dependent, repeatability introduction in humans, male masculinity is regarded as a signal of ‘genetic quality’. nevertheless, women do not always favor more masculine traits. indeed, women’s preferences for masculine characteristics varies strongly among individuals. while several studies have found indications for context-dependent effects on the strength of choice for more masculine traits, the generality of these results have been questioned more recently [1]. face when choosing a mate, where higher masculinity would reflect “good genetic quality” while lower masculinity would reflect “good parental quality”. the contextdependency is thought to have evolved to drive mate preferences being optimized in such a way that under conditions where ‘genetic quality’ matters most to increase women’s fitness, preferences are biased towards more masculine traits. support has been found for increased preferences for more masculine male faces when conception is likely [2], in the context of short term relationships [2,3,4], when women consider themselves relative attractive [5], when exposed to pathogen cues [6] and when women are more sensitive to pathogen disgust [7]. however, not all studies find strong and consistent context-dependent effects, and more recently, one study has suggested a much more prominent role for genetic variation where genetic variation accounted for 30 – 40% of the between-women variation in preferences for masculine faces compared to less than 1% contribution of the context-dependent factors [1]. in this study, the importance of context-dependent factors – including relational status, interest in long vs. short term relationships, pathogen exposure, pathogen disgust, self-perceived attractiveness, use of hormonal anticonception and willingness to engage in uncommitted sexual relationships – are investigated on the basis of a large scale online questionnaire with 1190 participants. in addition, we compare patterns for two types of stimuli, namely manipulated photos which are often used in this type of research (and which we obtained from the published literature), and manipulated surface scans of the face, without any texture skin color or details about eyebrows. the latter thus only focus on facial shape, while photos present a more natural looking stimulus for evaluation. we specifically estimate between-individual variation in preferences for masculinity and how much of this variation is explained by our context-dependent variables. patterns observed for photos and scans are compared. 1 all res. j.biol, 2019, 10, 1-6 materials and methods participants and background information: we invited students from the university of antwerp and ghent, to participate to the online questionnaire via email and advertisement on student web fora. in total 1190 heterosexual women participated. participants age (in years), relational status (in steady relationship or not), preferred type of relationship in the near future for women claiming not to be in a steady relationship (long term, short term or no relationship), use of hormonal anticonception (yes, no, no answer) and own attractiveness (on a scale from 1 to 10), were first obtained. figure 1: stimuli used from three published papers, where from top to bottom, the right, left and left photo is more masculine. preference for facial masculinity: preference for masculinity was measured with a standard forced choice test, a technique commonly used in this type of research [1]. participants were shown two stimuli of the same face side by side, one of which was manipulated to be more masculine, the other less masculine. the left-right order was randomized. participants were asked to rate which face they found more attractive on an 8-point scale (1= left is much more attractive, 8 = right is much more attractive; 4= left is slightly more attractive, 5 = right is slightly more attractive). two types of stimuli were shown, manipulated pictures available from the literature and manipulated surface scans. three sets of pictures were shown. we used a male set of pictures from [6,8,9] (figure 1 (top panel). in addition, participants rated manipulated facial surface scans. these scans were part of the caesars dataset (http://store.sae.org/caesar/). this dataset contains approximately 2000 european males and females aged between 18 and 65 years. first, a statistical shape model was built from 346 dutch males, 346 dutch females, 346 italian males and 346 italian females [10]. from this entire dataset, we then selected 364 males and 374 females aged between 18 and 30 years old having a normal bmi (20-25). next, the shape sexual dimorphism was modelled using a linear discriminant analysis, of which a heat map is shown in figure 2 (top panel), and a gender score was calculated for each subject in this dataset. in total, 6 scans with varying degree of masculinity were selected and manipulated to become more and less masculine with 0.5 standard deviation units using the linear discriminant function. an example of such manipulated scans is shown in figure 2 (bottom panel). figure 2: top: shape differences along the femininemasculine dimension on the basis of surface scans of the face. blueish color reflects small sexual dimorphism while the orange and reddish color reflect areas of high sexual dimorphism. bottom: example of manipulated scans presented to the participants, where the right scan is more masculine. 2 all res. j.biol, 2019, 10, 1-6 pathogen exposure, perceived vulnerability to disease and the sociosexual-orientation inventory: participants were randomly exposed to pictures holding cues of potential disease threat using the pictures in [11] just prior to scoring the stimuli of masculinized/feminized pictures and scans. at the end of the questionnaire, perceived vulnerability to disease was scored using a 15-item self-report instrument [12]. sociosexuality was assessed using the 9-item revised soi questionnaire [13]. we first tested for associations with the total score, and if this was statistically significant, the importance of the three sub-scales – sociosexual behavior, attitude and desire – was investigated as well. perceived vulnerability to disease has been shown to consist of two subscales, namely believes about one’s own susceptibility to infectious diseases (further called perceived infectability) and emotional discomfort in context that connote an especially high potential for pathogen transmission (further called germ aversion) [12]. to assess if in our population, these two subscales were also present, we performed a factor analysis and constructed a biplot. in this plot, the 15 different items are labelled on the basis of their established link with perceived infectability (pi) and germ aversion (ga) [12]. this analysis confirmed the existence of these two dimensions (fig.3) which will be used as explanatory variables modeling preferences for masculinity. figure 3: biplot of the factor analysis of the 15-item questionnaire on perceived vulnerably to disease. items were label with a prefix of pi for questions related to perceived infectability and ga for questions related to germ aversion [12]. to evaluate if the soi total score indeed provides information on the willingness of individuals to engage in uncommitted (short-term) relationships, we first compared soi total among the 4 possible relational categories (steady, not steady looking for long term, not steady looking for short term, not steady and not looking for relationship), expecting a higher score for women looking for short term relationships. there was a significant difference between the four groups (f3,1186=49.6, p<0.0001), with the highest score for women not in a steady relationship and looking for short term relationships (mean = 3.0, se=0.09), compared to the other groups (steady: mean = 2.29, se=0.03; not steady looking for long term: mean = 2.20, se=0.02; not steady not looking for relationship: mean = 2.12, se=0.04). thus, as expected, single women who claimed to be interested in a short term relationship scored higher on the soi total score. figure 4: association between women-specific preferences for facial masculinity on the basis of manipulated photos and scans. the association was significantly positive (r=0.19, t1188 = 6.48, p<0.0001)). marginal distributions are presented as histograms. statistical analyses: the scores for preference for masculinity were transformed such that high values indicate a preference for more masculine traits and that an average of zero indicates no preference. these scores were used as dependent variable in linear mixed models. analyses were performed separately for the photos and scans as stimuli and for all stimuli combined. in a first set of models (null models), individual was treated as random effects and no explanatory variables were added. on the basis of these models, we estimated the overall preference for masculinity (significance of intercept) and the repeatability of this preference (between-individual variation divided by the sum of the between-individual variation and residual variation). from these two null models of photos and scan stimuli, we obtained the individual-specific preferences as the best linear unbiased prediction (blup) estimates of the random effects. we tested for a correlation between these two to establish the repeatability across the two types of stimuli (photo and scan) using pearson’s correlation. 3 all res. j.biol, 2019, 10, 1-6 finally, all explanatory variables (age, own attractiveness, perceived infectability, germ aversion, soi total, hormonal anticonception use, relational status, and preferred type of relationship (nested within relational status)) were added to the null models and tested for their significance and adjusting pvalues for multiple testing using a bonferroni correction. the repeatability of these full models was also calculated to obtain an estimate of the amount of between-individual variation in preference for masculinity was explained by the contextdependency incorporated in the fixed effects part of the model. table 1: tests of associations between explanatory variables and women’s preference for masculine faces. the overall intercept estimates the average score (0 = no preference, positive values indicate preference for more masculine faces) without correcting for any of the covariates. between-women variation in preferences of the null and final model as well as the proportion of variation explained are provided. significant effects after bonferroni correction (i.e., multiply all p-values by 10, the number of tests) (*: p<0.05; **: p<0.01; ***: p<0.001) are highlighted in bold. explanatory variable photos scans all pathogen exposure (yes vs. no) 0.02 (0.05) -0.01 (0.02) -0.004 (0.021) own attractiveness 0.06 (0.02)* 0.01 (0.01) 0.006 (0.008) age 0.04 (0.01)*** 0.01 (0.01) 0.002 (0.004) perceived infectability -0.09 (0.03)* -0.03 (0.01)* -0.033 (0.014)* germ aversion 0.06 (0.03) 0.01 (0.01) 0.012 (0.013) soi total -0.08 (0.06) 0.05 (0.02) 0.053 (0.022) relation (no vs. yes) 0.09 (0.06) -0.01 (0.02) -0.077 (0.026) future relationship (short vs. long) -0.07 (0.14) -0.06 (0.06) -0.048 (0.054) future relationship (no vs. long) -0.00 (0.09) -0.01 (0.04) -0.017 (0.036) hormonal anticonception use (no vs. yes) -0.04 (0.07) 0.04 (0.03) 0.073 (0.026)* overall preference for masculinity (intercept of null model) -0.15 (0.03)*** -0.10 (0.01)*** -0.21 (0.07)*** repeatability null model 17.2% 2.05% 6.6% repeatability final model 16.1% 1.91% 6.4% proportion of betweenfemale variation explained by model 6% 7% 3% results descriptive statistics: of the 1190 heterosexual women participants, 657 (55%) reported to be in a steady relationship. of the 533 (45%) others, 333 (62%) reported to be looking for a long term relationship, 56 (11%) for a short term relationship and 144 (27%) were not looking for a relationship at the time of the questionnaire. overall, 863 (73%) of the woman participants reported using hormonal anticonception. this proportion was higher in women in a steady relationship (87%), compared to those not in a steady relationship (55%). the average age equaled 21.6 years (standard deviation=2.85) and ranged between 18 and 30 years. repeatabilities and rreferences for masculinity: for both stimuli (photos and scans) and all combined, women showed a slight but highly significant preference for less masculine traits (table 1). the effect was strongest when all stimuli were analyzed simultaneously. the repeatability was almost a tenfold higher for photos compared to scans (table 1). nevertheless, masculinity preferences of both types of stimuli were significantly correlated (fig.4) indicating that they reflect comparable underlying biological preferences of facial aspects. of all explanatory variables, age, own attractiveness and perceived significantly explained variation in preferences for masculinity in photos (table 1, fig. 5). for perceived infectability, the association was – contrary to a priori expectations – negative, where women with a low score showed no preference for more masculine or feminine faces, while women with a high score showed significant preference for less masculine traits (i.e., confidence band not including zero, fig.5). the association with age was positive where younger women showed preference for less masculine faces, while the older women showed a slight preference for the more masculine version of the photos (fig. 5). for own attractiveness, the association was as positive, as expected, with no significant preference for masculine traits in women with high perceived own attractiveness, and a significant preference for less masculine photos for women who experienced themselves as unattractiveness (fig. 5). all explanatory variables together explained 6% of the betweenindividual variation in preferences for masculinity (table 1). figure 5: graphic representation of the three significant associations between preference for masculinity in photos and context dependent explanatory variables (table 1). raw data and estimated regression lines with 95% confidence bands are 4 all res. j.biol, 2019, 10, 1-6 presented. strength of associations were 1.0% for perceived infectability, 1.4% for age and 0.7% for own attractiveness. variation in preferences for masculinity on the basis of the scans was only significantly explained by perceived infectability and the association was highly comparable to that found for photos (table 1, fig. 6). indeed, the association was again – contrary to expectations – negative, where women with a low score showed no preference for more masculine or feminine faces, while women with a high score showed significant preference for less masculine traits (fig.6). all explanatory variables together explained 11% in the betweenindividual variation of masculinity preferences (table 1). figure 6: graphic representation of the only significant association between preference for masculinity in scans and a context dependent explanatory variables (table 1). raw data and estimated regression line with 95% confidence bands is presented. the strength of association was 0.8%. figure 7: graphic representation of the only significant association between preference for masculinity and perceived infectability (table 1). raw data and estimated regression line with 95% confidence bands is presented. the strength of association was 0.95%. finally, for all stimuli combined, we found a significant contribution of perceived infectability and use of hormonal anticonception (table 1). women who did not use hormonal anticonception showed a less strong preference for less masculine traits (table 1). nevertheless, for women not taking hormonal anticonception, the preference score equaled -0.18 (se=0.06) and differed significantly from zero (t13=-2.26, p=0.02) indicating that these women also preferred the less masculine versions of the scans and photos. the association between perceived infectability and preference for masculinity was again negative (table 1) and women showed a significant preference for less masculine stimuli for all values of perceived infectability, yet a stronger preference for lower masculinity when perceived infectability was higher (fig. 7). discussion several studies have proposed an adaptive explanation for variation in women’s preferences for masculinity in facial traits, where women increase their preference for higher male masculine features in contexts where greater ‘genetic quality’ over greater ‘parental quality’ is desired to increase inclusive fitness. on the other hand, larger more recent studies question this theory [1]. in this study, studied context-dependent explained 3 to 6%. we found evidence for effects of perceived infectability, a measure of believes about one’s own susceptibility to infectious diseases [12], women’s own attractiveness and use of hormonal anticonception. in addition, no effect of pathogen exposure, germ aversion (a correlate of disgust) relational status, interest in short term relationships and sociosexuality was detected. as predicted, preferences for less masculine traits were observed in women who rated themselves as relatively unattractive, while women who rated themselves as attractive showed no preference. however, this effect could only be demonstrated when manipulated photos were rated, and not for the scans nor for all stimuli combined. nevertheless, associations were – albeit weakly – in the expected direction when scans were evaluated as well. the interpretation is thus that the context-dependency of the choice switches from no preference for less masculine stimuli in women who rate themselves as unattractive and thus presumed ‘better parental quality’. when analyzing all stimuli together, women not taking hormonal anticonception showed a preference for the more masculine versions of the scans and photos. the most consistent association between a context-dependent variable and preference for masculinity across photos and scans was an unexpected negative correlation with perceived 5 all res. j.biol, 2019, 10, 1-6 infectability. women who consider themselves as more likely to become infected prefered more feminine male faces. previous studies did not investigate perceived infectability, yet, found correlations with pathogen disgust [7]. however, our study did not show any associations between preference and germ aversion, a factor which showed relatively strong correlations with disgust [12]. together with the absence of any effect of exposure to pathogen cues on preference for masculinity in our study, our results do not support any evidence that pathogens influence female mate choice in our population. it is unlikely that the negative results are the result of a lack of power, since 1190 individuals participated in this study. indeed, the power to detect a correlation coefficient of 0.10 (i.e., coefficient of determination of 1%) with a sample size of 1190 equals 93%. it is important to note, however, that among-individual variation in preferences when manipulated surface scans of the face were used as stimuli was much smaller compared to the photos. this indicates that when presenting variation in shape only, the variation in masculinity is less obvious for the participants. this could also implicate that shape variation alone does not evoke the same mate preferences. alternatively, a manipulation of 0.5 standard deviation units may simply not generate the same visual differences compared to photos. nevertheless, the betweenwomen variation in masculinity preferences as estimated from scans and photos, did correlate positively – albeit fairly weakly – suggesting that, at least in part, the preferences measured by both stimuli did reveal similar mate preferences in these women. it thus seems important that future studies not only focus on the relative importance of different contextdependent factors but also on which aspects of morphological masculinity affect women mate preferences. acknowledgments we thank marino raes for setting up the online questionnaire. this work was supported by the agency for innovation by science and technology in flanders (iwt-sb 141520). references 1. zietsch bp, lee aj, sherlock, jm & jern p (2015) variation in women's preferences regarding male facial masculinity is better explained by genetic differences than by previously identified context-dependent effects. psychological science 26, 1440–1448. 2. penton-voak is, perrett di, castles dl, kobayashi t, burt dm, murray lk & minamisawa r. (1999). menstrual cycle alters face preference. nature, 399, 741– 742. 3. little ac, jones bc, penton-voak is, burt dm & perrett di (2002). partnership status and the temporal context of relationships influence human female preferences for sexual dimorphism in male face shape. proc.r.soc.b 269, 1095–1100. 4. little ac, cohen dl, jones bc & belsky j (2007). human preferences for facial masculinity change with relationship type and environmental harshness. behavioral ecology and sociobiology, 61, 967–973. 5. little ac, burt dm, penton-voak is & perrett di (2001). self-perceived attractiveness influences human female preferences for sexual dimorphism and symmetry in male faces. proc.r.soc. b, 268, 39–44. 6. little ac, debruine lm & jones bc (2011). exposure to visual cues of pathogen contagion changes preferences for masculinity and symmetry in opposite-sex faces. proc.r.soc b 278, 2032–2039. 7. debruine lm, jones bc, tybur jm, lieberman d & griskevicius v (2010). women’s preferences for masculinity in male faces are predicted by pathogen disgust, but not by moral or sexual disgust. evolution and human behavior, 31, 69–74. 8. allan k, jones bc, debruine lm & smith ds (2012). evidence of adaptation for mate choice within women's memory. evolution and human behavior, 33, 193–199. 9. smith fg, jones bc, debruine la & little ac (2009). interactions between masculinity–femininity and apparent health in face preferences. behav ecol, 20, 441– 445. 10. danckaers f, huysmans t, lacko d, ledda a, verwulgen s, van dongen s & sijbers, j (2014) correspondence preserving elastic surface registration with shape model prior. international conference of pattern recognition, 22, 2143-2148. 11. curtis v, aunger r & rabie, t. (2000). evidence that disgust evolved to protect from risk of disease. proc.r.soc.lond. b (suppl.), 271, s131-s133. 12. duncan la, schaller m & park jh (2009) perceived vulnerability to disease: development and validation of a 15-item self-report instrument. personality and individual differences, 47, 541 – 546. 13. penke l & asendorpf jb (2008) beyond global sociosexual orientations: a more differentiated look at sociosexuality and its effects on courtship and romantic relationships. journal of personality and social psychology, 9, 1113–1135. 6 microsoft word manuscript_le.docx sgk1 (glucose transport), dishevelled2 (wnt signaling), lc3/p62 (autophagy) and p53 (apoptosis) proteins are unaltered in lafora disease peixiang wang, lori israelian, yunlin xue, siyuan song, liliana attisano and berge a. minassian all res. j. biol., 2016, 7, 28-33 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 3, vol 7, 2016, 28-33 sgk1 (glucose transport), dishevelled2 (wnt signaling), lc3/p62 (autophagy) and p53 (apoptosis) proteins are unaltered in lafora disease peixiang wang1, lori israelian1,2, yunlin xue1, siyuan song3, liliana attisano3 and berge a. minassian1,2,4,5 1program in genetics and genome biology, the hospital for sick children, toronto, on, canada. 2institute of medical sciences, university of toronto, 1 king’s college circle, toronto, on, canada. 3donnelly centre for cellular and biomolecular research, university of toronto, toronto, on, canada. 4division of neurology, department of paediatrics, the hospital for sick children, toronto, on, canada. 5correspondence: berge.minassian@sickkids.ca graphical abstract: multiple disparate functions have been attributed to the lafora disease gene products laforin and malin with separate hypotheses of pathogenesis of neurotoxic polyglucosans (lafora bodies) and neurodegeneration in this disease. we tested four of these hypotheses in lafora disease mouse models. we were unable to confirm these hypotheses. abstract: glycogen forms through the concerted actions of glycogen synthase (gs) which elongates glycogen strands, and glycogen branching enzyme (gbe). lafora disease (ld) is a fatal neurodegenerative epilepsy that results from neuronal accumulation of hyperphosphorylated glycogen with excessively long strands (called polyglucosans). there is no gbe deficiency in ld. instead, the disease is caused by loss-of-function mutations in the epm2a or epm2b genes, encoding, respectively, a phosphatase, laforin, and an e3 ubiquitin ligase, malin. a number of experimentally derived hypotheses have been published to explain ld, including: the sgk1 hypothesis phosphorylated sgk1 (psgk1) raises cellular glucose uptake and levels, which would activate gs. based on observing increased psgk1 in ld mice it was proposed that raised psgk1 leads to polyglucosan generation through gs hyperactivation. the dishevelled2 hypothesis downregulating malin in cell culture was reported to increase levels of dishevelled2, which through the wnt/glycogen synthase kinase-3 pathway would likewise overactivate gs. the autophagic defect hypothesis polyglucosans may be natural 28 all res. j. biol, 2016, 7, 28-33     byproducts of normal glycogen metabolism. ld mice were reported to be autophagy-defective. ld would arise from failed autophagy leading to failed polyglucosan clearance. finally, the p53 hypothesis laforin and malin were reported to downregulate p53, their absence leading to increased p53, which would activate apoptosis, leading to the neurodegeneration of ld. in the present work we repeat key experiments that underlie these four hypotheses. we are unable to confirm increased psgk1, dishevelled2, or p53 in ld mice, nor the reported autophagic defects. our work does not support the above hypotheses in understanding this unique and severe form of epilepsy. keywords: lafora disease; polyglucosan; glucose transport; wnt pathway; autophagy; apoptosis introduction glycogen is synthesized by glycogen synthase (gs) and glycogen branching enzyme (gbe). gs activity is downregulated by phosphorylation, mainly by glycogen synthase kinase-3 (gsk3), and allosterically activated by the main intracellular form of glucose, glucose 6-phosphate (g6p). gbe deficiency results in an abnormal form of glycogen (called polyglucosan) which is poorly branched (i.e. has chains that are too long) and which precipitates, accumulates, and in neurons clogs axons causing adult polyglucosan body disease (which clinically resembles amyotrophic lateral sclerosis). gs overactivity likewise generates polyglucosans. in horses, an overactivating muscle gs gene mutation, and in mice, transgenic overexpression of muscle gs, lead to muscle polyglucosan accumulations. in humans, deficiency of the irreversible glycolytic enzyme muscle phosphofructokinase impairs glycolysis, which raises g6p levels, which hyperactivates gs and leads to muscle polyglucosans and disease1. lafora disease (ld) is an epilepsy that onsets in teenagers, progresses to intractability, and gradually to death within 10 years2. pathologically, ld is characterized by gradual accumulation in neuronal somatodendritic compartments of masses of polyglucosans (lafora bodies; lb) that have the particularity of being hyperphosphorylated3. lb are most likely pathogenic: it was shown that preventing their formation through knockout (ko) or downregulation of gs in ld mouse models eliminates lb and rescues the disease4-6. ld is caused by loss-of-function mutations of the epm2a or epm2b genes encoding the interacting laforin and malin enzymes respectively7, 8. laforin is a glycogen phosphatase, and malin a ubiquitin e3 ligase whose ubiquitination targets, at least in cell culture experiments, include gs3, 7-11. multiple hypotheses have been advanced to explain ld, including: (i) laforin deficiency leads to glycogen hyperphosphorylation, which, through unknown mechanisms, results in conversion of normal glycogen to polyglucosan3. (ii) malin deficiency leads to reduced ubiquitin-mediated proteasomal degradation of gs, and the resultant increased gs drives polyglucosan generation9, 11. (iii) laforin or malin deficiency lead, through unknown mechanisms, to increased phosphorylated serum/glucocorticoid-induced kinase-1 (psgk1), which increases glucose uptake, potentially hyperactivating gs to drive polyglucosan formation12. (iv) malin deficiency upregulates dishevelled2, which upregulates the wnt pathway, which downregulates gsk3, which would upregulate gs, leading to polyglucosans13. (v) polyglucosans may be byproducts of normal glycogen metabolism, which would need to be cleared, e.g. by autophagy. laforin or malin deficiency downregulates autophagy, which would prevent clearance of polyglucosans and allow their accumulation14-16. (vi) laforin or malin deficiency upregulates p53, which is proapoptotic and would lead to neurodegeneration17. the first two hypotheses garnered the greatest attention because of the direct mechanistic connections between the disease gene products and corresponding substrates, glycogen for laforin’s phosphatase activity and gs for malin ubiquitination. this led to substantial new glycogen metabolism knowledge, including the specific locations of phosphorylation in glycogen18, 19. however, both hypotheses were undermined by recent results. it was shown that expressing a laforin mutant lacking the laforin phosphatase activity in laforin ko mice rescues the disease, including eliminating lb20, suggesting that laforin’s glycogen phosphatase function is dispensable, and some other laforin function is critical in ld. secondly, it was shown that laforin or malin ko mice do not have increased amounts of soluble gs or gs activity3, 21, 22, bringing into question the relevance of the malin action on gs seen in cell culture experiments. given these new weaknesses of the leading hypotheses we decided to take a fresh look at the other four, and as a first step repeated experiments that had been fundamental to generating each of them, and performed new experiments. in each case we were unable to replicate 29 all res. j. biol, 2016, 7, 28-33     critical experiments, or obtain new results contradicting the proposed hypothesis, which we present below. results hypothesis iii (sgk1) authors of this hypothesis showed that in cell culture psgk1 increases following shrna knockdown of laforin or malin. they then tested whether psgk1 is increased in laforin ko ld mice and reported this to be the case. specifically, they show a western blot of sgk1 and psgk1 and quantify the psgk1/sgk1 ratio, which is seen to be twofold increased in the ko mice compared to wild-type (wt)12. we reproduce their figure of this result in figure 1a and note that: (1) they studied only laforin (and not malin) ko mice. (2) while there is variance in the measurements of the ko results, no variance is shown in the wt result (n=2 for each genotype). (3) they analyzed only skeletal muscle (and not brain). (4) they do not state the age of the mice, which is important given the progressive nature of the disease (for a finding to be pathogenic it should be present prior to disease onset). we performed sgk1 and psgk1 western blots on extracts from skeletal muscle and brain from 1 mo laforin23 and malin24 ko mice (lb are just starting to form at this age). we used the same antibodies as the authors of the hypothesis and the same laforin ko mice. we find no differences between wt and the ko animals (figure 1b).   figure 1. sgk1 and psgk1 are not increased in ld mice. (a) results from singh et al. reporting increased psgk1 in laforin knockout mice. (b) current results do not confirm elevated psgk1. anti-sgk1 and anti– phospho (thr-256) sgk1 were purchased from millipore, and the secondary antibody is from santa cruz biotechnology. wt, wild-type; mko, malin knockout; lko, laforin knockout. one month-old brain and muscle tissue lysates were used, respectively. the two samples from the same genotype are from two different animals. the western blot experiment was repeated at least three times. all western blot procedures are as described in turnbull et al, annals of neurology, 2010; 68:925-933. briefly, 40 μg of total proteins was applied to 10% sds-page, and transferred onto nitrocellulose membrane. after blocking with 5% milk in 1× tris-buffered saline and tween 20, the membrane was probed with primary antibody at 4°c for overnight, followed by secondary antibody conjugated with horseradish peroxidase as recommended by the manufacturer. proteins were visualized using a chemiluminescent detection kit. hypothesis iv (dishevelled2) noting interaction between malin and dishevelled2 in a yeast 2-hybrid screen, authors of this hypothesis overexpressed malin in cell culture and showed that this results in ubiquitination and degradation of dishevelled213. we tested whether dishevelled2 is reduced in malin ko animals and found this not to be the case (figure 2).   figure 2. dishevelled2 (dvl2) is not increased in ld mice, at both 1 month and 10 months of age. lanes 1 and 10 in the left panel confirm specificity of the antibody (dvl2 antibody from cell signaling); lane 1, non-activated dvl2 (mda-mb-123 cells grown in rpmi-1640 and 5% fbs); lane 10, activated dvl2 (same cells grown in wnt-3a conditioned media for 3 hours. bao et al: plos one 2012; 7(11):e48670). one month-old and 10 month-old muscle tissues, from wild-type, mko and lko mice, were used, respectively. hypothesis v (autophagy) when autophagy is active, the lc3 protein is lipidated (lc3-ii) and associated with autophagosomes. lc3-ii is the most commonly used autophagy marker, its increase indicating increased autophagy and decrease the opposite. two groups reported decreased lc3-ii in cell lines from laforin ko mice and ld patients14, 16. one reported reduced lc3-ii in laforin ko and malin ko mouse brains. a second autophagy marker, p62, was also affected. surprisingly, additional experiments showed that the apparent autophagy defect in laforin ko mice was mtor-dependent, while in malin ko mice it was not 15. we performed lc3 and p62 western blots in skeletal muscle and brain from laforin and malin ko mice and observe no changes in either (figure 3). 30 all res. j. biol, 2016, 7, 28-33       figure 3. lc3-ii and p62 are unchanged in ld mice (anti-lc3 antibody from novus biologicals, and anti-p62 from sigma). one month-old brain and muscle tissue lysates from wild-type, mko and lko mice, were used, respectively. hypothesis vi (p53) – based on cell culture experiments, and observations in laforin and malin ko mouse, the authors concluded that laforin or malin deficiency lead to increased levels of p53, which leads to apoptosis, which could explain the neurodegeneration of ld17. we cannot confirm increased p53 levels in laforin or malin ko mice (figure 4).   figure 4. p53 protein level is unchanged in ld mice (anti-p53 antibody from santa cruz biotechnology). western blot results for anti-p53 antibody were obtained from total protein and soluble protein of wild-type, mko and lko mice, respectively. conversion of glycogen to toxic polyglucosans in the diseases associated with gbe deficiency or phosphofructokinase deficiency is readily explained by classical glycogen metabolism. this is not so in ld, where heretofore veiled and until recently unsuspected facets of glycogen metabolism need to be uncovered. identification of the ld genes and enzymatic activities of their products, glycogen dephosphorylation (laforin) and gs ubiquitination (malin), were critical steps forward and gave rise to the leading ‘glycogen hyperphosphorylation’ and ‘gs hyperactivation’ hypotheses of ld. as mentioned, recent observations weakened these hypotheses and led us in this work to revisit four others. we are unable to confirm in ld mouse models the disturbance in dishevelled2 suggested by cell culture overexpression experiments and are unable to replicate results critical to the sgk1, autophagy, and increased p53-apoptosis hypotheses. a possible explanation for the difference between our mouse data with previous results is murine background. our malin ko mice24 were generated separately from others15. the laforin ko mice, on the other hand, are the same in all ld labs23, although they have been available in the different labs for over a decade and might have diverged in genetic background. the ‘defective autophagy’ hypothesis is additionally indirectly contradicted by the following. one of the groups that advanced this hypothesis found a disturbance in lc3 in cell lines but themselves could not confirm this in mouse brain16. secondly, a recent experiment showed that what disturbances there are in markers of autophagy in ld mice disappear with gs downregulation. in this experiment, malin ko mice were bred with gs ko mice to eventually generate malin ko animals heterozygous for gs (gs activity is reduced by 50% in these mice) or completely lacking gs. in both situations, lb were dramatically reduced, the ld neurological phenotype was completely rescued, and what autophagic marker dysregulations there were corrected4, confirming that autophagic disturbance in ld, if or when present, is secondary to the disturbance in glycogen metabolism, and is not a primary cause of ld4. but what then are the early pathogenic steps linking laforin or malin deficiency to polyglucosan formation and ld? there are additional ld hypotheses invoking laforin/malin actions via the neurodevelopmental protein neuronatin25 and others, all of which will need to be revisited. however, in our view, the original two major hypotheses should continue to be explored. after all, laforin is a confirmed glycogen phosphatase, glycogen in both laforin and malin ko mice is hyperphosphorylated, and this hyperphosphorylation correlates with polyglucosan accumulation and lb formation 3, 18, 19, 21, 24. as for malin, it is a confirmed ubiquitin e3 ligase whose substrates include gs, hyperactivity of which is a long-established cause of polyglucosan formation in phosphofructokinase deficiency and in various natural or engineered models of polyglucosan diseases1. we anticipate that future experiments will mitigate the recent observations weakening these gene function-based hypotheses, and will, in ways we cannot presently see, bring them together to explain the toxic glycogen structural transformation underlying ld. acknowledgments this work was funded by the ontario brain institute, genome canada and many families of patients with lafora disease. ba minassian holds the university of toronto michael bahen chair in 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(2007). laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo. proceedings of the national academy of sciences of the united states of america, 104:19262-6. 23. ganesh, s., delgado-escueta, a. v., sakamoto, t., avila, m. r., machado-salas, j., hoshii, y., akagi, t., gomi, h., suzuki, t., amano, k., agarwala, k. l., hasegawa, y., bai, d. s., ishihara, t., hashikawa, t., itohara, s., cornford, e. m., niki, h. & yamakawa, k. (2002). targeted disruption of the epm2a gene causes formation of lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice. human molecular genetics, 11:1251-62. 24. turnbull, j., wang, p., girard, j. m., ruggieri, a., wang, t. j., draginov, a. g., kameka, a. p., pencea, n., zhao, x., ackerley, c. a. & minassian, b. a. (2010). glycogen hyperphosphorylation underlies lafora body formation. annals of neurology, 68:925-33. 32 all res. j. biol, 2016, 7, 28-33     25. sharma, j., mukherjee, d., rao, s. n., iyengar, s., shankar, s. k., satishchandra, p. & jana, n. r. (2013). neuronatin-mediated aberrant calcium signaling and endoplasmic reticulum stress underlie neuropathology in lafora disease. the journal of biological chemistry, 288:9482-90. 33 microsoft word arj biol editted_layout.docx electroporation inefficiency in caulobacter henricii cb4 taylor howell carter all res. j. biol., 2022, 13, 1-6 the publication cost of this article might be covered by external sponsors. more info for sponsors at: innovacion@sacsis.es article issue 1, vol 13, 2022,1-6 electroporation inefficiency in caulobacter henricii cb4 taylor howell carter1 1university of south carolina, department of biology abstract: caulobacter is a well-studied model organism. the introduction of plasmids into caulobacter by way of electroporation or conjugation has been well-documented, as well as the barrier caulobacter’s s-layer has played in the efficiency of these techniques. standard electroporation and conjugation protocols with caulobacter henricii cb4 resulted in zero transformants. various parameters have been shown to increases electroporation efficiency and these parameters were adjusted for cb4. although, cb4 was not able to take up foreign dna though electroporation or conjugation, a variety of parameters which increases electroporation efficiency are now located in one location. keywords: electroporation, caulobacter, plasmids, eps, s-layer introduction caulobacter crescentus is a gram-negative alphaproteobacterium that divides asymmetrically, resulting in a stalked cell and a motile swarmer cell (1). in addition, it has a well-developed system of genetics (2) that makes it an excellent model system for the study of cellular differentiation, asymmetric division, and cell cycle transition (3,4). unlike the c. crescentus strains, the wild type strain c. henricii cb4 has received little attention even though it houses a 93 kb plasmid, which contains 21 heavy metal resistance genes (5,6, * submitted). cb4 was isolated from pond water in 1959 displaying a vibrioid morphology and forming bright yellow colonies (7). the nucleotide sequence of the cb4 genome and its plasmid has been determined (6), and an analysis of the results indicated that the plasmid contains genes involved in conjugation so it may be able to transfer itself to other strains of caulobacter. to facilitate a test for conjugal transfer, we attempted to add a kanamycin resistance gene to the plasmid by transposon mutagenesis. christen et al. (8) established the essential genome of caulobacter crescentus utilizing transposon mutagenesis from the pxyltn5 plasmid containing a xylose inducible tn5 transposase. the basic approach was to transfer the plasmid into c. crescentus from e. coli with selection for the kanamycin resistance conferred by the transposon. this technique proved to be an excellent way to get large numbers of c. crescentus mutants (8). we planned to use the pxyltn5 plasmid to transpose the gene for kanamycin resistance into the cb4 plasmid to provide a marker to select for the transfer of the cb4 plasmid transfer into a c. crescentus strain. a second approach was to modify the pbr322 plasmid so that it contained sequence homology to a copper atpase gene (copa) located on the cb4 plasmid. the resulting pbr322-copa plasmid contained the beginning and ending sequences of the copa gene flanking a tetracycline antibiotic gene. since this plasmid is not stable in caulobacters, once it was transferred to cb4, a homologous recombination event would be needed to disrupt the plasmid copper-translocating p-type atpase gene and confer tetracycline resistance to the host cell. preliminary experiments employing standard electroporation or conjugation techniques did not produce any antibiotic resistant cb4 colonies even though control experiments with c. crescentus cb15 were successful. although conjugation experiments generally work well, caulobacters are difficult to electroporate due to their paracrystalline protein surface layer, or s-layer (9). visual and centrifuge assays have shown the cb4 s-layer to be thicker than the cb15 s-layer and could be the reason for the unsuccessful electroporation. alternatively, cb4 could house an exopolysaccharide layer, or eps. in cb15 and cb2, the eps is a tetrasaccharide capsule which could not be easily removed by washing (10). the s-layer is comprised of a 98 kda rsaa protein that assembles into six subunit hexagons that combine with other hexagon subunits to create a two-dimensional hexagonal array (11). ca2+ is required for the proper crystallization of the rsaa protein, and the removal of ca2+ has been shown to disrupt the crystallization (12). s-layers are involved in cellular protection and stabilization (13). in caulobacter, the s-layer is hypothesized to be involved in both ion sensing and allowing caulobacter to live in calcium 1 all res. j. biol, 2022, 13, 1-6 deficient environments (14). s-layer deficient cb2 and cb15 caulobacter strains were found to have a 10 times higher electroporation efficiency compared to their s-layer containing counterparts (9). various techniques to destabilize the s-layer have been described. for example, a licl2 wash has been shown to decrease the calcium levels destabilizing the s-layer and increasing electroporation efficiency (15). increasing the resistance utilized during electroporation has been demonstrated to increase electroporation efficiency in s-layer caulobacter strains as well (9). unrelated to the s-layer, it has been shown that growing the bacterium in glycine can improve electroporation efficiency in various bacteria (16, 17). finally, unmethylated plasmids were shown to incorporate at a higher frequency than methylated plasmids (18). since our initial attempts to move plasmids into cb4 were not successful, these techniques were used in combination and independently in an attempt to improve electroporation efficiency in cb4 with no success. methods plasmids the pxyltn5 plasmid (8) was obtained from dr. beat christen (eth zurich). to construct the pbr322-copa plasmid (figure 1), primers for the two flanking regions of the copa cb4 plasmid gene were designed with restriction sites added on the end of the primers and used to amplify the beginning and end regions of the copa gene with pcr (supplementary table 1). concurrently, the pbr322 plasmid was cut with bsmi and avai (fig. 1) and the resulting fragments were separated by agarose gel electrophoresis. the larger section of the cleaved pbr322 plasmid was extracted, purified, and ligated to the copa bsmf2 and avar2 pcr fragment. the ligated plasmid was transformed into e. coli s17 (19) and then plated on lb (20) tetracycline (1 ug/ml) plates. a tetracycline resistant colony was purified, grown overnight in lb, and the pbr322 plasmid containing the distal part of the copa gene was isolated. the plasmid was digested with bsmi and avai to confirm the presence of the copa gene fragment. the newly constructed plasmid was then digested with ecor1 and clai, purified as above, and ligated with the copa ecof1 and clar1 pcr fragment. after the ligated construct was transformed into e. coli, purified and re-isolated, the proper configuration of the pbr322-copa plasmid was confirmed by pcr using the ecof1 and avar2 primers. the pbr322-copa plasmid also was transformed into a dcm-/dame.coli strain (zymo mix n go), and both the methylated and unmethylated forms of pbr322-copa were used for the remainder of the experiments. figure 1. structure of pbr322-copa plasmid. two ~250 bp segments from the cb4 plasmid copa gene were ligated in at ecori/clai and bsmi/avai cut sites. electroporation to prepare electrocompetent cells, cb4 and na1000 were grown in 100 ml pye (2) to mid-log phase. the samples were divided into 50 ml centrifuge tubes and spun at 7000 x g for 5 minutes, and the resulting cell pellets were resuspended in 20 ml of sterile deionized water. the samples were then spun and resuspended as before. the final pellets were resuspended in 2 ml of 10% glycerol, spun at 4000 x g for 5 minutes, and resuspended in 400 ul of 10% glycerol. finally, 200 ul aliquots were prepared with half used right away and half stored in the -70°c freezer. for electroporation, 40 ul of the cb4 electrocompetent cells were placed in a 0.5 ml tube, and 2 ul plasmid dna was added. subsequently, 40 ul of the cb4/plasmid mixture was placed in a 0.2 cm bio rad (hercules, ca) gene pulser cuvette and subjected to a 2.5kv shock with the capacitor set at 25 uf (microfarad) and the resistance at 200 ohms in a bio rad gene pulser electroporator. with a 0.2 cm cuvette gap, a 4.5 to 5 msec time constraint was expected at 200 ohms. after the shock, the contents of the cuvette were mixed with 1 ml pye, transferred to a sterile test tube, and incubated at 30°c for 2 hours before 200 ul of each sample was plated on pye plates containing the appropriate antibiotic. when using the pxyltn5 plasmid, the plates also contained 100 ul of a 10% xylose solution to induce transposition. altered electroporation parameters can be found in supplementary table 2. conjugation procedure the e. coli bc1490 which houses the pxyltn5 plasmid and cb4 cultures were grown overnight in lb or pye medium, respectively. to initiate conjugation, one ml of the cb4 culture was gently mixed with 0.1 ml of the e. coli bc1490 culture. after the mixture was filtered through a millipore ha 0.45 um filter, the filter was then placed on a pye plate and incubated overnight at room temperature. after the incubation, the filter was placed in sterile test tube with 500 ul pye, and the bacteria were resuspended by vortexing. subsequently 200 ul of the bacteria suspension was plated on each of two pye plates containing the pbr322 copa 5.4 kb ecori 2 all res. j. biol, 2022, 13, 1-6 appropriate antibiotic to select for the presence of the plasmid and nalidixic acid (20 ug/ml) to select against the e. coli donor strain. each mating experiment was performed twice for replication of results. plates were then incubated at 30°c for 2-3 days until colonies appeared. results the pbr322-copa plasmid contains two sections of the copper-translocating p-type atpase copa gene (approximately 250 bp each) that flank a tetracycline gene and are identical to the corresponding regions of the cb4 plasmid (fig. 1). since the pbr322-copa plasmid cannot replicate in caulobacter, homologous recombination would be needed for the host bacterium to acquire tetracycline resistance after the introduction of the plasmid. initially, the standard electroporation procedure was used in an unsuccessful attempt to get the plasmid into cb4. of note, cb4 tended to aggregate into clumps that were difficult to disperse during the preparation of the electrocompetent cells, in comparison to na1000 where the bacteria go back into solution from the pelleted state without much agitation. cb4 gave a time constraint of 3.4-3.6 msec at the standard electroporation settings compared to the 4.6-4.8 msec obtained with other caulobacter strains. each experiment was run in duplicate and then it was replicated 4 separate times. no cb4 transformants were obtained from any of these experiments. to further investigate, we attempted to transform the pxyltn5 plasmid into both cb4 and na1000. the standard protocol was followed and approximately 100 transformants were obtained with na1000, and no transformants were obtained with cb4. the experiment was replicated with the same results. since the standard procedures were not working, we decided to alter both the variables involved in the preparation of the electrocompetent cb4 cells and the electroporation procedure itself as described below. varying resistance during electroporation gilchrist and smit (9) demonstrated that increasing the resistance from 200 or 400 to 600 or 800 ohms, respectively, increased the number of transformants for slayer containing caulobacters. also, spath et al. (18) showed that the use of an unmethylated plasmid can increase transformation efficiency. therefore, cb4 electrocompetent cells were prepared according to standard protocol and electroporation experiments were performed at 200, 400, 600, and 800 ohms. for each level of resistance, transformations with both a methylated and an unmethylated plasmid were attempted in duplicate, and the time constraints did increase in relation to the increase in resistance. although increasing the resistance had previously increased the number of transformants in s-layer containing caulobacters (9), no transformants were observed for any of the 16 samples. as a control an additional aliquot was plated on antibiotic-free control plate to verify bacterial viability. licl2 wash a licl2 wash has been shown to disrupt the ca2+ component of the s-layer and increase electroporation efficiency (15). therefore, a licl2 wash was added after the initial pelleting of cb4. the pelleted cb4 cells were resuspended in 5 ml of 5 m licl2 and held on ice for 30 minutes, pelleted, then resuspended in 5 m licl2 and held on ice for an additional 30 minutes. the licl2 procedure called for the use of smeb as the resuspension and electroporation buffer instead of using the 10% glycerol as called for by the standard procedure. using the smeb after the licl2 resulted in a 0.1 msec time constraint and an electrical arc of the cuvette. one cause of this is can be a high salt concentration. to bypass this problem, a 10% glycerol solution was used during the wash and final preparation of the electrocompetent cells. this modified procedure resulted in the expected time constraints at the 200 and 400 ohms resistance. again, the experiment was performed with both a methylated and unmethylated plasmid at both 200 and 400 ohms, and no transformants were obtained. to confirm cell viability, 400 ul of one of the licl2 washed electroporation samples was plated on a pye with no antibiotics and cell growth occurred at the expected rate. glycine growth media in other bacteria, adding glycine to the growth medium has been shown to increase electroporation efficiency (16,17). cb4 was unable to grow in the previously established 1% glycine concentration which had been shown to increase electroporation efficiency. the highest concentration of glycine that allowed cb4 to grow was 0.1%, and electrocompetent cells were prepared from cb4 cells grown in the presence of 0.1% glycine. for electroporation, both the methylated and unmethylated pbr322-cop plasmids were used at 200 and 400 ohms resistance. the experiment was performed in duplicate and the expected time constraints were obtained, yet none of the runs resulted in any transformants. eps caulobacter can possess an exopolysaccharide, or eps layer than acts as another physical barrier to the cell (10). electron microscopy showed that an eps layer was present on cb4 cells, as did the clumping of cells during the cell preparation procedure (fig. 2). growing bacteria in the presence of 0.7 mm edta has been shown to disrupt the eps and increase electroporation efficiency in other gramnegative bacteria (21), but cb4 did not grow at that concentration of edta. caulobacter requires 0.5 mm ca++ for optimal growth (22) and is sensitive to calcium levels when the s-layer is disrupted (14). cb4 was able to grow at 0.3 mm and 0.4 mm, but not 0.7 mm, edta. therefore, 30 ml cultured of cb4 with 0.3 mm or 0.4 mm edta were grown to 30 kletts, spun down, and washed with 10% glycerol four times. the cells were then pelleted and resuspended in 160 ul of 10% glycerol. subsequently, 40 ul of the electrocompetent cells were mixed with 1 ul of methylated or unmethylated pbr322-copa and electroporated with the standard procedure. all runs resulted in zero transformants. a control experiment with electrocompetent cells created from cb4 grown in both pye and pye with 0.4 mm edta resulted in 1.25x105 cfu/ml 3 all res. j. biol, 2022, 13, 1-6 of the pye-grown cb4 competent cells, and no colonies were observed when 20 ul of the edta-grown cb4 competent cells were spread on pye plates. therefore, we concluded that growth in edta lowered the survival rate during the preparation of electrocompetent cells to the point that no surviving cells were present. figure 2. electron microscope images of c. henricii cb4. fig. 2a) the eps layer surrounding the cb4 bacterium is visible as a halo. fig. 2b) a clump of cb4 cells. electroporation variables description source increased resistance increasing the resistance to 600 or 800 ohms has been shown to increase electroporation efficiency in caulobacters with s-layers. [9] unmethylated plasmids unmethylated plasmids (dcm-/dam-) have been shown to increase electroporation efficiency. [18] licl2 wash licl2 wash used to disrupt ca2+ and rsaa interaction and destabilize the s-layer [15] glycine growth media glycine added to growth media has been shown to increase electroporation efficiency. [16,17] eps disruption bacteria grown in the presence of edta has been shown to disrupt the exopolysaccharide layer. [21] conjugation experiments conjugation can also be used to transfer a plasmid from e. coli to caulobacter (23). therefore, conjugation experiments were carried out using the same na1000 and cb4 bacteria strains with e. coli bc1490 which houses the pxyltn5 plasmid. approximately 400 colonies were obtained with na1000 as a recipient and no colonies were obtained with cb4 as a recipient. this experiment was repeated with c. crescentus strain sc1004 and cb4 with similar results (fig. 3). alternatively, conjugation experiments were carried out with cb4 and e. coli hb101 which houses the prk290 plasmid. the prk290 plasmid houses a rk2 replicon which has been shown to be stable in caulobacter (25), but again, no colonies were obtained from the conjugation with cb4. figure 3. conjugation results with cb4 and bc1490 (a), and c. crescentus sc1004 and bc1490 (b). discussion electroporation is a useful technique for the introduction of foreign dna into a bacterium. previous work has demonstrated that getting dna into c. crescentus electroporation is more difficult due to its s-layer (9). the slayer is a crystalline layer consisting of rsaa protein subunits and ca2+ ions. gilchrist and smit (9) demonstrated that increasing the resistance increased electroporation efficiency in s-layer containing c. crescentus strains. however, our use of this procedure did not result in any cb4 transformants. other researchers increased bacterial electroporation efficiency by using unmethylated plasmids (17), or growth in glycine (16, 17). however, neither of these procedures was successful with cb4. also, a licl2 wash during the preparation of electrocompetent cells has been shown to disrupt the s-layer by removing the ca2+ ions (15), but this technique also did not result in any cb4 transformants. in addition, we were unable to transfer plasmids into cb4 using standard conjugation techniques. it is possible that the cb4 s-layer is thick enough so that these techniques do not disrupt it sufficiently to allow for the uptake of foreign dna. in addition, cb4 also has an extracellular polysaccharide (eps) layer that may block dna uptake as well. it also is possible that cb4 produces an endonuclease that cleaves extracellular dna during the electroporation procedure. however, the inability to transfer a plasmid via conjugation where exposure endonuclease activity should not occur indicates that the primary issue is likely some type of external barrier(s) that prevents both conjugation and electroporation from occurring. further evidence for this barrier is that cb4 tends to form clumps of cells during growth (fig. 2b), and cb4 cell pellets are difficult to resuspend after centrifugation. thus, cb4 cells tend to stick to each other in ways that are not observed with c. crescentus cells. the role of calcium in s-layer and eps disruption was also found to decrease cell viability after electroporation. this creates a difficult situation in which disrupting the eps and s-layer to allow foreign dna to enter decreases cell viability. further studies could be conducted to find an equilibrium of depleting the eps and s-layer while maintaining cell viability. a b b a b table 1. description of altered variables to increase electroporation efficiency. 4 all res. j. biol, 2022, 13, 1-6 references 1. skerker, j. m., & laub, m. t. (2004). cell-cycle progression and the generation of asymmetry in caulobacter crescentus. nature reviews microbiology, 2(4), 325-337. 2. ely, b. (1991). genetics of caulobacter crescentus. in methods in enzymology (vol. 204, pp. 372-384). academic press. 3. nierman, w. c., feldblyum, t. v., laub, m. t., paulsen, i. t., nelson, k. e., eisen, j., ... & fraser, c. m. (2001). complete genome sequence of caulobacter crescentus. proceedings of the national academy of sciences, 98(7), 4136-4141. 4. govers, s. k., & jacobs-wagner, c. (2020). caulobacter crescentus: model system extraordinaire. current biology, 30(19), r1151r1158. 5. schoenlein, p. v., & ely, b. (1983). plasmids and bacteriocins in caulobacter species. journal of bacteriology, 153(2), 1092. 6. scott, d., & ely, b. (2016). conservation of the essential genome among caulobacter and brevundimonas species. current microbiology, 72(5), 503-510. 7. poindexter, j. s. (1964). biological properties and classification of the caulobacter group. bacteriological reviews, 28(3), 231. 8. christen, b., abeliuk, e., collier, j. m., kalogeraki, v. s., passarelli, b., coller, j. a., ... & shapiro, l. (2011). the essential genome of a bacterium. molecular systems biology, 7(1), 528. 9. gilchrist, a., & smit, j, (1991). transformation of freshwater and marine caulobacters by electroporation. journal of bacteriology, 173(2), 921-925. 10. herr, k. l., carey, a. m., heckman, t. i., chávez, j. l., johnson, c. n., harvey, e., ... & marks, m. e. (2018). exopolysaccharide production in caulobacter crescentus: a resource allocation trade-off between protection and proliferation. plos one, 13(1), e0190371. 11. smit, j., engelhardt, h., volker, s., smith, s. h., & baumeister, w. (1992). the s-layer of caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy. journal of bacteriology, 174(20), 6527-6538. 12. walker, s. g., smith, s. h., & smit, j. (1992). isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters. journal of bacteriology, 174(6), 1783-1792. 13. sleytr, u. b., & beveridge, t. j. (1999). bacterial slayers. trends in microbiology, 7(6), 253-260. 14. herrmann, j., jabbarpour, f., bargar, p. g., nomellini, j. f., li, p. n., lane, t. j., ... & wakatsuki, s. (2017). environmental calcium controls alternate physical states of the caulobacter surface layer. biophysical journal, 112(9), 18411851. 15. walker, d. c., aoyama, k., & klaenhammer, t. r. (1996). electrotransformation of lactobacillus acidophilus group a1. fems microbiology letters, 138(2-3), 233-237. 16. hermans, j., boschloo, j. g., & de bont, j. a. m. 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(1951). studies on lysogenesis i.: the mode of phage liberation by lysogenic escherichia coli1. journal of bacteriology, 62(3), 293. 21. fournet-fayard, s., joly, b., & forestier, c. (1995). transformation of wild type klebsiella pneumoniae with plasmid dna by electroporation. journal of microbiological methods, 24(1), 49-54. 22. ely, b., & johnson, r. c. (1977). generalized transduction in caulobacter crescentus. genetics, 87(3), 391-399. 23. ely, b. (1979). transfer of drug resistance factors to the dimorphic bacterium caulobacter crescentus. genetics, 91(3), 371-380. 24. o'neill, e. a., berlinberg, c., & bender, r. a. (1983). activity of plasmid replicons in caulobacter crescentus: rp4 and cole1. genetics, 103(4), 593-604. 5 all res. j. biol, 2022, 13, 1-6 supplementary table primer nucleotide sequence ecof1 ttttttgaattccaatcgacgagtctatggtcac clar1 ttttttatcgataggatcgccgagaggataa bsmf2 ttttttgaatgcgggttgacgaaaccgatctactc avar2 ttttttcccgagggcctgttccaccatgaa supplementary table 1. primers for flanking regions of copper-translocating p-type atpase cb4 plasmid gene parameters methylated/ unmethylated plasmids control bacterial lawn transformants increased resistance 200 ohms, 400 ohms, 600 ohms, 800 ohms + + 0 licl2 wash two 30-minute washes with 5.0ml of 5.0m licl2 + + 0 glycine wash .5% glycine growth media + + 0 eps disruption .3-.5mm edta added to growth media + 0 supplementary table 2. parameters changed during electroporation. (+) is present and (-) is not present. each variable was replicated at least 4 times. 6 microsoft word template_authors_arjournals_biol_2.doc insufficient antifungal potential of crude extracts of carissa carandas linn. & nerium oleander linn meenakshi fartyal*, padma kumar all res. j. biol., 2016, 7, 47-54 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 4, vol 7, 2016, 47-54 insufficient antifungal potential of crude extracts of carissa carandas linn. & nerium oleander linn meenakshi fartyal*1, padma kumar1 1laboratory of plant tissue culture and secondary metabolites, department of botany, university of rajasthan, bapu nagar, jaipur302004, india. email*: mksh35@gmail.com graphical abstract: abstract: objective: to evaluate the antifungal potential of crude extracts from different parts of carissa carandas linn. (leaf, stem & root) and nerium oleander linn. (leaf, stem & root). material & methods: different parts of plants were collected, dried and then extracted by using the soxhlet extraction method in different polar and non-polar solvents (water, methanol & petroleum ether). extracts were then screened for antifungal activity using a ‘disc diffusion assay’ against candida albicans (yeast), aspergillus flavus & tricophyton mentagrophyte (fungi). minimum inhibitory concentration, minimum fungicidal concentration & total activity were studied. the mean and standard deviation were calculated. results: the results indicate that all the tested extracts were found to have no antifungal activity against the tested microorganisms. conclusion: the tested extracts did not have, or had too little, antifungal activity. hence, may not be explored as promising sources of new antimicrobial drugs. keywords: disc diffusion assay, minimum fungicidal concentration, minimum inhibitory concentration, polar and non polar solvents, total activity. soxhlet  extraction  for  extraction  of   extracts  from  plant  parts     extracts  dried  and  collected  in  vials    disc  diffusion  assay  for  antifungal   screening  of  extracts     micro  broth  dilution  for  mic  &   mfc  determination       insufficient  antifungal  potential  of   tested  extracts   47 all res. j.biol, 2016, 7, 47-54     introduction india with its richness and diversity can be considered as the paradise of medicinal plants. since prehistoric times, nearly all cultures, both ancient and modern, have used plants as natural resources for medicinal purposes. who [1] reported that 80% of the global population relies on traditional therapies which involve the use of plant extracts or their active constituents. developing countries depend on plants as the source of medicine thus; traditional medicine plays a major role in health care [2]. since there has been an increase in the use of synthetic drugs leading to many side effects and undesirable hazards, there is a worldwide trend to go back to natural resources (mainly traditional plants) which are both culturally acceptable and economically viable. medicinal plants dominate indigenous/alternative systems of medicine and are common elements in ayurveda, chinese, homeopathy, naturopathy, oriental and native american medicine. in the present investigation, carissa carandas & nerium oleander have been selected for the study.   carissa carandas (common name karaunda) is a perennial shrub belonging to the family apocynaceae [3]. it grows naturally in the himalayas at elevations of 300 to 1800 meters, in the siwalik hills, the western ghats and in nepal and afghanistan. it flourishes well on land with high temperatures. presently it is grown on a limited scale in the rajasthan, gujarat, bihar and uttar pradesh regions of india. various medicinal properties like stomachic, anthelmintic, cardiotonic, and lowering blood pressure are attributed to this plant. other properties attributed are strengthening tendons, effectivity against remittent fever, earache and syphilitic pain [4].   nerium oleander (common name kaner) is an evergreen shrub belonging to the family apocynaceae [5]. it is native to southern europe and is widely cultivated and naturalized in asia, europe and north america. it is four meters in height, occurs along watercourse, harsh and damp ravines; is widely cultivated particularly in warm temperate subtropical regions where it grows outdoors in parks, gardens and along roadsides. various medicinal properties like cardiotonic, analgesic, antidiabetic, anti-inflammatory, antibacterial, anticancer/antineoplastic, antifungal, depressant, antimitotic, insecticidal, and larvicidal are attributed to this plant. other properties attributed are the inhibition of nuclear factorkappa b (nf-/cb) activation, muscle stimulation, effective against asthma, seizures, cancer, menstrual pain, skin problems, warts, epilepsy, leprosy, malaria, ringworm, indigestion, venereal diseases, and causing abortions [6].   the microorganisms selected for the study are candida albicans, aspergillus flavus & tricophyton mentagrophyte. candida albicans is a major model of pathogenic yeast which is found in the mouth, throat, intestine and genitourinary tract of humans and is considered a common constituent of bowel flora together with many bacterial species e.g. e. coli, s. aureus and p. mirabilis. it lives in 80% of the human population with no harmful effects, although overgrowth results in candidiasis which is often observed in immunocompromised individuals such as patients of cancer, transplants and aids. it is a causal agent of opportunistic oral and genital infections in humans [7]. superficial and mycosis infections cause local inflammation and discomfort in human beings [8]. candidiasis also known as ‘thrush’, usually occurs in immunocompromised people [9]. aspergillus flavus is the second leading cause of invasive and non-invasive aspergillosis [10]. the presence of aspergillus in the air is a major risk factor for both invasive and allergic aspergillosis [11]. a. flavus can cause storage problems in stored grains. it also causes diseases in economically important crops, such as maize and peanuts and produce potent mycotoxins. it can also be a human pathogen, associated with aspergillosis of the lungs and sometimes causing corneal, otomycotic and naso orbital infections.   tricophyton mentagrophyte is a cosmopolitan dermatophyte, belonging to a homogeneous group of fungi called the dermatophytes. the organism is found in soil, floors of swimming pools, hairs of wild boar, cats and dogs, farm animals, footwear and from human toe webs without clinical lesions. it requires keratin for growth and can cause a variety of cutaneous (hair, nail and skin) infections in humans and animals; hence it is considered to be anthropophilic or zoophilic in nature [12, 13]. it causes dermatophytosis in dogs, cats, cattle and especially in rodents [14, 15, 16].     antimicrobial activity against staphylococcus aureus, staphylococcus epidermidis, escherichia coli, aspergillus niger, candida albicans was seen in aqueous, ethanol, methanol, chloroform and acetone extracts of c. carandas [17]. unripe roots and fruits of c. carandas exhibited antimicrobial activity in their methanol and petroleum ether extract [18]. antimicrobial activity of ethanolic extracts of the fruits of c. carandas have been reported against staphylococcus aureus, staphylococcus epidermidis, streptococcus pneumoniae, bacillus subtilis, escherichia coli, proteus vulgaris and proteus mirabilis [19].   antimicrobial activity of methanolic and aqueous extracts of nerium sp. has been reported against escherichia coli, streptococcus uberi and staphylococcus aureus [20]. ethanol, methanol and acetone extracts of leaves of nerium sp. exhibited antimicrobial activity against klebsiella, pseudomonas, alkaligen excluding acinetobacter sp.[21, 22]. antimicrobial activity of aqueous and ethanolic extracts of nerium sp. has been reported against various pathogenic micro-organisms [23]. chloroform, ethanol and methanol extracts of root, bark and leaves of nerium oleander exhibit antimicrobial activity against bacillus pumulis, bacillus subtilis, staphylococcus aureus, escherichia coli and aspergillus niger[24]. antimicrobial activity has been screened in nerium flower (essential oil) against various pathogenic organisms [25]. aqueous extract of nerium sp. exhibits antimicrobial activity against bacillus subtilis, staphylococcus aureus, escherichia coli, proteus vulgaris, salmonella typhi, pseudomonas aeruginosa, and candida albicans [26].   considering the rich diversity of plants, it is expected that screening and scientific evaluation of plant extracts for their 48 all res. j.biol, 2016, 7, 47-54     antimicrobial activity may provide new antimicrobial substances. review of the current literature reveals that not so much work has been carried out for extraction and screening of specific compounds from selected plants. hence, in the present work an extraction and screening for antifungal activity of crude extracts of c. carandas & n. oleander has been undertaken.   materials and methods different parts of c.carandas (leaf, stem and root) & n. oleander (leaf, stem, root and flower) were collected in the months of april to june from the western parts of india (jaipur, rajasthan). plants were identified by a senior taxonomist at the department of botany, university of rajasthan and voucher specimen no: rubl 21130 (c. carandas) & rubl 21176 (n. oleander) were submitted to the herbarium, botany department, university of rajasthan.   preparation of extracts:   extraction in polar and non polar solvents:   powder of all the plant parts were prepared in different round bottom flasks in different solvents. 20 g of powder was put in each flask and water, methanol and petroleum ether were used as solvents. dried material and solvents were taken in a 1:10 ratio. those were kept at the soxhlet unit for 24 hours. then the extracts were filtered. the filtrates were subjected to evaporation to obtain dried extract. the percentage yield of each dried plant extract was calculated.   selected test microorganisms:   three pathogenic bacteria were screened: candida albicans (mtcc no. 183), aspergillus flavus (mtcc no. 277) and tricophyton mentagrophyte (mtcc no. 7687). the pathogens were procured from ‘the institute of microbial technology’ imtech (chandigarh, punjab, india). fungal strains were grown and maintained on sabouraud dextrose (sd) agar medium. antimicrobial assay: ‘disc diffusion assay’ was performed for screening [27]. sabouraud dextrose agar base plates were seeded with fungal inoculum (1×107 cfu/ml). sterile filter paper discs of whatmann no.1 (6mm in diameter) were impregnated with 100µl each of the extract of a 10mg/ml concentration giving a final concentration of 1mg/disc. discs were left to dry in vacuo so as to remove residual solvent, which might have interfered with the determination of antimicrobial activity. discs with extract were then placed on the corresponding seeded agar plates. each extract was tested in triplicate along with ketoconazole (1mg/disc) for t. mentagrophyte and terbinafine for c. albicans and a. flavus as standard drugs. the plates were kept for 1h at 4oc for extract diffusion and were incubated thereafter at  27oc  (c. albicans and a. flavus for 48 h & t. mentagrophyte for 5-7 days). inhibition zone (iz) values were measured & activity index (ai) for each extract was calculated by the standard formula: activity index = iz produced by the extract/ iz produced by standard where, iz = inhibition zone (in mm) mean and standard deviation was also calculated for each extract (n=2). [table i] determination of minimum inhibitory concentration (mic) & minimum fungicidal (mfc) concentration: the minimum inhibitory concentration (mic) was determined for each plant extract showing antimicrobial activity against the test pathogens. the ‘micro broth dilution’ method was followed for the determination of mic values [8]. plant extracts were resuspended in acetone (which has no activity against test microorganisms) to make a final concentration of 10mg/ml.  two fold serially diluted extracts were added to broth media in 96-wells of microtiter plates. thereafter 100µl of fungal inoculum (1×107 cfu/ ml) was added to each well. fungal suspensions were used as a negative control, while broth containing the standard drug was used as a positive control. micro titer plates were then incubated at 27◦c for 48 h. each extract was assayed in duplicate and each time two sets of microplates were prepared, one was kept for incubation while another was kept at 4◦c for comparing the turbidity in the wells of micro plates. the mic values were taken as the lowest concentration of the extracts in the well of the micro titer plate that showed no turbidity after incubation. the turbidity of the wells in the micro titer plate was interpreted as visible growth of microorganisms. the minimum fungicidal concentration (mfc) was determined by sub culturing 50 µl from each well showing no apparent growth. the lowest concentration of extract showing no visible growth on sub culturing was taken as mfc. [table ii].     total activity (ta) determination:   total activity is the volume up to which test extract can be diluted without losing the ability to kill microorganisms. it is calculated by dividing the amount of extract from 1g plant material by the mic of the same extract or compound isolated and is expressed in ml/g [29]. [table iii] total activity= amount of extract from 1gm of dry plant material/ mic of the same extract statistical analysis: the results were expressed as mean ± (standard deviation) sd (n = 2) 49 all res. j.biol, 2016, 7, 47-54 table i: antifungal activity of crude extracts of carissa carandas linn. & nerium oleander linn. against some pathogenic fungi p1, p2, p3, p4, p5, p6 = petroleum ether extract of respective plant parts, m1, m2, m3, m4, m5, m6 = methanolic extract of respective plant parts, w1, w2, w3, w4, w5, w6 = water extracts of respective plant parts, iz=inhibition zone in mm (value: including 6mm diameter of disc), mm= millimeters, ai= activity index (iz developed by extract/iz developed by standard), (-) = no activity, ±=sem. plant & plant parts extract microorganisms candida albicans aspergillus flavus tricophyton mentagrophyte iz(mm) ai iz(mm) ai iz(mm) ai 1. c. carandas leaf stem root 2. n. oleander leaf stem root p1 m1 w1 p2 m2 w2 p3 m3 w3 p4 m4 w4 p5 m5 w5 p6 m6 w6 10.5 8 7 10 1.31±0.01 1±0.01 0.47±0.01 0.67±0.01 50 all res. j.biol, 2016, 7, 47-54 table ii: mic and mfc of active crude extracts of carissa carandas linn. & nerium oleander linn. against some pathogenic fungi p1, p2, p3, p4, p5, p6 = petroleum ether extract of respective plant parts, m1, m2, m3, m4, m5, m6 = methanolic extract of respective plant parts, w1, w2, w3, w4, w5, w6 = water extracts of respective plant parts, mic= minimum inhibitory concentration, mfc= minimum fungicidal concentration, mg/ml= milligram per milliliter, (-) = no activity plants & plant parts extracts microorganisms and their mic & mfc candida albicans aspergillus flavus tricophyton mentagrophyte mfc(mg/ml) mic(mg/ml) mfc(mg/ml) mic(mg/ml) mfc(mg/ml) mic(mg/ml) 1. c. carandas leaf stem root 2. n. oleander leaf stem root p1 m1 w1 p2 m2 w2 p3 m3 w3 p4 m4 w4 p5 m5 w5 p6 m6 w6 0.625 1.25 1.25 0.625 0.312 0.625 0.625 0.312 51 all res. j.biol, 2016, 7, 47-54     table iii: quantity & total activity of crude extracts of carissa carandas linn. & nerium oleander linn. p1, p2, p3, p4, p5, p6 = petroleum ether extract of respective plant parts, m1, m2, m3, m4, m5, m6 = methanolic extract of respective plant parts, w1, w2, w3, w4, w5, w6 = water extracts of respective plant parts, ta= total activity (extract per g dried plant part/mic of extract), mg/g.d.wt.= milligram per gram dry weight of extract, ml/g= milliliter per gram. plants & plant parts extracts quantity of extract (mg/g.d.wt.) total activity(ml/g) candida albicans aspergillus flavus tricophyton mentagrophyte 1. c. carandas leaf stem root 2. n. oleander leaf stem root p1 m1 w1 p2 m2 w2 p3 m3 w3 p4 m4 w4 p5 m5 w5 p6 m6 w6 24.17 117.5 68.75 12.67 66 28 12.67 77 25 7 95 46 2.5 33.5 31.5 11 47.5 38 376.60 20.27 152 147.43 52 all res. j.biol, 2016, 7, 47-54 results: the study screened the antifungal activity of extracts of selected plant parts on candida albicans (yeast), aspergillus flavus & tricophyton mentagrophyte (fungi), using the in vitro technique, “disc diffusion assay”. the water, methanol and petroleum ether extracts (18 extracts) of the selected plant parts (6 parts) were assessed for their antifungal activity in terms of the zone of inhibition in mm against selected microorganisms [table i]. in the present study, all the tested extracts showed no activity or very little activity at the tested concentration of 1 mg/ml. nevertheless, the highest antifungal activities were recorded for methanolic extracts of leaves of c. carandas (iz= 10.5mm, ai= 1.31±0.01) followed by water extracts of leaves of n. oleander (iz=10mm, ai= 0.67±0.01) against c. albicans. petroleum ether extracts from the stem of c. carandas (iz= 8mm, ai= 1±0.01) & methanolic extracts of leaves of n. oleander (iz= 7mm, ai= 0.47±0.01) also showed some activities against c. albicans. such low values of iz indicate the insufficient antifungal potential of the extracts. the extracts that showed activity in the disc diffusion assay were evaluated for their mfc and mic values by using a 96-well broth dilution method and are tabulated in table ii. the lowest mfc & mic values of the extracts that showed confined activity were 0.625 mg/ml & 0.312 mg/ml, respectively. ta values were also calculated [table iii]. the highest ta value recorded for the methanolic extract of leaves of c. carandas (376.6ml/g), indicated low efficiency of the extract also in diluted form. the remaining extracts exhibited no activity. aspergillus flavus and tricophyton mentagrophyte were found to be completely resistant throughout the study.   discussion: due to indiscriminate use of antimicrobial drugs, the microorganisms have developed resistance to many antifungal drugs and very few successful drugs are now available for the treatment of fungal infections. this has created immense clinical problems in the treatment of infectious diseases. hence, continuous research for getting new & potent antifungal agents is the need of the present scenario, either by designing and synthesizing new agents, chemically or through the search of new natural sources. ever since the importance of the distribution of pharmacologically active principles in higher plants was understood and acknowledged, the importance of such plantderived medicines in modern therapeutic practice has paved the way for the development of new drug leads that are safe, costeffective and ecofriendly. the present investigation is an effort towards this direction. in the present study, c. carandas & n. oleander have shown insignificant antifungal potential against all the three tested microorganisms. this can be due to the lack of ability of the tested extracts to inactivate or suppress the mechanism followed by the particular fungus responsible for their activity or due to the absence of some specific compound in these extracts accountable to kill or inhibit the growth and   activity of fungus. however, the studied extracts had relatively high mic and mfc values and thus cannot be considered as good antifungal agents.   conclusion c. carandas & n. oleander crude extracts were found to be inefficient to work as antifungal agents with high values of mic and mfc or no activity. to conclude, it is necessary to emphasize the significance of more comprehensive studies that will detect new potent molecules underlying the action of crude extracts that allow for new discoveries. essentially, in this study, these extracts lacking of significant antifungal activity will be useful to avoid any study repeated in this direction in the future.     acknowledgement: the authors would like to extend their sincere thanks and appreciation to the department of botany, university of rajasthan for providing adequate laboratory facilities and providing the required materials needed for the study.   references: [1] world health organization. (1993). summary of who guidelines for the assessment of herbal medicines. herbal gram. 28, pp. 13-4. [2] zakaria, m. (1991). isolation & characterization of active compounds from medicinal plants. asia pac. j. pharmacol. 6, 15-20. [3] khare, c.p. (2007). indian medicinal plants: an illustrated dictionary springer berlin, pp.123. [4] balakrishnan, n., and bhaskar, v.h. (2009). karaunda (carissa carandas linn.)as a phytomedicine: a review. alternative medicine. 95. [5] sunset editors. (1995). sunset western garden book, pp.606–7. [6] duke, j.a. (1985). handbook of medicinal herbs. boca raton, fl: crc press. [7] houck, h.e., cooley, j.e., lowitt, m.h., and kao, g.f. (1996). tinea caput medusa; an unusual presentation of tricophyton mentagrophyte on the scalp. cutis. 58, 48-52. [8] enfert, c., and hube, b. editors. (2007). candida: comparative and functional genomics (caister academic press). [9] pappas, p.g. (2006). invasive candidiasis. infect. dis. clin. north am. 20(3), 485-506. [10] ryan, k.j., and ray, c.g. (2004). sherris medical microbiology (4th ed.) (mc graw hill). [11] hedayati, m.t., pasqualotto, a.c., warn, p.a., bowyer, p., and denning, d.w. (2007). aspergillus flavus: human pathogen, allergen and mycotoxin producer. microbiology. 153, 1677-92. [12] denning, d.w., venkateswarlu, k., oakley, k.l., anderson, m.j., manning, n.j., stevens, d.a., et al. (1997). itraconazole resistance in aspergillus fumigatus. antimicrob. agents chemother. 41, 13648. [13] sanchez-castellanos, m.e., mayrga-rodrigues, j.a., sandoval-tress, c., and hernandztorres, m. (2007). tinea incognito due to tricophyton mentagrophyte. mycoses. 50(1), 85-7. [14] van rooij, p., detandt, m., and nolard, n. (2006). tricophyton mentagrophyte of rabbit origin causing 53 all res. j.biol, 2016, 7, 47-54     family incidence of kerion: an environmental study. mycoses. 49(5), 426-30. [15] ajello, l., and cheng, s.l. (1967). the perfect state of t. mentagrophyte. sabouraudia. 5, 230-4. [16] george, l.k., roberts, c.s., menges, r.w., and kalpan, w. (1957). tricophyton mentagrophyte infection on dogs and cats. j. am. vet. med. assoc. 130(10), 427-32. [17] salar, r.k., and dhall, a. (2012). antimicrobial and free radical scavenging activity of extracts of some indian medicinal plants. j. med. plants res. 4(22), 2313-20. [18] mishra, c.k., pattnaik, a.k., rani, a., sasmal, d., and neema, r.k. (2009). antifungal and antibacterial activity of carissa carandas linn. int. j. plant sci. (muzaffarnagar, india). 4(2), 564-8. [19] israr, f., hassan, f., naqvi, b.s., azhar, i., jabeen, s., and hasan, s.m. (2003). studies on antibacterial activity of some traditional medicinal plants used in folk medicine. pak. j. pharm. sci. 25(3), 669-74. [20] gopinath, s.m., suneetha, t.b., and mruganka, v.d. (2011). chemical prophylaxis and antibacterial activity of methanolic and aqueous extracts of some medicinal plants against bovine mastitis. int. j. adv. biol. res. 1(1), 93-5. 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(2011). phytochemicals screening and antimicrobial activities of selected medicinal plants of khyber pakhtun khwa pakistan. afr. j. pharm. pharmacol. 5(6), 746-50. [27] andrews, j.m. (2001). bsac standardized disc susceptibility testing method. j. antimicrob. chemother. 4, 43-57. [28] basri, d.f., and fan, s.h. (2005). the potential of aqueous and acetone extracts of gall of quercus infectoria as antibacterial agents. indian j. pharmacol. 37, 26-9. [29] eloff, j.n. (2004). quantifying the bioactivity of the plant extracts during screening and bioassay-guided fractionation. phytomedicine. 11(4), 370-1 54 microsoft word siddique_proofreaded.docx                                                                                                                  issue 2, vol 6, 2015, 16-23 effect of withania somnifera leaf extract on the dietary supplementation in transgenic drosophila model of parkinson’s disease yasir hasan siddique*1, syed faiz mujtaba2, mohammad faisal3, smita jyoti1, falaq naz1 1drosophila transgenic laboratory, section of genetics, department of zoology, faculty of life sciences, aligarh muslim university, aligarh, uttar pradesh, india. 2photobiology division, csir-indian institute of toxicology research, lucknow, uttar pradesh, india. 3 forest entomology division, forest research institute, dehradun, 248006, uk, india. graphical abstract abstract: the role of withania somnifera l. leaf extract was studied on the transgenic drosophila model flies expressing normal human alpha synuclein (h-αs) in the neurons. the leaf extract was prepared in acetone and was subjected to gc-ms analysis. w. somnifera extract at final concentration of 0.25, 0.50 and 1.0 µl/ml was mixed with the diet and the flies were allowed to feed for 24 days. the effect of extract was studied on the climbing ability, lipid peroxidation and protein carbonyl content in the brains of transgenic drosophila. the exposure of extract to pd model flies did not show any significant delay in the loss of climbing ability nor reduced the oxidative stress in the brains of transgenic drosophila as compared to untreated pd model flies. the results suggest that w. somnifera leaf extract is not potent in reducing the pd symptoms in transgenic drosophila model of parkinson’s disease. keywords: withania somnifera; lipid peroxidation; protein carbonyl content; drosophila; climbing ability. 16 all res. j.biol, 2015, 6, 16-23     introduction parkinson’s disease (pd) is a chronic neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons of substantia nigra pars compacta in the ventral midbrain.1 one of the pathological features of pd is the presence of lewy bodies.2 the function of alpha synuclein (αs) is not fully understood but it has been reported to bind with lipid membranes, forming an amphipathic helix.3 it has been suggested that there may be synaptic role for αs.4 lewy bodies contain other proteins including neurofilaments and other cytoskeletal proteins, suggesting the presence of co-precipitants that might be important in aggregation.4 the aggregation of αs leads to the toxicity and oxidative stress,5 but it is still unclear whether misfolded proteins directly cause toxicity or damage cells via the formation of protein aggregates.6 the neurons being lost as the progression of pd have been reported to generate endogenous toxins (hydrogen peroxides) and free radicals that may further lead to the loss of neurons.7 in recent years, use of natural antioxidants from food and other biomaterials have been increased due to their presumed safety, nutritional and therapeutic values.8 ayurveda has been in practice in india for more than 3500 years, and traditional healers have used this system since time immemorial for the benefit of mankind.8 the dietary habits are specific to population and vary widely hence it is necessary to study the diseaseprevention potential of functional micronutrients in the regional diet.9 withania somnifera or ashwagandha is widely used in ayurvedic medicine and the traditional medical system of india.10 w. somnifera (indian ginseng) is a subtropical under shrub that belongs to the family solanaceae.11 its root powder has been reported to posses free radical scavenging,12 improvement of motor neurons function,13 formation of dendrities,14 stimulating thyroid function,15 inhibition of tumor cell lines16 catecholamines and physiological abnormalities in pd model mouse,13,17 inhibits amyloid-β fibril,18 anti-diabetic,19 antigenotoxic,20 and other pharmacological properties. most of the studies have been performed on the root extract. however, little is known about possible effects of leaf extract. hence, an attempt has been made to study the effect of the leaf extract of w. somnifera on the pd model transgenic flies. experimental preparation of leaf extract: the leaves of w. somnifera were collected from the nursery of forest research institute (fri), dehradun (accession no: 143201).the extract was prepared according to the protocol described in an earlier published work.21 analysis of w. somnifera extract through gc-ms: gcms analysis was performed using trace gc ultra gas chromatograph connected to a quantum xls mass spectrometer (thermo scientific, fl, usa). gc was equipped with tg-5ms capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) consisting of a stationary phase 5% phenyl and 95% methyl polysiloxane. the injection was carried out in ct splitless mode at an injector temperature of 260°c helium gas used as a carrier gas with a flow rate of 1.1 ml/min. the oven temperature programming was as follows: the initial oven temperature was held at 70°c for 2.0 min, and then increased to 210°c at a rate of 20°c/min and then increased to 290°c at a rate of 10°c/min held for 13.0 min. the ion source and transfer line temperature were 220°c and 290°c respectively. identification of the compounds was performed by comparing their mass spectra with the nist library available in the instrument. drosophila stocks: transgenic fly lines that expresses wildtype h-αs under uas control in neurons ‘‘(w[*]; p{w[+mc]=uas–hsap/snca.f}’’5b and gal4 ‘‘w[*]; p{w[+mc]=gal4elavl}’’3 were obtained from bloomington drosophila stock center (indiana university, bloomington, in). when the males of uas (upstream activation sequence)-hsap/snca.f strains are crossed with the females of gal4-elav. l (vice-versa) the progeny will express the human αs in the neurons.22 drosophila culture and crosses: the flies were cultured on standard drosophila food containing agar, corn meal, sugar and yeast at 25°c (24 ± 1).23 crosses were set up as described in an earlier published work.23 first, the climbing assay was 17 all res. j.biol, 2015, 6, 16-23     performed for the pd flies and uas-hsap/snca.f (control). the other group of pd flies was exposed separately to different doses of w. somnifera extract mixed in the culture medium. w. somnifera extract was added in the medium at final concentration of 0.25, 0.50 and 1.0 µl/ml. the pd flies were also exposed to 10-3 m of l-dopamine. the uashsap/ snc.f acts as a control. the control flies were separately exposed to the selected doses of w. somnifera extract. drosophila climbing assay: the climbing assay was performed as described by pendleton et al.24 ten flies were placed in an empty glass vial (10.5 cm × 2.5 cm). a horizontal line was drawn 8 cm above the bottom of the vial. after the flies had been acclimated for 10 min at room temperature, both controls and treated groups were assayed at random, to a total of 10 trails for each. the mean values were calculated and then averaged, and a group mean and standard error were obtained. all behavioral studies were performed at 25°c under standard lightning conditions. lipid peroxidation assay: lipid peroxidation assay in the brain homogenate was performed according to the procedure described by siddique et al.25 reagent 1 (r1) was prepared by dissolving 0.064 g of 1-methyl-2-phenylindole into 30 ml of acetonitrile to which 10 ml of methanol was added to bring the volume to 40 ml. the preparation of 37% hcl served as the reagent r2. the brain of flies was isolated under stereozoom microscope in ice cold tris hcl (20 mm) (10 brain/group; five replicates/group). homogenate was prepared in tris hcl and centrifuged at 3000g for 20 min and subsequently the supernatant was collected. in a microcentrifuge tube 1300 µl of r1 was taken. a volume of 1µl of supernatant was added along with 300 µl of r2 vortexed and incubated at 45°c for 40 min. after incubation, the tubes were cooled in ice and centrifuged at 15,000g for 10 min at 4°c. all samples were read at 586 nm. estimation of protein carbonyl content: the protein carbonyl content was estimated according to the protocol described by hawkins et al.26 the brain homogenate was diluted to a protein concentration of approx. 1mg/ml. about 250µl of each diluted homogenate were taken in eppendorf centrifuge tubes separately. to it 250µl of 10mm 2, 4dinitrophenyl hydrazine (dissolved in 2.5m hcl) was added, vortexed and kept in dark for 20 min. about 125µl of 50% (w/v) trichloroacetic acid (tca) was added, thoroughly mixed and incubated at -20°c for 15min. the tubes were then centrifuged at 4°c for 10 min at 9000 rpm. the supernatant was discarded and the pellet obtained was washed twice by ice cold ethanol: ethylacetate (1:1). finally, the pellets were re-dissolved in 1ml of 6m guanidine hydrochloride and the absorbance was read at 370nm. statistical analyses: the statistical analyses were done using statistica soft inc. the mean values of various fly groups were statistically compared using an unpaired group of the student ‘‘t’’-test. results and discussion the compounds present in acetone extract of leaves of w. somnifera were identified by gc-ms analysis (figure 1). figure 1. gc chromatogram of acetone extract of leaves of withania somnifera. 18 all res. j.biol, 2015, 6, 16-23     the gc-ms chromatogram shows the presence of 2 major compounds with highest concentrations. their retention times (rt), molecular formula and molecular weight (mw) in the leaves of w. somnifera are presented in table 1. table 1. the active principles in the leaf extract of withania somnifera with their retention time (rt), molecular formula and molecular weight. the gc-ms analysis revealed that the acetone extract is mainly composed by dibenzyl ether and arachidonic acid or phytanic acid. figures 2 a and b show the fragmentation pattern of mass spectrum and structures of the compounds extracted from the leaf extract and confirmed through nist library of gc-ms. those peaks matching similarity index (si) greater than 70% in nist library were assigned. some of the major peaks are either column bleeding or impurities in plant extract. mass spectra of two compounds extracted from plant leaf with similarity index (si) greater that 70% confirmed by nist library. the climbing response of control flies did not change over 24 days in a time course evaluation (figure 3). from the day 12 on, however, the response of the pd flies were significantly lower as compared to the control (p<0.05). the climbing assay was performed after 24 days of the exposure to various doses of w. somnifera. the exposure of pd flies to 0.25, 0.50 and 1.0 µl/ml of w. somnifera did not show any significant delay in the loss of climbing ability as compared to untreated pd flies (figure 4). the mean climbing ability of control flies was 9.6 ± 0.26 (figure 4). the pd flies and the pd flies with l-dopa were associated with the mean climbing ability of 3.2 ± 0.06 and 6.7 ± 0.17 respectively (figure 4). the control flies treated with 0.25, 0.50, and 1.0µl/ml of w. somnifera leaf extract were associated with mean climbing ability of 9.4 ± 0.28, 9.2 ± 0.23 and 9.5 ± 0.32, respectively (figure 4). no. rt name of the compound molecular formula mw 1 7.65 benzene, 1,1’-oxybis c14h14o 198.26 2 10.22 ethanol, 2-(9-octadecenyloxy)c20h40o2 312.53   figure 2. (i and ii) mass spectra of acetone extract with its products having the high concentration compared with nist library.   figure 3. climbing ability in parkinson disease (pd) flies and control for a period of 24 days. the values are the mean of five assays (asignificant with respect to control p < 0.05). 19 all res. j.biol, 2015, 6, 16-23     figure 4. effects of withania somnifera on the climbing ability. the flies were allowed to feed on the diet supplemented with withania somnifera for 24 days and then assayed for climbing ability. the values are the mean of five assays. (asignificant with respect to control p < 0.05; bsignificant with respect to pd flies p < 0.05). (pd – parkinson disease model flies). the mean absorbance values for the estimation of lipid peroxidation for the control, pd flies and pd flies with ldopa were, 0.10 ± 0.001, 0.98 ± 0.049 and 0.32 ± 0.029 respectively, (figure 5). the treatments of 0.25, 0.50 and 1.0 µl/ml of w. somnifera extract to pd were associated with the mean absorbance values of 0.94 ± 0.063, 0.99 ± 0.073 and 0.94 ± 0.076 and, with control, the values were 0.11 ± 0.001, 0.12 ± 0.003 and 0.10 ± 0.004, respectively (figure 5).  figure 5. effect of withania somnifera on lipid peroxidation measured in the brain of transgenic drosophila melanogaster after 24 days in various treated groups. [a) insignificant with respect to control p<0.05; b) significant with respect to pd model flies p<0.05]. no significant decrease in the mean absorbance values for lipid peroxidation was observed after the exposure to various doses of w. somnifera extract as compared to untreated pd flies (figure 5). the mean absorbance values for the estimation of protein carbonyl content are shown in figure 6. the control flies, pd flies and pd flies + l-dopa were associated with 0.114 ± 0.0017, 0.181 ± 0.0015 and 0.136 ± 0.0019 respectively (figure 6). the pd flies treated with 0.25, 0.50, and 1.0 µl/ml of w. somnifera leaf extract were associated with the mean values of 0.180 ± 0.0019, 0.183 ± 0.0020 and 0.181 ± 0.002, respectively (figure 6). the control flies treated with. 0.25, 0.50, and 1.0µl/ml of w. somnifera leaf extract were associated with the mean values of 0.112 ± 0.0011, 0.113± 0.0016 and 0.112 ± 0.001, respectively. no significant decrease in the mean absorbance value was observed as compared to pd flies, at the exposure to 0.25, 0.50 and 1.0 µl/ml of w. somnifera extract (figure 6). figure 6. effect of withania somnifera on protein carbonyl content measured in the brain of transgenic drosophila melanogaster after 24 days in various treated groups.[asignificant with respect to control p<0.05; bsignificant with respect to pd model flies p<0.05]. the results of the present study reveals that the leaf extract of w. somnifera is not potent in reducing the pd symptoms in the transgenic flies expressing human α-synuclein. the accumulation of lewy bodies together with the loss of dopaminergic neurons and the loss of climbing ability has been reported in the transgenic flies.22 20 all res. j.biol, 2015, 6, 16-23     in our present study the pd flies showed a progressive loss in the climbing ability as the age of the flies progresses. at 24th day the climbing ability of the flies were at the lowest, hence the duration of the treatments was selected for 24 days. the dietary supplementation of w. somnifera leaf extract did not show any significant delay in the loss of climbing ability, indicating that the extract is not potent in delaying the pd symptoms. the accumulation of the lewy bodies leads to the toxicity and oxidative stress6. the damaging neurons as result of pd have been reported to generate hydrogen peroxide and free radicals7. various studies on the experimental models suggest that the oxidative stress plays an important role in neurodegenerative diseases. hence, the lipid peroxidation (lpo) and protein carbonyl content (pc) was measured in the drosophila brains as a marker of oxidative stress. lpo represents a reliable marker of free radical generation and indicates the membrane damage4. oxidative stress leads to the damage of lipid, protein and dna27. the present method used for the estimation of lipid peroxidation is based on the reaction of malondialdehyde (mda) with 1-methyl-2phenylindole at 45°c. two molecules of 1-methyl-2phenylindole react with one molecule of mda to form a stable chromophore, having a maximal absorbance at 586nm28. the results obtained for lipid peroxidation showed no reduction in the pd model flies treated with w. somnifera extract. pc content indicates the protein oxidation by the free radicals or ros. the oxidation of protein results in the generation of carbonyl (co) groups on protein side chains and is widely used as a marker of oxidative stress. the treatment of w. somnifera did not show any reduction in the oxidative stress markers used in the present study, hence suggesting that w. somnifera leaf extract has no effect on the oxidative stress. the results of our present study support the study carried out by kaur et al29. the leaf extract was reported to be antiproliferative but not antioxidative. the therapeutic intervention that could effectively decelerate the rate of degeneration within the substantia nigra pars compacta could add years of mobility and reduce morbidity associated with pd7. hence the attention has been directed towards the identification of the inhibitors of αs aggregations or free radicals scavengers30. in this regard the leaf extract of w. somnifera did not show any protective effect against the pd symptoms in the transgenic model flies expressing hαs. in mouse model of parkinson’s disease the leaf extract has shown to increase the levels of superoxide dismutase, catalase and malondialdehyde, reduced levels of glutathione and glutathione peroxidase in the mid brain and corpus striatum thus suggesting it as an antioxidative and protective against pd symptoms13. there are various neurotoxins that have provided valuable information about the pathophysiology of pd but paraquat and rotenone failed to produce any obvious signs of dopaminergic damage31. the process of damaging the dopaminergic neurons by the various neurotoxins and α-synuclein aggregation is entirely different31. however, the roots of w. somnifera contain steroid lactones (withanolides), phytosterols and alkaloids (ashwagandhine, ashwaghandhinine, withasomine, visamine, somniferine, somniferinine)8, 32, 33. our gc-ms analysis reveals the absence of these compounds in the leaf extract of w. somnifera. this may be the possible reason of not showing any protective effects against the pd symptoms in the flies. the above compounds have been shown to have neuroprotective and neuro regenerative potential32. our earlier study with eucalyptus citriodora leaf extract showed a protective effect in the pd model flies expressing hαs34. the natural antioxidants such as apigenin,35 curcumin,28 nordihydroguaiaretic acid,23 ascorbic acid,34 and grape extract,36 are potent in delaying the loss of climbing ability and reduction in the oxidative stress in the brains of pd model flies. the dietary supplementation of the flavonoids showed improvement in cognitive function possibly by protecting vulnerable neurons, enhancing existing neuronal function or by stimulating neuronal regeneration.30 the complex brain of drosophila is capable of learning and memory. almost all the major classes of ion channels, receptors and neurotransmitters similar to humans composed of specialized cell types are found in drosophila brain22. there are reports for the presence of novel and known withanolides from w. somnifera leaf extracts16, 37. various pharmacological studies have attributed linkage of therapeutic actions to one or the other type of withanolides. a significant qualitative as well as quantitative difference 21 all res. j.biol, 2015, 6, 16-23     between the leaf and root tissue, particularly with respect to secondary metabolites, has been reported in the gc-ms analysis37. however, in our gc-ms analysis, no peaks corresponding to the withanolides were observed according to the nist library. c12h10o is arachidic acid, a saturated fatty acid38 and c20h40o2 is phytanic acid, a branched fatty acid, and its metabolites have been reported to bind or activate transcription factors39. the antioxidant and free radical scavenging potential have been reported due to the presence of the withanolides/withanone. but there are reports of having no antioxidant potential in the leaf extract of w. somnifera29. no one scientific approach is likely to single handedly solve all the mysteries of neurodegenerative disease and hence multidisciplinary research approaches utilizing many model systems will be required for elucidating the neuropathogenesis with the disease40. the complex brain of drosophila consists of all major classes of ion channels, receptors, and neurotransmitters found in humans40. the neural complexity and proteomic analysis have revealed that more than 70% of the disease related loci in humans have a clear ortholog in drosophila41. hence, the drosophila is the most ideal organism for studying the neurodegenerative diseases. the variation in the compounds in the gc-ms gram may be due to the type of solvent used in the preparation of extract or part from where the leaves were collected. age of the leaves may also have some effects. in our present study with leaf extract of w. somnifera, the extract was not found to be potent in reducing the pd symptoms, in the transgenic flies expressing hαs. acknowledgments we are thankful to the chairman of the department of zoology for providing the laboratory facilities. the flies for the experiments were purchased from the bloomington drosophila stock centre, department of biology, indiana university, bloomington, in, usa. references     1. kartika, b., muralidharan, p., and rahman, h. 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(2007). the neuroprotective effect of withania somnifera root extract in mptpintoxicated mice: an analysis of behavioural and biochemical variables. cell mol. bio. lett., 12, 473-481. 12. panda, s., and kar, a. (1997). evidence for free radical scavenging activity of ashwagandha root powder in mice. indian j. physio. pharmaco., 41, 424-426. 13. rajasankar, s., manivasagam, t., sankar, v., prakash, s., muthusamy, r., krishnamurti, a., and surendran, s. (2009). withania somnifera root extract improves catecholamines and physiological abnormalities seen in a parkinson’s disease model mouse. j. ethnopharmacol., 125, 369-373. 14. chihiro, t., tomoharu, k., and komatsu, k. (2000). dendrite extension by methanol extract of ashwagandha (roots of withania somnifera) in sk-nsh cells. neuroreport., 11, 1981-1985. 15. panda, s., and kar a. (1999). withania somnifera and bauhinia purpurea in the regulation of circulating thyroid 22 all res. j.biol, 2015, 6, 16-23     hormone concentrations in female mice. j. ethnopharmacol., 67, 233-239. 16. jayaprakasan, b., zhang, y., seeram, n. p., and nair, m. g. (2003). growth inhibition of human tumor cell lines by winthanolides from withania somnifera leaves. lif. sci., 74, 125-132. 17. ahmad, m., saleem, s., ahmad, a. s., ansari, m. a., yousuf, s., hoda, m. n., and islam, f. (2005). neuroprotective effects of withania somnifera on 6hydroxydopamine induced parkinsonism in rats. human exp. toxicol., 24, 137-147. 18. kumar, s., harris, r. j., seal, c. j., and okello, e. j. (2012). an aqueous extract of withania somnifera root inhibits amyloid β fibril formation in vitro. phytothera. res., 26, 113-117. 19. udayakumar, r., kasthurirengan, s., mariashibu, t. s., rajesh, m., anbazhagan, v. r., kim, s. c., ganapathi, a., and choi, c. w. (2009). hypoglycaemic and hypolipdaemic effects of withania somnifera root and leaf extracts on alloxan-induced diabetic rats. intern. j. mol. sci., 10, 23672382. 20. rani, g., kaur, k., wadhwa, r., kaul, s. c., and nagpal, a. c. (2005). evaluation of antigenotoxicity of leaf extract of ashwagandha. food chem. toxicol., 43, 95-98. 21. siddique, y. h., ara, g., beg, t., faisal, m., ahmad, m., and afzal, m. (2008). antigenotoxic role of centella asiatica l. extract against cyproterone acetate induced genotoxic damage in cultured human lymphocytes. toxicol. in vitro., 22, 10-17. 22. feany, m. b., and bender, w. w. (2000). a drosophila model of parkinson’s disease. nature., 404, 394-398. 23. siddique, y. h., ara, g., jyoti, s., and afzal, m. (2012). the dietary supplementation of nordihydroguaiaretic acid (ndga) delayed the loss of climbing ability in drosophila model of parkinson’s disease. j. diet. suppl., 9, 1-8. 24. pendleton, r. g., parvez, f., sayed, m., and hillman, r. (2002). effects of pharmacological agents upon a transgenic model of parkinson’s disease in drosophila melanogaster. pharmacol. exp. therap., 300, 91-96. 25. siddique, y. h., ara, g., and afzal, m. (2012). estimation of lipid peroxidation induced by hydrogen peroxide in cultured human lymphocytes. dose resp., 10, 110. 26. hawkins, c. l., morgan, p. e., davies, m. j. (2009)quantification of protein modification by oxidants. free rad. bio. med., 46, 965-988. 27. ryter, s. w., kim, h. p., hoetzel, a., park, j. w., nakahira, k., wang, x., and choi, a. m. k. (2007). mechanisms of cell death in oxidative stress. antioxi. redox signal., 9, 49-89. 28. siddique, y. h., ara, g., jyoti, s., and afzal, m. (2012). protective effect of curcumin in transgenic drosophila melanogaster model of parkinson’s disease. alter. med. stud., 2, e3. 29. kaur, k., rani, g., widodo, n., nagpal, a., taira, k., kaul, s. c., and wadhwa, r. (2004). evaluation of the antiproliferative and anti-oxidative activities of leaf extract from in vivo and in vitro raised ashwagandha. food chem. toxicol., 42, 2015-2020. 30. amer, d. a. m., irvine, g.b., and el-agnaf, o. m. a. (2006). inhibitors of α-synuclein oligomerization and toxicity: a future therapeutic strategy for parkinson’s disease and related disorders. exp. brain res., 173, 223-233. 31. rojo, a.i., cavada, c., de sagarra, m. r., and cuadrado, a. (2007). chronic inhalation of rotenone or paraquat does not induce parkinson's disease symptoms in mice or rats. exp. neurol., 208, 120-126. 32. tohda, c., kuboyama, t., and komatsu k. (2005). search for natural products related to regeneration of the neuronal network. neurosignals., 14, 34-45. 33. tohda, c., kuboyama, t., and komatsu, k. (2000). dendrite extension by methanol extract of ashwagandha (roots of withania somnifera) in sk-n-sh cells. neuroreport., 11, 1981-1985. 34. siddique, y. h., mujtaba, s. f., jyoti, s., naz, f. (2013). gc–ms analysis of eucalyptus citriodora leaf extract and its role on the dietary supplementation in transgenic drosophila model of parkinson’s disease. food chem. toxicol., 55, 2935. 35. siddique, y. h., jyoti, s., naz, f., and afzal, m. (2011). protective effect of apigenin in transgenic drosophila melanogaster model of parkinson’s disease. pharmacologyonline., 3, 790-795. 36. long, j., gao, h., sun, l., liu, j., and zhao-wilson, xi. (2009). grape extract protects mitochondria from oxidative damage and improves locomotor dysfunction and extends lifespan in a drosophila parkinson's disease model. rejuven. res., 12, 321-331. 37. chatterjee, s., srivastava, s., khalid, a., singh, n., sangwan, r. s., sidhu, o. p., roy, r., khetrapal, c. l., and tuli, r. (2010). comprehensive metabolic fingerprinting of withania somnifera leaf and root extracts. phytochemistry., 71, 1085-1094. 38. beare-rogers, j. l., dieffenbacher, a., and holm, j. v. (2001). lexicon of lipid nutrition (iupac technical report). pure app. chem., 73, 685-744. 39. gloerich, j., van vlies, n., jansen, g. a., denis, s., ruiter, j. p. n., van werkhoven, m. a., duran, m., vaz, f. m., wanders, r. j. a., and ferdinandusse, s. (2005). a phytol-enriched diet induces changes in fatty acid metabolism in mice both via pparα-dependent and independent pathways. j. lipid res., 46, 716-726. 40. palladino, m. j., and celotto, a. m. (2005). drosophila: a “model” model system to study neurodegeneration. mol. interv., 5, 292-303. 41. reiter, l. t., potocki, l., chien, s., gribskov, m., and bier, e. (2001). a systematic analysis of human diseaseassociated gene sequences in drosophila melanogaster. genome res., 11, 1114-1125.   23 microsoft word 112-609-3-layoutediting.docx analysis of the effects of tissue inhibitor of metalloproteinases-1, -2 and -3 nand cterminal domains on signaling markers during x. laevis development m. a. nieuwesteeg, j. a. willson, m. cepeda, m. a. fox, and *s. damjanovski all res. j. biol., 2014, 5, 30-36 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                                    issue 4, vol 5, 2014, 30-36 analysis of the effects of tissue inhibitor of metalloproteinases-1, -2 and -3 n and c-terminal domains on signaling markers during x. laevis development m. a. nieuwesteeg, j. a. willson, m. cepeda, m. a. fox, and *s. damjanovski *corresponding author: dr. sashko damjanovski, sdamjano@uwo.ca department of biology, university of western ontario, 1151 richmond street, london ontario, canada, n6a5b7 abstract: extracellular matrix (ecm) remodeling is accomplished largely by matrix metalloproteinases (mmps), which cleave individual components of the ecm to affect many cellular functions. tissue inhibitors of metalloproteinases (timps) are secreted mmp inhibitors, which, along with the cell surface mmp inhibitor reversion-inducing cysteine-rich protein with kazal motifs (reck), bind to mmps and inhibit their activity. although timps were originally characterized based on their mmp-inhibitory activities, timps are now known to be multifunctional proteins, with structurally and functionally distinct nand c-terminal domains. timp n-terminal domains bind to and inhibit mmps, while their c-terminal domains have demonstrated in vitro cell signaling activity in apoptosis, cell proliferation, and cell migration pathways. this study utilized timp nand c-terminal domain constructs to examine individual domain functions related to cell proliferation, apoptosis and reck expression in xenopus laevis embryos. western blot analysis revealed that none of the timp constructs altered cell proliferation as measured by phospho-histone-3 levels. conversely, timp-1 full-length and c-terminal domain constructs both elevated both reck and apoptosis levels, the latter as measured by caspase-3. conversely timp-2 c-terminal and timp-3 n-terminal domain constructs both decreased reck levels. keywords: timp, xenopus laevis, reck, apoptosis introduction extracellular matrix (ecm) remodeling is accomplished largely by matrix metalloproteinases (mmps), which cleave individual components of the ecm to facilitate cell migration. although tissue inhibitors of metalloproteinase (timp) family members were originally identified and characterized based on their abilities to bind to and inhibit mmps, it is now acknowledged that timps are multifunctional proteins, which can have pleiotropic effects on ecm remodeling and cell behavior1. in vivo, timp knockout mouse models display developmental deficiencies, demonstrating the importance of timps in regulating normal development. in particular, timp-1 and -2 knockouts have been associated with defects in brain and neural development. timp-1 null mice had diminished neuronal development and impaired learning and memory2, while timp-2 null mice showed decreased neurite outgrowth, delayed neuronal differentiation and considerable motor dysfunction3. in contrast, timp-3 null mice predominantly showed increased apoptosis in cells of the mammary glands, and defective alveolar and lung development4,5, both of which have been attributed to enhanced mmp activity6. in x. laevis, ectopic expression of timp-3 has been associated with perturbation of head and neural structures7, a finding that we have corroborated using full-length and n-terminal domain timp-3 constructs (but not c-terminal timp-3), also indicating that the role of timp-3 in development is dependent on its mmp-inhibitory activities8. while the timp n-terminal domains have been shown to impede mmp activity in many cell types and model organisms, their c-terminal domains have demonstrated cell signaling activity in specific cell types in vitro. characterization of timp c-terminal domain function has been difficult as their roles in cell signaling vary, not only between the different timps, but also depending on the cell line under study1. the seemingly contradictory roles of timps in up-regulating or down-regulating apoptosis and cell proliferation pathways indicate that timps may have cell or tissue-specific roles, where timp function may depend on both the availability of cell surface receptors and the stoichiometry of free timps and mmps within the ecm. timp-1 inhibits apoptosis in burkitt’s lymphoma cell lines, and this inhibition was demonstrated to occur through the timp-1 c-terminal domain binding to the cd63-β1-integrin 30 all res. j.biol, 2014, 5, 30-36     complex in mcf10a cells,10. timp-1 has also been shown to have both cell growth-promoting and cell growth-inhibiting activities in various cell lines, which are independent from the n-terminal domain-mediated inhibition of mmps11,12. likewise, there are several conflicting reports in the literature regarding the role of timp-2 in mediating cell growth and apoptosis pathways. in the mcf-7 breast cancer cell line timp-2 demonstrated cell growth promoting activity, which was mediated by its c-terminal domain interacting with mt1-mmp on the cell surface to activate the mapk pathway13. in contrast, in cultured human endothelial cells timp-2 inhibited cell growth by binding to α3β1 integrin receptors, leading to upregulation of the cyclin dependent kinase inhibitor p27kip114. in addition to both promoting and inhibiting cell growth, timp-2 has been shown to both suppress apoptosis (in melanoma cells), and promote apoptosis (in human t-lymphocytes)15,16. in contrast to timp-1 and -2, which are peri-cellular, timp3 is sequestered in the ecm away from the cell surface and consequently may have limited direct ability to participate in cell surface signaling events1. however, timp-3 is a good inhibitor of adam family proteases, particularly adam-17 (also known as tace [tnf-α converting enzyme]). adam17 facilitates the shedding of transforming growth factor-α (tnf-α) as well as various cell surface receptors including fas and tnf-related apoptosis inducing ligand receptor 1 (trail-r1)17. thus, some timp-3 effects are thought to occur indirectly as a result of adam inhibition, rather than as a result of direct signaling activity1. in addition to linking timps with both apoptotic and cell growth pathways, in vitro studies have also connected timp functions to that of reck (a cell surface mmp inhibitor)18. timp-1 overexpression can down regulate reck in both xenopus embryos and mouse cells19. timp-2 has been shown to inhibit endothelial cell migration by elevating reck levels20, while in melanomas timp-2 down regulated reck21. furthermore, mouse timp-3 and reck share a common regulatory mechanism involving microrna (mirna-712)22. taken together timps 1, 2 and 3 can have differing roles in cell proliferation, apoptosis and regulation of reck. we have previously demonstrated that ectopically injected full length and individual nor c-terminal domains of timp-1, -2 and -3 had varying effects on xenopus embryo morphology and survival8,23. in general, injection of individual nor c-terminal domains had a more deleterious effect than injection of the full length protein, with timp-1 c, timp-2 c and n, and timp-3 n having the most damaging effects. while all timp n-terminal domains bind to and inhibit mmps, different timps have varying effects on apoptosis16. although specific c-terminal interactions have been associated with cell proliferation10,13 and cell migration20, the purpose of this investigation was to examine whether timp c-terminal domains have unique abilities to alter these signaling pathways in vivo in frog embryos. specifically, changes in apoptosis were assayed using an active caspase-3 antibody, proliferation via a phosho-histone3 (ph3) antibody, and reck protein levels using a reck antibody. we show that no timp construct altered phosphohistone-3 levels. only timp-1 could regulate caspase-3 levels, while different timp-1, timp-2 and timp-3 domains could regulate reck levels. results the presence of equal levels of ectopic proteins for all constructs has been previously confirmed in embryonic protein extracts using an ha-antibody8,23 . as a control for rna injection artifacts, injection of equivalent levels of gfp mrna and production of high levels of gfp protein did not have any developmental effects, while the phenotypic consequences of timp-1 and -2 and -3 mrna overexpression has been previously reported8,23. ph3 levels were not altered by the overexpression of any timp construct in x. laevis embryo protein lysates in order to characterize the roles of the timp-1, -2 and -3 n and c-terminal domains in regulating cell proliferation levels, phospho-histone-3 was used as an indicator. western blot analysis of stage 30 whole embryo protein lysates showed that the overexpression of full-length, n-terminal or c-terminal domain timp constructs did not significantly alter ph3 levels when compared to whole embryo lysates from control embryos (timp-1, fig 1a; timp-2, fig 1b; timp-3, fig 1c). this suggests that timps do not significantly impact cell proliferation in early x. laevis embryos. only overexpression of full-length and c-terminal timp-1 constructs increased active caspase-3 levels in x. laevis embryo protein lysates. active caspase-3 was used as an indicator of apoptosis to measure differences in the abilities of timp constructs to alter this pathway during development. western blot analysis from stage 30 whole embryo protein lysates demonstrated increased caspase-3 levels only following overexpression of full-length (t1fl) and c-terminal (t1c) timp-1 constructs (asterisks fig 2a, p < 0.05). the n-terminal timp-1 construct (t1n) did not significantly alter caspase-3 (fig 2a). similarly, western blot analysis demonstrated that no timp-2 or timp-3 construct significantly altered caspase-3 levels relative to control embryos (figs 2b, 2c). 31 issue 4, vol 5, 2014, 30-36 figure 1. injection of timp constructs did not alter ph3 levels in x. laevis embryo protein extracts. embryos were injected at the 1 cell stage with 4 ng of each timp full-length, n-terminal domain, or c-terminal domain mrna construct. western blot analysis was performed to measure changes in phospho-histone-3 (ph3) levels from stage 30 whole embryo protein lysates. ph3 levels were measured relative to β-actin. results are displayed as mean ± se using protein isolated from 10 injected embryos from 3 independent experiments. no significant changes in ph3 levels relative to control uninjected (cont) embryos were observed following overexpression of any timp construct, as analyzed by oneway anova and dunnett’s multiple comparisons test (p > 0.05). constructs are; full length, n-terminal domain and c-terminal domain of a; timp-1 (t1fl, t1n, t1c), b; timp-2 (t2fl, t2n, t2c) and c; timp-3 (t3fl, t3n, t3c), respectively. figure 2. only t1fl and t1c constructs increased active caspase-3 levels in x. laevis embryos. embryos were injected at the 1 cell stage with 4 ng of each timp full-length, n-terminal domain, or c-terminal domain mrna construct. western blot analysis was performed to measure changes in caspase-3 levels from stage 30 whole embryo protein lysates. caspase-3 levels were measured relative to β-actin. results are displayed as mean ± se using protein isolated from 10 injected embryos from 3 independent experiments. only embryos injected with tifl and t1c constructs significantly increased caspase-3 levels relative to control uninjected (cont) embryos, as analyzed by one-way anova and dunnett’s multiple comparisons test *(p < 0.05). constructs are; full length, n-terminal domain and c-terminal domain of a; timp-1 (t1fl,t1n, t1c), b; timp-2 (t2fl, t2n, t2c) and c; timp-3 (t3fl, t3n, t3c) respectively. overexpression of several timp constructs altered reck protein levels in x. laevis embryo protein lysates. western blot analyses from stage 30 embryos showed that reck expression significantly increased following overexpression of tifl and t1c constructs (asterisks fig 3a, p < 0.05). in contrast, while the full-length (t2fl) and n-terminal domain timp-2 (t2n) constructs did not significantly alter reck levels, the c-terminal domain timp-2 (t2c) construct resulted in significantly decreased levels of reck relative to control embryos (asterisk fig 3b, p < 0.05). interestingly, with timp-3, the full-length (t3fl) and c-terminal domain (t3c) constructs did not result in altered reck levels compared to controls, whereas the nterminal domain (t3n) construct significantly decreased reck levels compared to controls (asterisks fig 3b and c, p < 0.05). figure 3. t1fl and t1c increased reck, while t2c and t3n decreased reck in x. laevis embryos. embryos were injected at the 1 cell stage with 4 ng of each timp full-length, n-terminal domain, or c-terminal domain mrna construct. western blot analysis was performed to 32 all res. j.biol, 2014, 5, 30-36     measure changes in reck levels from stage 30 whole embryo protein lysates. reck levels were measured relative to β-actin. results are displayed as mean ± se using protein isolated from 10 injected embryos from 3 independent experiments. embryos injected with tifl and t1c, constructs significantly increased reck levels relative to control uninjected (cont) embryos, whereas embryos injected with t2c and t3n constructs decreased reck levels relative to control (cont) uninjected embryos. all data were analyzed by one-way anova and dunnett’s multiple comparisons test *(p < 0.05). constructs are; full length, n-terminal domain and c-terminal domain of a; timp-1 (t1fl, t1n, t1c), b; timp-2 (t2fl, t2n, t2c) and c; timp-3 (t3fl, t3n, t3c), respectively. discussion we have previously shown that overexpression of both timp-1 cand timp-2 c-terminal domains (but not the timp-3 c-terminal domain) resulted in severe developmental defects. furthermore, we demonstrated that the timp-2 c-terminal domain altered mmp levels independent of mmp inhibition8. in order to better understand the unique contributions the timp c-terminal domains may have on the regulation of signaling pathways during development, we overexpressed timp-1, -2 or -3 fulllength, n-terminal and c-terminal domain constructs in x. laevis embryos, and examined changes in levels of ph3 as an indicator of cell proliferation and active caspase-3 as an indicator of apoptosis. additionally, we examined changes in reck levels, as the timp-2 c-terminal domain has previously been shown to increase reck protein levels in cultured human endothelial cells20. ectopic expression of timp constructs did not alter ph3 levels in x. laevis embryos overexpression of timp-1, -2 or -3 full-length, n-terminal or c-terminal domain constructs did not result in significantly altered cell proliferation in x. laevis embryos, as indicated by changes in the levels of ph3 (fig 1a, b and c), suggesting that there may be no direct or indirect role for timps in regulating this pathway during x. laevis development, during a time were very rapid early embryonic cleavage cycles are autonomously controlled. interestingly, even the c-terminal domain constructs of timp-1 and -2 (which have been demonstrated in various cancer and fibroblast cell lines to either promote or inhibit cell proliferation as reviewed in stetler-stevenson 20081), did not alter the rapid proliferation in injected relative to control embryos. as past in vitro proliferation studies with timps were performed using homogenous populations of differentiated cells, this mechanism or pathway may not be maintained in the undifferentiated rapidly dividing cells of an embryo. many pluripotent and multipotent cells in the embryo divide autonomous such that timps may not have the ability to alter the proliferation of significant cell numbers in the whole embryo, at least as demonstrated by the ph3 assays. the timp-1 c-terminal domain increased active caspase-3 levels in x. laevis embryos to investigate whether any of the x. laevis timps had the ability to alter cell death during development (particularly through their c-terminal domains), we examined changes in active caspase-3 as an indicator of apoptosis following overexpression of each timp construct. the t1fl and t1c constructs increased caspase-3 levels relative to control embryos (fig 2a). as the c-terminal domain construct has no direct mmp-inhibitory activity, these data suggest that these subtle changes in apoptosis are mediated by another mechanism. while the timp-1 c-terminal domain has been linked to changes in apoptosis in both human lymphoma and mammary epithelia cell lines10,24, these in vitro studies are associated with suppression of apoptosis, rather than an increase in apoptosis observed here. although the importance of the timp-1 c-terminal domain in regulating caspase-3 levels seems to be maintained in x. laevis, the effects of this protein may be different during development. interestingly, a recent study using hematopoietic cells in culture has reported that timp-1 can indeed lead to increased activation of caspase-3; however, this activation was associated with cell differentiation through activation of mek1, mek6, and p38α25. nevertheless, the role of timp-1 in regulating the amount of active caspase-3 during x. laevis development seems to be specific to the timp-1 c-terminal domain, as both the t1c and t1fl constructs (which contain a functional c-terminus) increased apoptosis (but not the t1n construct). furthermore, none of the timp-2 or -3 constructs resulted in significantly altered levels of caspase-3 relative to control embryos, demonstrating that this effect is unique to timp-1. the timp-1 and -2 c-terminal domains had opposing effects on reck protein levels in x. laevis embryos reck is a potent cell surface inhibitor of mmps, thus, alterations in reck expression may have considerable effects on cell migration during embryogenesis. as timp-2 has previously been demonstrated to increase reck expression in vitro, here we investigated whether the x. laevis timps or their individual domains may contribute to the regulation of reck expression in vivo, during development. intriguingly, the timp-1 full-length and cterminal domain constructs resulted in significantly increased levels of reck protein when quantified using western blot analysis (fig 3a). the t1n construct, which only functions in inhibition of mmp activity, did not result in changes in reck levels relative to control embryos. thus, these data suggest that the observed increases in reck following overexpression of t1fl and t1c constructs may be due to a c-terminal domain specific mechanism. while no previous 33 all res. j.biol, 2014, 5, 30-36     reports have linked the individual timp-1 c-terminal domain to the regulation of reck levels, reck levels have been linked to the expression of timp-122 thus suggesting that they share a regulatory mechanism. following overexpression of timp-2 full-length, n-, and cterminal domain constructs, we found that only the t2c construct significantly decreased reck expression relative to controls (fig 3b). this finding is in contrast to the report by oh et al.20, which showed that the timp-2 c-terminal domain increased reck expression through binding to α3β1 integrin receptors on the surface of human microvascular endothelial cells. as the effect of timp-2 on the regulation of reck expression has currently only been examined using cultured human endothelial cells, it is possible that timp-2 may have varying effects on reck levels, or may alter reck expression through different pathways, depending on the requirements of different tissues and organisms. indeed, recently published work suggests that timp-2 levels drop as reck levels decrease21. we demonstrated here that timp-2 has the ability to alter reck through its c-terminal domain, independent of mmp inhibition, during x. laevis development. overexpression of full-length and c-terminal domain timp3 did not alter reck levels relative to control embryos, suggesting that the timp-3 c-terminal domain does not participate in signaling events that regulate reck expression in x. laevis embryos. the t3n construct, however, did result in significantly decreased reck levels relative to control embryos when analyzed by western blot (fig 3c). as the timp n-terminal domains are involved in mmp inhibition and have not been associated with direct signaling activity, it is unlikely that the t3n construct decreased reck levels through a typically associated cell-surface signaling mechanism. under normal conditions, full-length timp-3 is sequestered in the ecm away from the cell surface, a function that is mediated by its c-terminal domain26. thus, whereas the t3fl and t3c constructs may remain bound away from the cell, this ability would be impaired with the t3n construct. the t3n construct is free to act pericellularly, and therefore may result in inappropriate feedback to the cell to downregulate expression of other mmp inhibitors, like reck. conclusions we have shown for the first time that the timp-1 and -2 cterminal domains have the ability to influence caspase-3 and reck levels in x. laevis embryos. this research indicates that timps may be important not only for directly mediating mmp activity during development, but independent of their mmp inhibitory role, timps may regulate cell signaling pathways that influence apoptosis and/or cell differentiation through caspase-3, and cell migration through reck. materials and methods animals adult x. laevis were purchased from xenopus i inc (dexter, mi), fertilized and reared in accordance with standard protocols, and staged according to nieuwkoop and faber27. animals were housed and treated in accordance with uwo and ccac guidelines. generation of timp constructs for microinjection briefly, pcr was used to produce ha-tagged full-length, nterminal and c-terminal domain constructs for timp-1, -2 and -3. details regarding timp-2 and-3 construct generation can be found in 8. timp-1 was cloned from total cdna from stage 35 embryos based on unannotated x. laevis clone aai41767.1. forward and reverse primers (5’gacagaaggactgcccagcc, 5’caaaacacttctccttcgag) were used, and the fulllength x. laevis timp-1 sequence was confirmed at the robarts research institute dna sequencing facility at the university of western ontario, and submitted as genbank id: 1628144. using the full-length timp-1 clone as a template, pcr was used to generate full-length, n-terminal or c-terminal timp-1 (hemagglutinin) ha-tagged constructs as described previously8. this unobtrusive ha-tag allows the use of sensitive and specific ha antibodies to be used for the detection of ectopic protein production. respective pcr forward and reverse primers were as follows: t1fl 5’agatctatgttgtaccttgtggttgtg, 5’cagtctgctgccacaacacaatacccatacgatgttcca gattacgctactagt; t1n 5’agatctatgttgtaccttgtggttgtg, 5’gtgtatcgcaaagcctgttcctacccatacgatgttcca gattacgctactagt t1c signal sequence and reverse link 5’agatctatgttgtaccttgtggttgtg, 5’ctcagccaggaggtgttggggtgcaacatcgtcccctgc tat, t1c-terminal link 5’tgcaacatcgtcccctgctat, 5’cagtctgctgccacaacacaatacccatacgatgttcca gattacgctactagt. microinjection of timp-1, -2 and -3 mrna constructs in x. laevis embryos fertilized x. laevis embryos were microinjected with timp1, -2 or -3 full-length, n-terminal or c-terminal domain mrna constructs. embryos were injected at the one cell stage with 4 ng of mrna in a volume of 2.3 nl, using 10 µm diameter glass. embryos were maintained in 1x (marc’s modified ringers) mmr containing 4% ficoll for 5 hours 34 all res. j.biol, 2014, 5, 30-36     following injection, then transferred to 0.1 x mmr solution for rearing. protein preparations and western blotting protein was extracted from stage 30 embryos injected with the various timp constructs, or uninjected control embryos. protein extraction, quantification and western blotting were performed from 10 pooled embryos from 3 independent experiments, as previously described8. primary antibodies used were reck (h-300) (rabbit polyclonal, 1:200 dilution; santa cruz), p-histone h3 (ser-10) (rabbit polyclonal, 1:200 dilution; santa cruz), anti-active caspase-3 (rabbit polyclonal, 1:500 dilution; abcam), and anti-β-actin (c4) (mouse monoclonal, 1:1000 dilution; santa cruz). secondary antibodies used were goat anti-rabbit hrp (1:5000 dilution; life technologies) or goat anti-mouse hrp (1:5000 dilution; bio-rad). western blots were visualized and photographed using bio-rad quantity one 4.4.0 software. densitometry was performed using imagej software, where levels of ph3, reck or caspase-3 were standardized to β-actin, and plotted as mean ± se based on 3 independent experiments. statistical analysis western blot densitometry data were log transformed (log10) to achieve a normal distribution. all statistical analysis was performed using the ibm spss statistic 19 program. results were presented as mean ± se. statistical significance was determined using one way analyses of variance followed by dunnett’s multiple comparisons test. differences were considered statistically significant when p < 0.05. acknowledgements supported by a discovery grant to s.d. from the natural sciences and engineering research council of canada. references 1. stetler-stevenson, w.g., (2008) tissue inhibitors of metalloproteinases in cell signaling: metalloproteinase-independent biological activities. science signaling 1, re6. 2. chaillan, 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(north holland publication, amsterdam). 36 microsoft word 137-797-1-le.docx identification of endogenous bacteria in micropropagated helleborus ×nigercors lindsay caesar, m. melissa hayes, and jeffrey adelberg all res. j. biol., 2016, 7, 41-46 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 4, vol 7, 2016, 41-46 identification of endogenous bacteria in micropropagated helleborus ×nigercors lindsay caesar a,*, m. melissa hayes b, and jeffrey adelberg a a department of plant and environmental sciences, clemson university, clemson sc b department of animal and veterinary sciences, clemson university, clemson, sc 2332 hiawatha drive, greensboro nc 27408, *corresponding author email: lindsaycaesar@gmail.com graphical abstract: abstract: during micropropagation of helleborus ×nigercors, plantlets were observed to be bacterially contaminated. to determine the identity of contaminants, bacteria resistant to surface sterilization were isolated and gram stained. polymerase chain reaction (pcr) and 16s rrna sequencing were used to identify bacterial isolates h7g and h7s as belonging to the paenibacillus and luteibacteri genera, respectively. strain h7r had highest sequence similarity to the pseudomonas, stenotrophomonas, and lysobacter genera. strains h7r and h7s were unable to grow in the absence of plant tissue and other bacterial species. paenibacillus sp. h7g was screened using combinations of antibiotics including streptomycin sulfate, gentamicin sulfate, and cefotaxime, and was only eradicated by concentrations of gentamicin sulfate above phytotoxic levels. this is the first documented exploration of bacterial endophytes associated with helleborus species. keywords: endophytes; helleborus ×nigercors; micropropagation; antibiotics introduction an endophyte is an organism living within plant tissue. the term is associated with microbial organisms that are symbionts, often mutualists, which provide plants with a benefit of some kind, such as protection against pathogens or aid in rooting (wilson 1995). endophytic organisms are of special interest to the scientific community due to the close biological complexes they develop with their hosts. because of these close interactions, they may possess a range of biological activities that contribute to the plant’s success in nature (strobel 2003). endophytes found in micropropagated plantlets have in some cases been shown to lower plant quality due to the lack of competition that the ‘biological vacuum’ within sterile, carbohydrate-rich vessels promotes (cassells 1997). however, in other cases, bacterial endophytes have contributed to plant growth by promoting hormone production and aiding in rooting (dias et al. 2009; lata et al. 2006). during the multiplication stage, micropropagated plantlets of helleborus ×nigercors were observed to be visibly contaminated with bacteria as indicated by the presence of a gray halo around the base of plantlets. following sterilization with sodium hypochlorite, three bacteria remained prevalent 41 all res. j. biol, 2016, 7, 41-46     in culture. these three bacteria were likely harbored within the plant tissue, allowing them to survive surface sterilization. to better understand the biological complex found in hellebore cultures, putative endophytic bacteria were isolated and characterized. although fungal endophytes have been isolated from other members of the helleborus genus (spadaro et al. 2014), this is the first study in which bacterial endophytes have been identified. materials and methods plant material and reinitiation procedure. plant material for micropropagation was obtained from pine knot farms in clarksville, virginia, where the original cross was completed. parent plants were grown in containers with a combination of native soil characterized by silt loam on top of fine, kaolinitic red clay, permatill® (arden, nc), and metro mix® 852 heavyweight bark mix (bfg supply co.; burton, oh). shoot tips were initially sterilized at the institute for sustainable and renewable resources© (isrr; danville, virginia), following which they were transferred to clemson university for propagation. initiated plantlets were multiplied for several cycles before contamination was evident by clouding of the media. thirtytwo contaminated plantlets were disinfested using four possible treatment combinations: high or low bleach concentrations (0.83% and 0.41% sodium hypochlorite, respectively), and short and long time intervals (1 and 4 min). after soaking in the bleach solution, plantlets were rinsed twice with sterile deionized water and placed into smithersoasis flexible tissue culture vessels (smither-oasis, kent, oh). vessels contained 25 ml of liquid wpm salts (lloyd and mccown 1980) at ph 5.7, 3% sucrose, and 9 µm tdz. plants were placed under 28 µmol m-2 s-1 delivered from monochromatic led lights (33.3% blue, 66.7% red) at 10-13 °c on a rocker shaker (ew-51301-00, cole-parmer® portable rocker shaker, vernon hills, il) set to 3 rpm. isolation and characterization of endogenous bacteria. slices of callus from reinitiated plantlets were streaked onto petri plates containing tryptic soy agar (tsa) (470193-226, vwr scientific products, suwanee, ga) and incubated for 24 h at 30 °c to detect contamination. to establish purity of culture, individual colonies were subcultured, incubated for 24 h, and stored in a refrigerator at 10 °c in darkness. individual colonies from subcultured plates were restreaked to maintain vigor. individual colonies were selected at random from the agar plates for genetic analysis. gram reactions and colony and cell morphological characteristics were repeated in triplicate. polymerase chain reaction (pcr) was conducted on culture isolates using a transfer loop to inoculate each culture into a sterile eppendorf tube (20901-547, vwr scientific products, suwanee, ga) containing 50 µl of promega nuclease free water (pap1195, vwr scientific products, west chester, pa). tubes containing the culture and water mixtures were placed into boiling water for 10 min. after boiling, 12.5 µl of the mixture was transferred to a sterile pcr tube to be used as a dna template. two primers were added to the tube: 1 µl of the forward oligonucleotide primer (16s rrna for, 5’agagtttgatcctggctcag 3’, readymadetm primers, integrated dna technologies, coralville, ia), and 1 µl of the reverse oligonucleotide primer (16s rrna rev,5’acggctaccttgttacgactt 3’, readymadetm primers, integrated dna technologies, coralville, ia), as well as 10.5 µl of promega gotaq® green master mix (pam7122, vwr scientific products, west chester pa). each pcr reaction tube containing the dna template, primers, and gotaq® green master mix was placed in a thermocycler (icycler io, biorad laboratories, inc., richmond, ca). the thermal cycle program consisted of 1 cycle of 95 °c for 2 min, followed by 30 repeating cycles of 94 °c for 30 s, 50.6 °c for 30 s, 72 °c for 1 min, and a final extension at 72° c for 5 min. the cycling program ended by holding the tubes at 4 °c until removal from the thermocycler (hayes et al. 2012; promega corporation 2012). prior to sequencing, the concentration and 260:280 ratio of the pcramplified products was measured using a nanodrop 2000 spectrophotometer (nd-2000, thermo fischer scientific, pittsburg, pa). the pcr-amplified products were observed by gel electrophoresis in 1.5% agarose gels. ten µl of each pcr product and the hyladdertm molecular mass marker (denville scientific inc., cb4225-2, metuchen, nj) were examined using agarose gel electrophoresis with subsequent ethidium bromide staining (97064-970, vwr scientific products, suwanee, ga). the amplified dna fragments were visualized by uv illumination. purification of pcr products was completed with promega wizard® sv gel and pcr clean-up system (paa9281, vwr scientific products, suwanee, ga). the 16s rrna sequencing was completed through the clemson university genomic institute© (cugi). bioedit (version 7.2.5) was used to align sequences and to compile consensus sequences from forward and reverse primers. sequences were examined using the national center for biotechnology information (ncbi) blast database (altschul et al. 1997). the top blast nucleotide/16s rrna database results with maximum identity greater than 97% were reported. choice of antibiotics and susceptibility tests. minimal bactericidal concentrations of single antibiotics and antibiotic combinations were determined using a tube dilution method. original bacterial isolates were transferred into broth cultures containing 3% sucrose and ½ strength ms medium (murashige and skoog 1962). following clouding of medium, gram stains were done to confirm the presence of original strains. twelve single antibiotic treatments were tested in triplicate: streptomycin sulfate (s; 1000, 500, 250, or 125 µg ml⁻1), cefotaxime (c; 500, 250, 125, or 62.5 µg ml⁻1), and gentamicin sulfate (g; 50, 25, 12.5 or 6.25 µg ml⁻1). in addition, twelve combination treatments were prepared: streptomycin sulfate + cefotaxime (250 + 125, 250 + 62.5, 125 + 125, and 125 + 62.5 µg ml⁻1), streptomycin sulfate + gentamicin sulfate (250 + 12.5, 250 + 6.25, 125 + 12.5, and 125 + 6.25 µg ml⁻1), and cefotaxime + gentamicin sulfate (125 + 12.5, 125 + 6.25, 62.5 + 12.5, and 62.5 + 6.25 42 all res. j. biol, 2016, 7, 41-46     µg ml⁻1). antibiotics were diluted in 5 ml of deionized water containing 3% sucrose and ½ strength ms liquid medium (murashige and skoog 1962). following preparation of media, 1 ml of broth cultures containing bacterial isolates were added to each treatment and inoculated in flat-bottomed vials (o.d. x h.: 29x94 mm; fisher glass shell vials, thermo fisher scientific inc., pittsburg, pa) for 7 days at 25 °c. after 7 days, 1 ml of the inoculated antibiotic cultures was transferred into flat-bottomed vials containing the same media formulation without antibiotics. these tubes were inoculated at 25 or 35 °c for an additional 7 days. following inoculation, growth was assessed. tubes showing no growth were designated as bactericidal. the lowest concentration in each treatment showing no growth was termed the minimal bactericidal concentration (mbc). phytotoxicity tests and antibiotic treatment of plantlets. h. ×nigercors plantlets were treated with single antibiotics (ug ml-1): 12.5 (g) and 50 (g), and combinations of two antibiotics: 125 (s) + 12.5 (g), 250 (s) and 25 (g), 62.5 (c) + 12.5 (g), and 125 (c) + 25 (g). plantlets of h. ×nigercors known to be contaminated with three endogenous bacteria were totally submerged in ½ strength liquid ms medium with and without the aforementioned antibiotic treatments in magenta™ ga7 boxes (magenta® corp., chicago, il) and placed at 10-12 °c under 28 µmol m⁻2 s⁻1 monochromatic led lights (33.3% blue, 66.7% red) for 12 days on a rocker (ew-51301-00, cole-parmer® portable rocker shaker, vernon hills, il) set to 3 rpm. each of the four treatments contained three boxes, with three plantlets per box. following antibiotic treatment, bases of plants were streaked onto tsa medium and plantlets were transferred to a multiplication medium. phytotoxicity was ranked on a subjective numerical scale from 0-4, with a score of 0 indicating no phytotoxicity symptoms and a score of 4 indicating severe phytotoxicity or death. plantlets showing phytotoxicity symptoms were judged on the presence of chlorosis, tissue browning, softening of tissue, and morphological changes. plantlets were subcultured in fourweek cycles, and plant condition and bacterial contaminants recorded during transfer. results and discussion genetic identification and morphological characteristics of bacterial isolates. after plantlet reinitiation and subsequent streaking of helleborus ×nigercors callus on tsa plates, bacterial colonies were visible after three days. all bacterial isolates were gram-negative and individual cells were rod-shaped. using 16s rrna sequence analysis, gram stain results, and colony morphology, isolates were identified to the genus level (table 1). strain h7g (genbank accession no. ku721939) was identified as a member of the paenibacillus genus and was characterized by colonies that were grey, round, and opaque. members of this genus have been found as endophytes in plant species including pine, coffee, poplar (ulrich et al. 2008), and barley (rasimus et al. 2012). many endophytic paenibacilli produce compounds that aid in plant growth, such as auxins, cytokinins, and antibiotics (ulrich et al. 2008; mcspadden gardener 2004). most paenibacilli grow in cool temperatures (rasimus et al. 2012), and some have been isolated from alaskan tundra (nelson et al. 2009). the coldloving tendencies of this genus are consistent with their growth and persistence within tissues of the frost-tolerant and winter-flowering helleborus ×nigercors. strain h7s (genbank accession no. ku721940), with small, light yellow colonies, was identified as a member of the luteibacter genus. this genus was discovered in 2005 from the rhizosphere of spring barley (johansen et al. 2005). all members of this genus are yellow-pigmented, aerobic, gramnegative rods (kämpfer et al. 2009), which is consistent with the morphological characteristics of strain h7r. luteibacter rhizovicinus, the first species discovered from the genus, has been isolated from micropropagated malus domestica “golden delicious” plantlets (guglielmetti et al. 2013), where it lowered shoot regeneration abilities (piagnani et al. 2007), and from micropropagated barley plantlets, where rooting was stimulated (guglielmetti et al. 2013). strain h7r (genbank accession no. ku729138) had high sequence similarity (> 97%) with three genera ubiquitous in soils: stenotrophomonas, pseudomonas, and lysobacter (table 1). because these genera also share many genotypic and phenotypic similarities, 16s rrna is often inadequate to differentiate between members of these genera (svenssonstadler et al. 2012; hayward et al. 2010). the genus stenotrophomonas is comprised of several species of bacteria that show a range of activities including plant-growth promotion (zhu et al. 2012), antibiotic production (hayward et al. 2010), and pathogenicity (ryan et al. 2009; nyč and matĕjková 2010). endophytic strains of stenotrophomonas maltophilia have been isolated from cucumber, oilseed rape, potato, strawberry, alfalfa, maize, sunflower, rice, wheat, willow, and poplar (ryan et al. 2009), but some have developed antibiotic resistance to chloramphenicol and quinolone antibiotics (nyč and matĕjková 2010; alonso and martinez 1997). members of the pseudomonas genus include endophytic species such as those isolated from tulip poplar trees (liriodendron spp.) and willows (salix gooddingii), where they may produce rooting hormones through multiple pathways (taghavi et al. 2009). lysobacter species have been isolated from the rhizosphere of rice (aslam et al. 2009) and ginseng (srinivasan et al. 2010). lysobacter spp. are often studied as biological control agents due to their predatory activity against gram-negative and gram-positive bacteria, blue-green algae, yeasts, fungi, and nematodes (hayward et al. 2010; sullivan et al. 2003). choice of antibiotics and susceptibility tests. streptomycin sulfate and gentamicin sulfate are aminoglycoside antibiotics that show effectiveness in controlling gram-negative bacteria. cefotaxime, a beta-lactam antibiotic, was chosen in addition to these two aminoglycosides due to the effectiveness of beta-lactams against resistant strains of 43 all res. j. biol, 2016, 7, 41-46     table 1. endophytic bacteria isolates identified using gram reaction and cell morphology and 16s rrna sequencing analysis.   a bacterial candidates not fitting the gram reaction and morphology observed were not included in the top identity matches b percentages from 16s rrna database listed first, followed by percent matches from the nucleotide database, if applicable stenotrophomonas maltophilia, a candidate genus for strain h7r (alonso and martinez 1997). before antibiotic treatment, however, broth cultures containing bacteria were gram stained to confirm monocultures. luteibacter h7s and strain h7r were not included in the remainder of the experiments due to lack of growth in control tubes after three weeks. possibly, these strains showed initial growth in culture due to storage of a vital nutrient obtained directly from the plant tissue. following isolation and initial proliferation, the amount stored may have become inadequate for growth. initial experiments indicated that paenibacillus sp. h7g was not affected by streptomycin sulfate, cefotaxime, or combinations of the two. gentamicin sulfate was most effective, with an mbc of 12.5 µg ml⁻1. mbcs were used to determine plant treatments. since only treatments containing at least 12.5 µg ml⁻1 of gentamicin sulfate were bactericidal, only these treatments were were evaluated for phytotoxicity and effective elimination of bacteria from plant material. shoot tips of h. ×nigercors showed some symptoms of phytotoxicity (browning of outer leaves and minor chlorosis) immediately after treatment with single treatments and combinational treatments containing 12.5 µg ml⁻1 gentamicin sulfate. interestingly, control plants often had minor symptoms, indicating that submersion in media, even without antibiotics, causes some stress to plantlets. plantlets treated with 25 µg ml⁻1 gentamicin sulfate and 125 µg ml⁻1 cefotaxime or 25 µg ml⁻1 gentamicin sulfate and 250 µg ml⁻1 streptomycin sulfate showed more severe phytotoxicity, with shoots showing tissue browning and tissue softening. plantlets in the 50 µg ml⁻1 gentamicin treatment were affected most severely, and 22% of the plantlets did not survive the treatment. after a month of growth in nonantibiotic containing media, plantlets remained low quality. all plantlets from the 50 µg ml⁻1 treatment and 33% of isolate gram reaction and cell morphology colony color (on tryptic soy agar) 16s bacteria identification (>97% top identity matches) a 16s rrna/ nucleotide database % match b isolate 1 (h7g) negative rod grey paenibacillus xylanexedens paenibacillus tundrae paenibacillus amylolyticus paenibacillus taichungensis 98%, 99% 99%, 99 98%, 98% 98%, n/a isolate 2 (h7s) negative rod yellow luteibacter rhizovicinus luteibacter anthropi luteibacter yeojuensis 98%, 98% 98%, 98% 97%, n/a isolate 3 (h7r) negative rod beige stenotrophomonas maltophilia stenotrophomonas pavanii stenotrophomonas chelatinphaga stenotrophomonas humi stenotrophomonas terrae stenotrophomonas nitritireducens stenotrophomonas panacihumi stenotrophomonas ginsengisoli stenotrophomonas rhizophila stenotrophomonas daejeonensis stenotrophomonas acidiminiphilia stenotrophomonas koreensis pseudomonas geniculata pseudomonas hibiscicola pseudomonas pictorum lysobacter enzymogens lysobacter soli lysobacter rushenii lysobacter oryzae lysobacter yangpyeongensis 100%,100% 100%, n/a 100%, n/a 100%, n/a 99%, n/a 99%, n/a 99%, n/a 99%, n/a 99%, n/a 99%, n/a 99%, n/a 98%, n/a 100%, 100% 99%, 100% 99%, 100% 99%, n/a 98%, n/a 98%, n/a 98%, n/a 98%, n/a 44 all res. j. biol, 2016, 7, 41-46     plantlets from the 25 µg ml⁻1 gentamicin sulfate and 250 µg ml⁻1 streptomycin sulfate died, showing a failure to reacclimate after antibiotic treatment. surviving plantlets were very low quality, showing tissue blackening, shoot tip necrosis, and tissue softening (table 2). table 2. phytotoxicity rankings and survival percentages of h. ×nigercors plantlets treated with two single treatments of gentamicin sulfate (g), two combination treatments of gentamicin sulfate (g) and cefotaxime (c), and two combination treatments of gentamicin sulfate (g) and streptomycin sulfate (s) for 12 days. antibiotic treatment (µg ml⁻1) average phytotoxicity ranking a survival percentage b control 1.00 100% g(12.5) 1.67 100% g(50) 3.67 0% g(12.5) + c(62.5) 1.67 100% g(25) + c(125) 2.00 100% g(12.5) + s(125) 1.67 100% g(25) + s(250) 3.00 67% a phytotoxicity scores were subjectively determined from 0-4, with a score of 0 indicating no phytotoxicity symptoms and a score of 4 indicating extremely severe symptoms, including blackening of plant tissue and death. b results taken after the second cycle as a percentage of the original number of plantlets placed in treatment. original treatments used 3 magenta ga7 boxes with 3 plantlets/box. antibiotic effectiveness on plant tissue. initial experiments indicated that the 12-day antibiotic treatments of all single and combinational treatments containing 12.5 µg ml⁻1 gentamicin were ineffective for eliminating bacteria from culture, with all treatments showing the growth of at least one species. mbcs are used as starting points for determining effective treatments, and treatment concentrations required to penetrate plant tissues and maintain bactericidal effects are often two to four times higher than the mbcs (leifert et al. 1991). when concentrations of gentamicin sulfate were quadrupled (50 µg ml⁻1), up to 44% of plantlets showed no bacterial growth after the first 4-week cycle of treatment. after the second cycle, bacterial growth resumed completely, indicating that initial success was due to a slowing of bacterial growth rather than colony death (table 3). table 3. ratio of bacteria-free hellebore shoots to contaminated shoots in plantlets treated with two single treatments of gentamicin sulfate (g), two combination treatments of gentamicin sulfate (g) and cefotaxime (c), and two combination treatments of gentamicin sulfate (g) and streptomycin sulfate (s) for 12 days. treatment (µg ml⁻1)   cycle 1 cycle 2 bacteria-free plants/treated plants   g(12.5) 0/9 - g(50) 0/9 - g(12.5) + c(62.5) 0/9 - g(25) + c(125) 4/9 0/9 g(12.5) + s(125) 0/9 - g(25) + s(250) 3/9 0/9 conclusions the establishment of aseptic culture is one of the major challenges associated with micropropagation of h. ×nigercors. antibiotic treatments were unsuccessful at eliminating these endogenous bacteria due to the severe phytotoxicity associated with high antibiotic concentrations. combinational treatments showed initial success, but proved to have bacteriostatic properties rather than bactericidal properties when penetrating plant tissues. this is the first study in which endophytic bacteria have been characterized from members of the helleborus genus. while the evidence points to internal habitation by these strains, further studies are necessary to determine if these bacteria have beneficial, neutral, or negative effects on the growth of h. ×nigercors. since antibiotic treatment was unsuccessful, plants containing endogenous bacteria could not be compared to aseptic plants. as such, additional studies are 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(2012). genome sequence of stenotrophomonas maltophilia rr-10, isolated as an endophyte from rice root. j. bacteriol. 194, 1280-1281. 46 microsoft word 132-752-1-layoutediting.docx relationship of angiotensinase and vasopressinase enzymatic activities between hypothalamus and plasma in aged rats by high-fat diet germán domínguez-vías*, ana belén segarra, manuel ramírez-sánchez, sara jiménez-serrano all res. j. biol., 2016, 7, 20-27 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                  issue 2, vol 7, 2016, 20-27 relationship of angiotensinase and vasopressinase enzymatic activities between hypothalamus and plasma in aged rats by high-fat diet germán domínguez-vías1*, ana belén segarra1, manuel ramírez-sánchez1, sara jiménezserrano1 1unit of physiology, university of jaén, jaén, spain. *correspondence to: dr. germán domínguez-vías, physiology unit, university of jaén, 23071 jaén, spain, e-mail: german.dominguez@uca.es graphical abstract abstract: high-fat diets are associated with the development of hypertension. however, a high intake of monounsaturated fat has been proposed to be a dietary factor that can decrease the incidence of hypertension. the renin-angiotensin system (ras) and vasopressin interact to regulate blood pressure at central and peripheral level. in this study, we investigated the effect of different degrees of dietary fatty acid saturation in the control of ras and vasopressin on brain-blood. to improve our understanding of their interaction and their relationship, we analyzed angiotensinand vasopressin-metabolizing activities in hypothalamus and plasma, collected from old wistar rats fed for 24 weeks with diets enriched with extra virgin olive oil (monounsaturated fat) or butter plus cholesterol (saturated fat) compared with a standard diet. we found no angiotensinase and vasopressinase activities in hypothalamus and plasma, with no significant correlations between enzymatic activities in either region. therefore our results do not support the beneficial influence of extra virgin olive oil to regulate blood pressure on the central or systemic levels. furthermore, substrates downstream of vasopressin and the ras may be similarly unaffected, and given the previously described potential of the mediterranean diet as a tool in the treatment of hypertension, further studies need to be done in order to clarify the therapeutic mechanism. keywords: angiotensinases, vasopressinase, renin-angiotensin system, brain-blood connection, dietary fat 20 all res. j. biol, 2016, 7, 20-27     introduction dietary fat intake determines the fatty acid composition of cell membranes and plays a well-recognized role in cardiovascular risk and the development of cardiometabolic disease1. moreover, diet composition may also influence brain activity as the dietary fat modifies the brain membrane fluidity2,3. a high intake of monounsaturated fat has been proposed to decrease the incidence of hypertension4-6. in addition, increasing saturation of dietary fat resulted in increasing plasma total cholesterol concentration7 and systolic and diastolic blood pressures8. the systemic and the local renin-angiotensin system (ras), and vasopressin in brain9, interact together to regulate blood pressure (bp) by various endocrine and autonomic mechanisms10. the hypothalamic stimulatory effect of angiotensin (ang) iii (ang iii) on vasopressin release has been clearly demonstrated11. the hypothalamus is known to integrate behavioral, endocrine, neuroendocrine, and autonomic responses (including those that regulate cardiovascular function) to maintain homeostasis12. we hypothesized that the direct and/or indirect relationship between the hypothalamus and plasma, both of which regulate bp, may be mediated to some extent by angiotensinase and vasopressinase enzymatic activities and the metabolism of their corresponding peptidergic substrates. in order to analyze the link between ras and vasopressin in the control of bp, as well as the relationship between the hypothalamus and plasma, we have studied the metabolism of (a) ang i to ang 2-10, (b) ang ii to ang iii, (c) ang iii to ang iv and (d) the metabolism of vasopressin by measuring the activities of (a) aspartyl aminopeptidase (aspap), (b) glutamyl aminopeptidase (gluap), (c) alanyl aminopeptidase (alaap), arginyl aminopeptidase (argap), and (d) cystinyl aminopeptidase (cysap), respectively13,14. additionally, we observed the activities of angiotensinase and vasopressinase in plasma and their soluble and membrane-bound fractions from the anterior hypothalamus. in our model, aged male wistar rats were fed with high-fat diets (hfd) supplemented with different degrees of dietary fatty acid saturation, such as olive oil rich in monounsaturated fatty acids (mufas), or butter plus cholesterol rich in saturated fatty acids (safas). enzymatic activities were determined by fluorimetry using arylamide derivatives as substrates, to determine the effect of dietary fat on ras, vasopressin, and ultimately blood pressure regulation. experimental methods animals and treatments 17-week-old male wistar rats were purchased from harlan ibérica (barcelona, spain). the animals were allowed free access to food and water during 24 weeks and were maintained on a 12 hours light/dark cycle in a controlled temperature (20-25ºc) and humidity (50 ± 5%) environment. mean body weight was ~495 g at beginning of the study. experimental procedures for animal use and care were in accordance with european communities council directive 2010/63/ue and spanish regulation rd 53/2013. rats were randomly assigned into three groups (5-6 animals per group) as follows. in standard diet (s) group, rats were fed with a commercial diet for experimental control. in hfd groups, one group of mice (voo) was fed with a diet supplemented with 20% monounsaturated fat (virgin olive oil, voo), and the last group (bch) was fed a diet supplemented with 20% saturated fat (butter) plus 0.1% cholesterol . the hfd groups were isocaloric. food composition in different groups and nutritive value are shown in supplemental tables (appendix 1 and 2). twenty four weeks after the feeding period, a sample of blood was collected for peptidase activities assay, then the animals were sacrificed under equithensin (2ml/kg body weight) and perfused with saline solution through the left cardiac ventricle. the blood was centrifuged for 10 min at 2,000 g to obtain the plasma, which was stored at -20ºc. hypothalamus tissue was also collected and dissected as previously described10,15. briefly, the brain was quickly removed (less than 60 s) and cooled in dry ice. the hypothalamus (pooled left and right) was dissected according to the stereotaxic paxinos and watson atlas16. the selected area was between 7.7 mm and 3.7 mm anterior to the interaural line. sample preparation for enzyme activity assay samples from plasma were directly used for the peptidase activity assay. samples from the hypothalamus were quickly removed and frozen in dry ice. to obtain the soluble fraction, tissue samples were homogenized in ten volumes of hypoosmolar medium (10 mm hcl-tris buffer, ph 7.4) and ultracentrifugated (100,000 g for 30 min at 4ºc). the resulting supernatants were used to measure soluble (sol) angiotensinase and vasopressinase activities and protein content, assayed in triplicate. to solubilize membrane proteins, pellets were rehomogenized in hcl-tris buffer (ph 7.4) plus triton x-100 (1%). after centrifugation (100,000 g for 30 min at 4ºc), supernatants were used to measure membrane-bound (mb) activity and proteins, also in triplicate. to ensure the complete recovery of activity, detergent was removed from the medium by adding to the samples adsorbent polymeric biobeads sm-2 (100 mg/ml) purchased from bio-rad (richmond, va, usa) and shaking for 2 h at 4ºc17. peptidase activities assay sol and mb angiotensinase and vasopressinase activities were determined fluorometrically using the arylamide derivatives aspartyl-, glutamyl-, alanyl-, arginyl-, and cystinyl-βnaphthylamide (-β-nnap) as substrates according to the method of ramírez18. briefly, for alaap and argap assay, 10 µl of each supernatant were incubated for 30 min at 37ºc with 100 µl of substrate solution (100 µm alanylor arginyl-β-nnap), 1.5 mm albumin seric bovine (bsa), and 0.65 mm dithiothreitol (dtt) in 50 mm phosphate buffer ph 21 all res. j. biol, 2016, 7, 20-27     7.4). for aspap assay, 10 µl of each supernatant were incubated for 30 min at 37ºc with 100 µl of substrate solution (100 µm aspartyl-β-nnap), 1.5 mm albumin seric bovine (bsa), and 3·10-8 mm cacl2 in 50 mm tris-hcl buffer ph 7.4). for gluap assay, 10 µl of each supernatant were incubated for 30 min at 37ºc with 100 µl of substrate solution (100 µm α-glutamyl-β-nnap), 1.5 mm albumin seric bovine (bsa), 0.65 mm dtt, and 50 mm mncl2 in 50 mm tris-hcl buffer ph 7.4). finally, for cysap assay, 10 µl of each supernatant were incubated for 30 min at 37ºc with 100 µl of substrate solution (100 µm cystinyl-βnnap), 1.5 mm albumin seric bovine (bsa) and 0.65 mm dtt in 50 mm tris-hcl buffer ph 6.0). all the reactions were stopped by adding 100 µl of 0.1 m acetate buffer (ph 4.2). the amount of β-nnap released as a result of enzymatic activity was measured fluorometrically at 412 nm emission wavelength with 345 nm excitation wavelength. activities of specific peptidases were expressed as pmol of β-nnap hydrolyzed per minute and per milligram of protein. fluorogenic assays were linear with respect to time of hydrolysis and protein concentration. protein concentration was determined colorimetrically according to bradford method19 with bsa as standard. all chemical products were supplied by sigma (st. louis, mo usa). statistical analysis statistical analysis was performed by one-way anova followed by tukey post-hoc test for multiple comparisons. relations between variables were determined by pearson’s coefficients of correlation analysis. statistical significance was evaluated with sigmaplot v.11 software (systat software, inc., san jose, ca, usa). a p-value below 0.05 was considered to be statistically significant. all results were expressed as mean ± standard error. results results are represented in figure 1-6. in spite of the importance of mufas to maintain normal total body weight (figure 1a), plasma triglycerides (data not shown) and cholesterol levels (figure 1b) and, fundamentally, bp (figure 2), there were no significant differences in systemic angiotensinase (alaap, argap, aspap, gluap) and vasopressinase (cysap) activities in plasma (figure 3 and 4). likewise, there were no significant differences in either soluble nor membrane bound angiotensinase (figure 5) and vasopressinase (figure 6) activities in hypothalamus. therefore, the type of fat added to the diet – monounsaturated or saturated – did not seem relevant in the metabolic control of central and systemic ras and vasopressin. besides, significant correlations were not observed between fractions of angiotensinase and vasopressinase activities from hypothalamus with their homologs in plasma. additionally, the hydrolysis of amino acid naphthylamides by aminopeptidases from hypothalamus and plasma also did not show significant correlation with plasma cholesterol levels. figure 1. a. total body weight after feeding period. b. blood cholesterol ratio between total cholesterol and high density lipoprotein-cholesterol plasma fraction (hdl-c) after feeding period. a higher ratio means a higher risk of heart disease. ** (p <0.001) versus standard diet (s), # (p < 0.05) versus virgin olive oil diet (voo). figure 2. systolic blood pressure (sbp) after feeding period. * (p <0.05) versus standard diet (s), # (p < 0.05) versus virgin olive oil diet (voo). 22 all res. j. biol, 2016, 7, 20-27     figure 3. systemic angiotensinase activities. no significant differences were found in plasma (a) alaap, (b) argap, (c) aspap, and (d) gluap between dietary groups after the feeding period. figure 4. systemic vasopressinase activity. no significant differences were found in plasma cysap between dietary groups after the feeding period. discussion in this study we analyzed the potential role dietary fatty acid saturation in the control of ras and vasopressin on brainblood connection. after 24 weeks of treatment with diets supplemented with monounsaturated fat (olive oil, rich in mufas) or saturated fat (butter plus cholesterol, rich in safas), our results did not show diet-specific differences in angiotensinase and vasopressinase activities in either plasma or the hypothalamus. the two evaluated diets did not significantly change aminopeptidase (ap) activity as compared to the standard diet, and likewise there were no significant correlations between metabolic activities in the brain and blood, suggesting that the fatty acid composition of the diet might not have influence on the function of the hypothalamus-plasma connection. previous studies have also determined no changes in global ap/neuropeptidase (e.g.: aspap and gluap) activity in frontal cortex3,20 in adult male rats whose diets were supplemented with fatty acids with varying degrees of saturation, such as fish oil (rich in polyunsaturated fatty acids, pufas), olive oil (rich on monounsaturated fatty acids, mufas), and coconut oil (rich in saturated fatty acids, safas). the authors proposed that the types of lipids in the diet affect the fluidity of the membrane, with increase or loss of membrane-associated enzymes20. they observed that the diet composition affects fatty acid distribution in the brain. the change in fluidity may also affect the tertiary structure of the enzyme embedded in the membrane. this may affect the binding of the enzyme with its substrate2,20, which may explain the positive or negative correlation between fatty acid content and membrane-bound and soluble fraction of the angiontensinand vasopressin-degrading enzymes observed in the previous work; however it was not possible to clarify it in this current work. 23 all res. j. biol, 2016, 7, 20-27     figure 5. soluble (sol) and membrane-bound (mb) fractions of central angiotensinase activities. no significant differences were found in hypothalamus (a) alaap, (b) argap, (c) aspap, and (d) gluap between dietary groups after thefeeding period. figure 6. soluble (sol) and membrane-bound (mb) fractions of central vasopressinase activity. no significant differences were found in hypothalamus cysap between dietary groups after feeding period. many studies reveal that a hfd can modify bp by dysregulation of ras and their regulatory enzymes: the effect of the diet on peripheral enzymatic activity was clearly demonstrated21,22. our results showed a decrease of bp that correlated with mufas, although that physiologic response was not linked to enzyme activities in hypothalamus and plasma. other works23,24 described significant changes in their angiotensinase and vasopressinase activities in central and visceral tissues such as pituitary gland25, heart25, aorta25, adrenal gland25, kidney25, liver26, and testis27,due to fat saturation in the diet, but seemingly did not investigate enzyme activity in the hypothalamus and plasma. the bch diet was the only treatment which determined increase in body weight28, high serum triglyceride and cholesterol levels28, systolic blood pressure28, serum nitrates and nitrites28, and hepatic inducible nitric oxide (no) synthase (inos) expression28, however, these metabolic changes produced by a diet supplemented with saturated fat did not show an expected hypothalamus-plasma imbalance on the ras-vasopressin nexus. no significant correlations were observed between the ap values and lipid profile; nevertheless, previous results have suggested that cholesterol influences serum ap activities29,30, including the enzymes of study, raising the possibility that these compounds create a biochemical environment that regulates the activity of these enzymes. therefore, the present results may be partially or indirectly due to an increase in plasma total cholesterol, as a result of increasing saturation of dietary fat. other authors15,31-33 showed that an increase in aspap and gluap suggest a heightened metabolism of ang ii, which leads to an increase in ang iii formation. therefore, if both angiotensinases are modified according to the degree of saturated fat in the diet, their substrates, such as ang i and ang ii, and their metabolic products, such as ang iii and des-asp-ang i, may also be modified. consequently, their roles in the control of bp and other physiologic functions may be similarly affected. it has been demonstrated that the fat saturation of the diet also influences other enzymes, such as dipeptidyl peptidase-iv (dpp-iv)34,35 and gamma24 all res. j. biol, 2016, 7, 20-27     glutamyl transpeptidase (ggt)25,36,37. taken together, these results suggest that dietary fat saturation has a wide range of effects on various enzyme systems, despite not having been demonstrated in the hypothalamus and plasma. it is well established that the ras over-activity is connected to the hypertension produced by saturated fat. hypertension models have demonstrated a concomitant reduction of aspap and therefore reduced formation of ang 2-10, which has been suggested to counteract ang ii31. thus, the reduction of ang 2-10, together with a higher release of vasopressin due to the increased availability of ang iii, may contribute to the higher bp in l-name treated spontaneously hypertensive rats (shr)38. despite this, it is known that voo treatment of shr delays the decrease of systolic bp, and also presents with decreased levels of no and 8-isiprostatones assayed in urine21. coupled with these results, a highly significant down-regulation in aspap and gluap stimulates a higher formation of angiotensin 2-10 in the renal cortex21, as well as, a higher availability of ang ii in the renal medulla of animals fed a voo diet than in animals fed a standard diet21. our results showed that rats in the bch group, but not the voo group, had increased bp28; however, the lack of changes in hypothalamus and plasma activities in rats treated with hfd suggested no involvement of hypothalamus/plasma ang iii and ang iv and determined unaltered vasopressin. on the other hand, previous work observed hypercholesterolemia and increased body weight, as well as significantly increased serum argap22 and decreased gluap24, in rats fed with a diet supplemented with voo as compared to the standard diet. likewise, sol alaap and argap activities were increased in the brain31. these findings show that a diet supplemented with olive oil modifies certain ap activities in brain and serum, and these results may reflect functional modifications in susceptible endogenous substrates. oxytocin, together with vasopressin, is the principal substrate of cysap. the zorad group showed that obesity is associated with reduced plasma oxytocin due to increased peptide degradation by liver and adipose tissue rather than changes in hormone synthesis39. the use of polyunsaturated fatty acid, such as fish oil, demonstrates higher levels of cysap activity in mice than in those that were fed diets containing saturated oils (lard or coconut)40. our previous results also highlighted an increase mb cysap in the liver with voo26, but no evidence of changes in vasopressinase activity was found in the hypothalamus and plasma. oxytocinase/vasopressinase inhibition has been suggested as a candidate approach in the therapy of obesity39,41-43. conclusion all enzymatic activities found in hypothalamus and plasma showed no significant differences in our three experimental diet groups, which might indicate that if membrane changes occur, they could not significantly be involved in angiotensin-/vasopressin-degrading activities. therefore, we propose that the absence of correlations between these enzymes and the type of fatty acids indicates that they might not be involved in the modulation of ras and cognitive functions and, consequently, in the onset of hypertension and possible neurodegenerative disorders as causes of this disorder. finally, our results do not support the beneficial influence of virgin olive oil on central and systemic cardiovascular function, despite previous evidence that the mediterranean diet plays a major beneficial role in lowering bp. the present observation should be taken into account in strategies for the prevention of such diseases and as future diagnostic tools. acknowledgments we thank to rocío díaz-ríos (writer’s first aid) for english language revision of this manuscript. this work was supported by university of jaén through “plan de apoyo a la investigación 2006-2008”. appendix (supplemental tables) appendix 1: food composition in three different diets. chow composition ingredients (g/kg) s voo bch casein methionine 162 3 162 3 sucrose starch maltodextrin 288 160 100 288 160 100 virgin olive oil butter cholesterol 200 234 10 mineral-vitamin correcting choline chloride 43 4 43 4 fiber (cellulose) 40 40 total 1000 1000 lipid profile (%) saturate monounsaturated polyunsaturated 25 21 54 13.5 73.7 8.4 50.5 23.4 3 appendix 2: nutritive value of different diets. nutritious value of the diet (%) diet type s voo bch g kcal g kcal g kcal proteina 16.5 20 16.5 14 16.5 14 carbohydrates 60 72 55 48 55 48 fats 3 8 20 38 20 38 total energy (kcal/g) 3.410 4.740 4.740 25 all res. j. biol, 2016, 7, 20-27     references 1. haag m, dippenaar ng. 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(1997). comparative vasoconstrictor effects of angiotensin ii, iii, and iv in human isolated saphenous vein. j cardiovasc pharmacol 29: 451-456. 37. porta m, pumarega j, guarner l, malats n, solà r, real fx; pankras ii study group. (2012). relationship of hepatic and pancreatic biomarkes with the cholestatic syndrome and tumor stage in pancreatic cancer. biomarkers 17(6): 557-565. 38. villarejo ab, prieto i, segarra ab, banegas i, wangensteen r, vives f, de gasaro m, ramírezsánchez m. (2014). relationship of angiotensinase and vasopressinase activities between hypothalamus, heart, an plasma in l-name-treated wky and shr. horm metab res 46: 1-7. 39. gajdosechova l, krskova k, segarra ab, spolcova a, suski m, olszanecki r, zorad s. (2014). hypooxytocinaemia in obese zucker rats relates to oxytocin degradation in liver and adipose tissue. j endocrinol 220(3): 333-343. 40. segarra ab, arechaga g, prieto i, ramírezexpósito mj, martínez-martos jm, ramírez m, alba f, ruiz-larrea mb, ruiz-sanz ji. (2002). effects of dietary supplementation with fish oil, lard, or coconut oil on oxytocinase activity in the testis of mice. arch androl 48(3): 233-236. 41. altirriba j, pataky z, golay a, rohner-jeanrenaud f. (2015). oxytocin: metabolic effects and potential use for obesity treatment. rev med suisse 11(456457): 97-100. 42. lawson ea, marengi da, desanti rl, holmes tm, schoenfeld da, tolley cj. (2015). oxytocin reduces caloric intake in men. obesity (silver spring) 23(5): 950-956. 43. altirriba j, poher al, caillon a, arsenijevic d, veyrat-durebex c, lyautey j, dulloo a, rohnerjeanrenaud f. (2014). divergent effects of oxytocin treatment of obese diabetic mice on adiposity and diabetes. endocrinology 155(11): 4189-201. 27 microsoft word 133-786-1-le.doc do morphometric measurements allow sex discrimination in mockingbirds (mimus sp)? daniela v. fuchs*, diego montalti all res. j. biol., 2016, 7, 34-40 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 3, vol 7, 2016, 34-40 do morphometric measurements allow sex discrimination in mockingbirds (mimus sp)? daniela v. fuchs *1, 2, diego montalti 2, 3 1 centro de investigaciones científicas y transferencia de tecnología a la producción, dr. materi 50, e3105bwa-diamante, entre ríos, argentina. 2 conicet. 3 sec. ornitología, div. zoología vertebrados, museo de la plata, paseo del bosque s/n, b1900fwa-la plata, argentina. * email: danifuchs1@gmail.com abstract: sexual dimorphism in birds may be expressed as differences in body size, plumage, color and/or behavior. many species are monomorphic in color, making sex determination difficult in the field. an example of the latter are mockingbirds, which are passerines of the genus mimus, endemic to the americas. in order to distinguish between male and female mockingbirds using external body measurements that are easy to take, the objective of this work was to quantify morphometric differences between sexes in adults of the following species: m. thenca (45 specimens), m. patagonicus (95), m. saturninus (88), m. triurus (152), and m. dorsalis (7). we measured the following variables: culmen length, bill height and width, tarsus length, middle toe length, wing chord and tail length. measurements were generally larger in males than in females except for bill width in m. saturninus and m. triurus, culmen length in m. thenca and m. dorsalis, and bill height in m. dorsalis. there were significant differences between sexes in wing chord for m. patagonicus, m. saturninus and m. triurus; tail length for m. patagonicus and m. triurus; tarsus length for m. patagonicus; and in middle toe length for m. triurus. no significant differences in measurements were found between sexes for m. thenca. significant discriminant functions were obtained for m. patagonicus, m. saturninus and m. triurus, with a percentage of correct classification less than 80%. only a few variables were useful for sex determination in the studied mimus species, i.e. wing chord, tail length, middle toe length and tarsus length for three, two, one and one species, respectively. keywords: mimidae; mockingbirds; morphometrics; sexual dimorphism; southern south america. 34 all res. j. biol, 2016, 7, 34-40     introduction sex identification in animals is relevant for the understanding of many behavioral and ecological aspects1, and is particularly useful for management and conservation2. in many bird species, sex identification can be achieved by direct observation of differences in plumage, structural traits such as colored soft-tissue, behavior, or by morphometric characters3.   in passerines, sex might be determined by the presence/absence (female/male, respectively) of an incubation patch4. the external morphology cannot be used for sexing birds of monomorphic species, and differences in behavior between sexes are often restricted to the breeding season5. in these cases, the sex of birds can be determined by laparotomy6, 7, 8 or molecular genetic analysis of blood or feather samples9, 10, 11. as an alternative to avoid destructive or invasive techniques, the use of external morphometrics to sex birds is of great value, being inexpensive and immediate in sex determination12, 13. in particular, external measurements are used in discriminant function analysis (dfa)14 to distinguish the sex of numerous taxa in the field16, 17, 18, 19, 20, 15. in birds, the dfa has been effectively applied to a broad taxonomic range of species including: penguins21, 22, divers23, petrels24, 25, cormorants26, 27, vultures28, gulls29, skuas30, 31, moorhens32, rooks33, flamingos34, 11, 12, owls35 and passerines36. although external morphometric indices have been widely used in sexing birds, data of passeriformes from the neotropical region are scarce and fragmented37, 38, 39, 40, 41, 42, 36. bird species that are sexually monomorphic for plumage but dimorphic for size may have differences in wings, tarsi or body mass43, 36, 44. according to gill45, sexual differences in size among birds may have resulted from the evolution of small females. in turn, these would have been favored by sexual selection, as they can accumulate more energy reserves required for egg formation and early breeding. however, females of some species are larger than males46; a situation known as reversed sexual dimorphism (rsd)47. this is common in raptorial birds such as hawks and owls of the orders falconiformes and strigiformes, respectively, and in the families stercorariidae and fregatidae45. the family mimidae comprises 34 species of oscine songbirds, which are broadly distributed throughout the americas, from southern canada to the southern extreme of south america. these species mainly occupy scrublands and arid areas48. southwestern north america is the center of diversity and radiation of the family, from where it expanded its range to islands of the pacific and the atlantic oceans49. in temperate and subtropical south america, the only genus of the family is mimus (mockingbirds), with 6 species48. three mimus species, distributed from central to northern argentina, have partly overlapping ranges but different habitats. in central argentina, the white-banded mockingbird (m. triurus) lives in sympatry with the patagonian mockingbird (m. patagonicus), but while the former is typical of low chaco woodland and monte scrub areas, the latter prefers arid lower desert scrub and steppe. moreover, in the northeast region of argentina the whitebanded mockingbird is extensively sympatric with the chalkbrowed mockingbird (m. saturninus), which is typical of a more open, savanna-like country. the brown-backed mockingbird (m. dorsalis) occurs east of the andes in arid foothill scrub similar to that occupied by the patagonian and white-banded mockingbirds. however, they are not in contact because m. dorsalis has a more northerly range from bolivia south to the province of tucumán, in northwest argentina49. these four species together with the chilean mockingbird (m. thenca) form a monophyletic group50. these birds are territorial and sexually monomorphic in plumage51, 52, 53, 49. some passerine species are known to be sexually monomorphic in plumage but dimorphic in size, with males usually larger than females44. in this regard, deloach54 suggested that the larger size of males of the northern mockingbird mimus polyglottos is the result of evolutionary forces acting on male-male interactions or on female choice of mates. in this study, we provide external morphometric data of five mimus species. in four of these species we analyzed the differences in measurements between sexes to obtain discriminant functions that best distinguish the sexes. materials and methods measurements were made on adult males and females of five mimus species from the following museum collections: museo de la plata (la plata), fundación miguel lillo (tucumán), museo argentino de ciencias naturales (buenos aires) and museo nacional de historia natural (montevideo). we studied the following species: chilean mockingbird (23 males and 22 females), patagonian mockingbird (54 males and 41 females), chalk-browed mockingbird (41 males and 47 females), white-banded mockingbird (70 males and 82 females), and brown-backed mockingbird (2 males and 5 females). we took seven external measurements following baldwin et al.55 and considering the recommendations of winker56: 35 all res. j. biol, 2016, 7, 34-40     length of exposed culmen (cu) from the anterior end of the nostril to the tip of the bill; bill height (bh) and bill width (bw) at the base of the bill; tarsus length (ts) from the notch on the back of the intertarsal joint to the ventral surface of the foot with toes extended; middle toe length (mt); wing chord (wc) from the distal portion of the carpus to the tip of the longest primary feather; and tail length (ta) from the base of the tail to the tip of the longest rectrix. the first five measurements were made using a caliper accurate to 0.01 mm and the last two with a metal ruler to the nearest 1 mm. to avoid bias all measurements were taken by the same person (d.m.). in addition, all measurements were made on the right side of each bird because some species show bilateral asymmetry (one side of the body is larger than the other)57. we recorded the sex and age of each specimen from the museum tag.  since specimens were prepared to enter the ornithological collections, sex determination was made at the time of this procedure, through direct anatomical examination. the dimorphism index (di58) was calculated for all the variables. the following formula was used: di = (a/b)*100 where: a = mean value of variable “a” in females – mean value of variable “a” in males. b = (mean value of variable “a” in males/2) + (mean value of variable “a” in females/2) all variables were measured in millimeters. a positive index value indicates that the female is larger than the male and a negative value indicates the opposite. in addition, we used the student’s t-test to evaluate differences in body measurements between males and females59 in four species (m. dorsalis was excluded from the analysis due to the small sample size). normality and homoscedasticity were tested using the shapiro-wilk’s test and levene’s test, respectively14, 59. discriminant function analysis (dfa) was used to develop the classification functions for sex assessment in mockingbirds60. m. dorsalis was excluded as described above.   discriminant functions were performed with all possible combinations of all measured variables. we also evaluated the performance of each single-variable as discriminant (univariate discriminant analysis). forward discriminant analyses were applied to obtain combinations of characteristics (discriminant functions) that best distinguished the sexes. the associated cutting point value was calculated following phillips and furness61. the effectiveness of the discriminant analyses was checked in terms of the proportion of birds that were classified correctly and by a jackknifed validation62, 63, 61. results the student’s t-test indicated that the measurements of male mockingbirds were, in general, larger than those of females (table 1), except for the following variables: bill width for m. saturninus and m. triurus, culmen length for m. thenca and m. dorsalis, and bill height for m. dorsalis. table 1. morphometric results of the five species of mockingbirds (mimus spp.) presented as mean ± standard deviation (sd) and range. significant differences between sexes (student’s ttest) are indicated in bold. ns: p>0.05, *: p<0.05, **: p<0.01. mean ± sd range n mean ± sd range n t p m . thenca culmen length 17.78±0.86 16.66-19.30 21 17.82±0.84 16.55-20.22 22 0.13 ns bill height 6.20±0.34 5.34-6.70 22 6.17±0.36 5.68-7.21 22 -0.24 ns bill width 5.87±0.54 4.82-6.6 22 5.84±0.40 5.12-6.75 22 -0.22 ns tarsus length 38.73±1.03 36.98-41.71 22 38.13±1.67 34.88-41.30 21 -1.39 ns middle toe length 23.35±1.14 21.82-25.46 11 22.88±0.85 21.76-23.91 10 -0.86 ns wing chord 118.13±5.44 110.00-129.00 23 117.14±4.71 110.00-127.00 22 0.03 ns tail length 122.87±7.40 111.00-138.00 23 120.41±7.38 100.00-134.00 22 -1.12 ns m . patagonicus culmen length 16.56±1.24 14.30-20.25 52 16.19±1.47 13.70-20.00 39 -1.29 ns bill height 5.86±0.46 5.20-7.30 52 5.69±0.42 5.10-6.80 39 -1.77 ns bill width 5.59±0.38 4.80-6.50 54 5.48±0.34 4.80-6.30 40 -1.41 ns tarsus length 35.61±1.70 29.10-38.00 54 34.60±1.54 31.30-38.80 40 -2.94 ** middle toe length 19.62±1.05 18.24-22.50 31 18.99±1.59 13.70-21.80 26 -1.40 ns wing chord 109.59±4.61 99.00-121.00 54 104.58±4.14 97.00-112.00 41 -5.47 ** tail length 106.43±6.22 92.00-121.00 54 100.12±5.85 90.00-110.00 40 -4.98 ** m . saturninus culmen length 18.25±1.48 15.40-21.40 37 18.17±1.55 15.50-23.00 41 -0.21 ns bill height 6.69±0.41 5.82-7.50 37 6.62±0.50 5.50-7.90 43 -0.66 ns bill width 6.16±0.57 5.50-7.95 39 6.28±0.43 5.22-7.30 44 1.60 ns tarsus length 36.81±1.97 33.21-41.20 39 36.57±2.23 32.62-41.50 47 -0.53 ns middle toe length 21.55±2.28 17.10-26.80 13 20.12±2.12 16.30-24.60 20 -1.83 ns wing chord 119.28±4.68 110.00130.00 40 115.17±5.28 104.00125.00 46 -3.79 ** tail length 120.71±5.85 109.00-135.10 34 117.74±7.90 85.00-133.00 39 -1.53 ns m . triurus culmen length 15.55±1.00 14.00-20.90 80 15.25±0.99 13.10-17.90 67 -1.51 ns bill height 5.97±0.33 5.10-6.70 75 5.90±0.32 5.00-6.70 65 -1.24 ns bill width 5.62±0.40 4.45-6.50 82 5.67±0.37 4.61-6.50 69 0.46 ns tarsus length 32.65±1.25 29.60-36.87 82 32.22±1.58 26.60-36.20 70 -1.81 ns middle toe length 19.00±0.76 17.40-21.66 60 18.66±0.80 16.50-21.09 54 -2.33 * wing chord 107.08±3.72 99.00-117.00 81 102.98±4.00 94.00-115.00 70 -7.54 ** tail length 110.11±5.60 85.00-129.00 77 104.98±5.02 93.00-115.00 66 -5.74 ** m . dorsalis culmen length 20.45±0.59 20.04-20.87 2 20.74±1.24 18.60-21.68 5 bill height 6.30±0.06 6.26-6.35 2 6.35±0.30 6.10-6.80 5 bill width 5.98±0.21 5.83-6.13 2 5.86±0.53 5.30-6.65 5 tarsus length 37.28±2.01 35.85-38.70 2 37.05±2.03 35.36-40.21 5 middle toe length 20.22±0.51 19.86-20.58 2 19.23±1.05 18.14-20.30 5 wing chord 118.00±2.83 116.00-120.00 2 117.80±5.02 113.00-125.00 5 tail length 122.00±5.66 118.00-126.00 2 115.60±4.04 111.00-120.00 5 species morphometric characters males females these results were consistent with the negative values obtained from the analysis of the di (table 2). table 2. dimorphism index (di) for body measurements of five species of mockingbirds (mimus spp.). the di is positive if the female is larger and negative if the male is larger. species culmen length bill height bill width tarsus lengthmiddle toe lengthwing chord tail length m. thenca 0.22 -0.48 -0.51 -1.56 -2.03 -0.84 -2.02 m. patagonicus -2.26 -2.94 -1.99 -2.88 -3.26 -4.68 -6.11 m. saturninus -0.44 -1.05 1.93 -0.65 -6.86 -3.51 -2.49 m. triurus -1.95 -1.18 0.89 -1.33 -1.81 -3.9 -4.77 m. dorsalis 1.41 0.79 -2.03 -0.62 -5.02 -0.17 -5.39 dimorphism index 36 all res. j. biol, 2016, 7, 34-40     the comparison between males and females yielded significant differences in wing chord and tail length for m. triurus and m. patagonicus, in tarsus length for m. patagonicus, in wing chord for m. saturninus, and in middle toe length for m. triurus (table 1). no significant differences in any of the studied variables were observed between sexes for m. thenca. the di values of the five mimus species studied ranged between -6.86 and 1.93, indicating low-tomoderate sexual dimorphism (table 2). discriminant function analysis using all variables resulted in a significant discriminant function equation for m. patagonicus and m. triurus. the significant discriminant function for m. patagonicus, m. saturninus and m. triurus included the following predictors: culmen length, tarsus length and wing chord. the dfa using the variables that had statistical significance with the student's t-test resulted in a discriminant function for m. patagonicus (tarsus length, wing chord and tail length) and m. triurus (middle toe length, wing chord and tail length). in all cases, the percentage of correct classification was less than 80% (table 3). the jackknifed validation provided the same classifications as those produced by the discriminant functions. table 3. significant classification functions generated by discriminant function analysis for 4 species of mockingbirds, and percentage of correct classification. cu: culmen length, bh: bill height, bw: bill width, ts: tarsus length, mt: middle toe length, wc: wing chord, ta: tail length. ns: p>0.05, *: p<0.05, **: p<0.01. species function cutting point total male female wilks’ λ f df1, df2 p m . patagonicus d1 = 0.302 cu 1.022 bh + 0.234 bw 0.510 ts + 0.131 mt 0.056 wc 0.066 ta + 28.146 0.13 78.0 82.8 71.4 0.70 2.55 7, 42 * d2 = -0.101 cu + 0.168 ts + 0.204 wc -26.164 -0.08 75.6 82.7 65.8 0.74 9.54 3, 85 ** d3 = 0.101 ts 0.137 wc 0.069 ta + 25.497 0.97 76.3 85.2 64.1 0.73 10.69 3, 89 ** m. saturninus d1 = 0.325 cu 0.012 ts + 0.229 wc 20.570 0.006 67.6 64.7 70.0 0.81 5.61 3, 70 ** m . triurus d1 = 0.146 cu 0.711 bh + 0.722 bw + 0.273 ts 0.252 mt 0.254 wc 0.009 ta + 25.68 1.04 75.0 72.5 77.8 0.61 7.49 7, 86 ** d2 = 0.151 cu 0.040 ts + 0.259 wc 28.163 -21.64 76.7 79.7 73.1 0.72 17.84 3, 141 ** d3 = 0.116 mt 0.231 wc 0.024 ta + 29.064 0.034 76.63 76.78 76.47 0.65 18.38 3, 103 ** correct classification % discussion in this study, we investigated whether the sex of five mimid species can be determined by external morphological characters. our results show that measurements were larger for male than female mockingbirds, except for bill width in m. saturninus and m. triurus, culmen length in m. thenca and m. dorsalis, and bill height in m. dorsalis. montalti et al.36, who studied other sexually monochromatic neotropical passeriformes, found significant differences between sexes in wing chord for the brown-and-yellow marshbird pseudoleistes virescens; middle toe length for the great kiskadee pitangus sulphuratus; tarsus length for the freckle-breasted thornbird phacellodomus striaticollis and p. virescens; tail length for the rufous-collared sparrow zonotrichia capensis and culmen length for p. virescens. moreover, the culmen of rufous horneros furnarius rufus, p. sulphuratus, and the tropical kingbird tyrannus melancholicus was longer in females than in males. in our study, the dimorphism indices for the five mimus species revealed low-to-moderate sexual dimorphism in most of the analyzed variables. we found sexual dimorphism in tarsus length, middle toe length, wing chord and tail length depending on the species, while m. thenca was sexually monomorphic for all the studied characters. deloach54 found that males of m. polyglottos showed significantly higher differences in wing chord and weight as compared with females. this author suggested that differences between sexes may have resulted from selective pressure driven by male-male interactions in the context of territorial competition or by female choice of larger mates. our results are similar to those found by deloach54, since we noted that males have larger wings than females in m. patagonicus, m. saturninus, and m. triurus. although the dfa produced significant discriminant functions separating males from females based on the morphometry of some mockingbird species, the percentages of correct classification were low thus indicating that the measurements used are not reliable enough for sexing these birds using this technique. however, we found significant differences in some measurements between sexes for three of the studied species. acknowledgments we thank the curatorial staff of fundación miguel lillo tucumán; museo argentino de ciencias naturales, buenos aires; museo de la plata, la plata argentina, and museo nacional de historia natural, montevideo, uruguay. we also would like to thank silvana finocchiaro; and the editor and two anonymous reviewers for their constructive comments, which helped us to improve the manuscript. references 1. ruckstuhl, k.e., and clutton-brock, t.h. 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(1996). using multivariate statistics. 3rd edition. harper collins publishers, new york, usa. 40 microsoft word template_authors_arjournals_biol-1.doc challenge towards plant recombinant protein expression: instability in nuclear and chloroplast transformation   mahshid amiri, mokhtar jalali-javaran*, parastoo ehsani, raheem haddad all res. j. biol., 2016, 7, 13-19 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 1, vol 7, 2016, 13-19 challenge towards plant recombinant protein expression: instability in nuclear and chloroplast transformation   mahshid amiri1, mokhtar jalali-javaran*1, parastoo ehsani2, raheem haddad3 1  department of plant breeding & biotechnology, faculty of agriculture, tarbiat modares university(tmu), tehran, iran; molecular biology department, pasteur institute of iran, tehran, iran department of agricultural biotechnology, imam khomeini international university, qazvin, iran corresponding author: mokhtar jalali-javaran: department of plant breeding & biotechnology, faculty of agriculture, tarbiat modares university(tmu), tehran, iran; jalali.mokhtar@gmail.com,tel: 098-2148292104       grafical abstract         abstract: it is crucial to maintain the stability of transgene and its expression level. it seems the transformation method and the target organ can influence this instability. to this aim, two transformation systems, agrobacterium-mediated and particle bombardment systems which have been applied to introduce tissue plasminogen activator (tpa) into nuclear and chloroplast respectively, have been compared to determine transformation efficiency, tpa expression, and stability. the presence of the tpa gene in transformants has been confirmed by pcr analysis. the gene expression in nuclear transformants and homoplasmy in transplastomic plants have been assayed by elisa and southern blot analyses, respectively. some of the agrobacteriumderived transformants have shown the heritability and stability of the integrated t-dna harboring the transgene, which encodes the tissue plasminogen activator and instability of its expression in the t1 generation. using southern blot analysis of bombardment-mediated transformants has surprisingly led to detecting the inheritability of tpa. there are several factors lead to the silencing of in transgenic plants, which should be considered. possible reasons for these silencing are likely vector designing, methylation, copy number, and genome rearrangement. keywords: recombinant protein, tissue plasminogen activator, agrobacterum, particle bombardment, instability   introduction the pharmaceutical industry has needed the production of recombinant proteins in sufficient quantity in order to keep up with demands 1, 2. there are other factors, besides the quantity, which can be obtained by recombinant technology such as increased quality, enhanced safety, and decreased cost 3. some living cells have been engineered as heterologous expression platforms to produce recombinant protein, including bacteria, animals, and plants 4. higher gene transformation system agrobacteriummediated particle bombardment t1 generation i n s t a b i l i t y t1 generation vector designing methylation copy number genome rearrangement 13 all res. j.biol, 2016, 7, 13-19     plants offer some advantages 4 which emphasize the practicality of utilizing them as green factories to express useful recombinant protein 5: lowering costs of production, no human pathogens, synthesizing protein with correct folding, and post-translational modification like glycosylation 6.   considering these advantages, up to date, several biopharmaceutical proteins have been produced in the plants 7, so, plant molecular farming has impacted positively on the important pharmaceuticals 8. plant molecular farming has enrolled genetically engineered plants as vehicles for expression of recombinant protein and provided an attractive perspective to produce these important proteins in a large scale at low costs 9.   this plant engineering refers to the introduction and integration of "interested" dna in plant cells, which can lead to transient or stable expression of interested dna 10. through either nuclear or plastid genomes, stabletransformed plants can be obtained 11, although, chloroplast transformation offers several advantages in comparison with nuclear transformation such as minimizing or avoiding the gene escape, eliminating the position effect, and higher expression level 12. as a consequence of stable transformation, the interested dna is integrated into the host dna and eligibly predicted to be passed on to the next generation 10. after about two decades, in which molecular farming is coming of age 7, the concentration has moved away from technical and principal studies towards a serious attention of necessity for sustainable production of recombinant protein 9.   in fact, it can be remarked that the stability is as important as the expression level of transgene for the large-scale commercialization of transformants 13, it is clear that the expression and stability are not guaranteed over generations 14. transgene instability can be defined as the loss of a transgene or its expression in genetically engineered organisms15; methylation, genome rearrangement, and the site of insertion found to be responsible for the phenomenon 16. the instability has been reported in both biolistic bombardment and agrobacterium mediation 17.   tissue plasminogen activator (tpa), a serine protease, hydrolyses the plasminogen to convert it to plasmin 18. tpa and plasminogen bind to the fibrinogen and fibrin to modulate proteolytic activity, enable the dissolution of blood clots. it has been found that it is useful to treat myocardial infarction, thrombosis, and stroke 19. it should be noted that there is a single-chain nonglycosylated form of tpa called reteplase, with a longer plasma half-life, better diffusion, and higher fibrinolytic activity 20. as utilizing tpa one hour after heart attack can lead to increasing the survival chance, there has been an interest to produce tpa in large scale 21. attempts have been made to produce the protein in several expression systems like saccharomyces cerevisiae 22, aspergillus nidulans 23, escherichia coli 24, chinese hamster ovary (cho) cells 25, leishmania 26, bowes melanoma cell line 27, mammalian cell lines 28, 29,and insect cells 30, however the aforementioned disadvantages lead to recent interests in plants. it has been expressed in tobacco 21, 31, 32 and oriental melon 33.   in this study, we compared the stability and inheritance of two forms of transgene in transformants transformed by different methods (agrobacterium-mediated transformation and particle bombardment technique) in different organs (nuclear and chloroplast, respectively). in fact, transgenic plants were investigated to determine the transgene and expression stability over generations because the probability of instability/ transgene silencing, through generations, increases 34. our attempts were made to find the stable transgenic plants and discuss the stability and expression level of transgene.     methods and materials   seed culture   two groups of transgenic plants were considered to assay. nicotiana tabacum cv. xhanti were transformed by the agrobacterium-mediated method and particle bombardment technique.   nuclear primary transformants (t0) created through agrobacterium-mediated transformation harboring pbit-pa construct containing the kozak sequence before the start codon and a kdel sequence before the stop codon, nptii gene was controlled by 35s promoter of the cauliflower mosaic virus (camv) and nos terminator of the agrobacterium tumefaciens 32. the seeds of t0 were cultivated to obtain t1 seeds. pkczk2s carrying k2s and aada gene controlled by prrn as the rrna operon promoter and rbcl3ʹ′chl of the chlamydomonas rbcl gene as a terminator was applied to make chloroplast transgenic plants. after several selection rounds and four rounds of regeneration, plants were allowed to produce t1 seeds 21. all seeds were grown in the pots containing a homogeneous mixture of perlite and peat moss (1:3 ratio of perlite:peat moss).   pcr analysis   genomic dna was extracted from fresh leaves of transgenic and non-transgenic plants utilizing the ctab method 35 and used as the template in the pcr assays. pcr was conducted to confirm the presence of both tpa and k2s genes (a truncated form of tpa) in the nuclear and chloroplast genome, respectively. two primer sets were used on each dna template: (1) one to amplify a 1.7 kb tpa (forward: 5ʹ′ gagtctagataaacaatggatgcaatgaagagagg g-3ʹ′; reverse: 5ʹ′ atagtcaactcatagctcatctttcggtcgcatgtt g-3ʹ′) and (2) another to amplify a 1.2kb k2s (forward: 5´ggaaacagtgactgctactttgggaatgg-3´ and revers 5´tcacggtcgcatgttgtcacgaatccag3´).   southern blot   14 all res. j.biol, 2016, 7, 13-19     to confirm the homoplasmy, a southern blot analysis was conducted on chloroplast transgenic plants. high-quality genomic dna was isolated from the fresh leaves of t1 pcrpositive plants by the ctab method 36. high quality genomic dna of transgenic and non-transgenic plants were digested by hindiii, fractionated at 20v through a 1% agarose gel for 16 h, and transferred onto nitrocellulose membranes (biorad, usa) utilizing a traditional wet system. fragments designed based on a flank region in the chloroplast genome, specifically amplified using the digdna labeling and detection kit (roche, germany) and the primer set (p-forward 5´atgtgtaatgattcccccattc-3´ and p-reverse 5´cttctctcccacttcacacctc-3´), were utilized as hybridization probes, producing a 1kb size fragment in nontransgenic(non-transplastomic) plants and a 2.8 kb in transgenic plants (transplastomic) along with a positive control(pkczk2s vector). based on the manufacturer’s protocol, the probe was hybridized and resolved on the membrane.   elisa   the level of specific tpa in leaves of t1 pcr-positive transgenic plants was estimated by enzyme-linked immunosorbent assay (elisa) on protein extracted just before the assay. tpa was assayed by an indirect elisa procedure as previously described 21. result   analysis of transformed plants by pcr   pcr was carried out with two primers sets to confirm the presence of recombinant tpa gene in transformants. the presence of 1.7 kb in some nuclear transformed plants, about 20 plants out of 200 plants, has showed the inherited integration of the transgene in these plants (fig.1). dna from some chloroplast transformed plants has represented the expected size of amplified product to be 1.2 kb with k2sgene-specific primers and some of them have shown no band.(fig.2). untransformed plants (negative control) have shown no pcr product.   figure  1 pcr  amplification  of  a  1.7  kb  fragment.  m:  1  kb  molecular  weight   marker   (fermentas),   c-­‐:   negative   control,   c+:   positive   control   (pbit-­‐pa   vector   contain   tpa   gene),   wt:   non-­‐transformed   plant,   1-­‐6:   transformed   plants.     figure  2 pcr  amplification  of  a  1.2  kb  fragment.  m:  1  kb  molecular  weight   marker   (fermentas),   c-­‐:   negative   control,   c+:   positive   control   (pkczk2s   vector  contain  k2s  gene),  wt:  non-­‐transplastomic  plant,  1-­‐5:  transplastomic   plants.   southern blot analysis   after confirming the presence of interested gene k2s in the chloroplast transformed plants by pcr, it was subjected to the southern blot analysis. as it was expected to observe just a 2.8 kb band on the membrane revealing homoplastic plants and a 1kb size band on the me mbrane indicating a heteroplasmic plant, we just observed the 1kb size band in all of our t1 pcr-positive plants (fig.3). our analyses which represented the heteroplasmic plants were not suitable for more analysis like elisa.   elisa   an elisa was conducted to quantify protein by comparing the absorbance readings of the three replicates of t1 pcrpositive plants of nuclear transformation with known quantities of the commercial tpa protein (alteplase). after estimating, it was found there was not any difference between transgenic and non-transgenic plants in tpa protein content (data was not shown).   discussion   as it can be observed in the fig.1 and fig.2 the positive-pcr transgenic plants have been obtained in both nuclear and chloroplast transformations, about 10% plants in nuclear and, surprisingly, some chloroplast transformants. considering the advantages of chloroplast genetic engineering over nuclear ones, which was mentioned above; due to the lack of gene silencing 37; it is assumed to see all chloroplast transformants represent the expected 1.2 kb band. so, by this experiment, we can observe the genomic instability of chloroplast transgenic plants causing the loss of the transgene. to assess the homoplasty, the southern blot was conducted on the positive-pcr transgenic plants transformed by particle bombardment. plants show only the expected size for the wild-type plastom band while no integrated transgene bands can be observed. it seems this lack of the 2.8 kb transgene band is precise and needs to be considered.   it can be inferred this phenomenon is the consequence of the intramolecular recombination event resulting in keeping the wild-type plastome copies during the chloroplast segregation procedure 38. to find out more about this phenomenon, a detailed study has been made on the cloning procedure and 15 all res. j.biol, 2016, 7, 13-19     the vector structure. pkczk2s vector was used to create transgenic plants producing tpa. the problem faced in plants transformed by pkcz has been the same in their next generation 38–40; plants have never met the genetically uniform lines, and comes back to the intramolecular recombination via the repeated sequence element, especially regulatory elements controlling foreign gene expression. as it is visible in the zou (2003) study, there are three copies of plastid promoter prrn governing both selection markers (aada) and reporter gene(uida), two prrn copies are oriented as the direct repeats; specific to transplastomic aada and endogenous rrn16 (16s rrna) genes. the result of intramolecular recombination of these direct repeats of prrn leads to the removal of ~10kb including all rrna (5s, 4.5s, 23s and 16s), the whole uida cassette, and several trna genes and keeps the aada cassette resulting in aberrant transplastomes  which are spectinomycin resistant.   figure   3 sothern   blot   analysis   of   transplastomic   t1   plants   with   hind   iii   enzyme.   m:   1   kb   molecular   weight   marker   (fermentas),   wt:   non-­‐ transplastomic  plant,  c+:  positive  control  (pkcz  vector  contain  k2s  gene),  1-­‐ 5:  transplastomic  plants.   abdoli-nasab et al (2013) decided to add the tpa cassette before aada one to overcome this problem, so, in their opinion, transplastomes could express both tpa and aada simultaneously in spectinomycin resistant plants. they remained unaware that the removal of all rrna and several trna genes were lethal for transplastomes, however aada was expressed resulting in the resistance to the spectinomycin. in their analysis, on the one hand, they did not address the issue as they analyzed transformants in the early stages of growth in t0 generation. the average of transgene loss was estimated about 5%, but it was a continuous process and did not stop until all intramolecular recombination via the direct repeats happened, resulting in all rrna and several trna genes to be removed 41. on the other hand, the intramolecular recombination procedure under selection stress seemed to be a slow process, but accelerated in the absence of antibiotics like growing in the soil, so, it seemed to be a plausible interpretation as to why we met the loss of transgene in the t1.   intramolecular recombination via direct repeats is not always a potentially lethal adverse phenomenon in transformation; this strategy has been helpfully used to create antibiotic marker-free transplastomic plants, 41 while one of the direct repeats has been left in the chloroplast genome after the recombination.   to applicate transformation technologies commercially, it is extremely important that transformants express the recombinant protein over generations 42. the evaluation of genetic instability seems to be obvious via a loss or sudden change in the phenotype of agronomically engineered plants 43 while more studies need to be considered in nonagronomically engineered ones. in our study, as shown in fig.1, we have found 20 plants out of 200 plants have shown positive in pcr. these pcr-positive plants have not represented any difference among transgenic and nontransgenic plants in tpa protein content.   the transgene instability can increase over generations especially when it advances to stabilize plants in the homozygous level 44.the instability of transgene inheritance, in the form of transgene lost or rearrangement, has been reported in the peg-mediated direct gene transfer, bilolistic bombardment or agrobacterium mediation 17. although agrobacterium-mediated transformation shows less rearrangement, co-suppression and instability in subsequent generations in comparison with bombardment transformation 44. there are several reasons which should be considered as the answer for this event; several factors are generally accepted to be responsible for transgene expression instability such as methylation, copy number, genome rearrangement, integration site in genome, and endogenous gene homology to the transgene 16.   it is crucial to produce single-copy number inserted transformants, because it is known that the loss of transgene expression and the expected phenotype can also result from multiple transgene copy numbers 45, 46. in addition, the tandem copies of t-dna are often transferred at a single locus in the agrobacterium-mediated transformation and it is found that the t-dna repeats which stand inverted and headto-head around the right border are often associated with transgene silencing 47.   epigenetic interactions as another potential source of transgene instability have been known as gene silencing system in plants 48, resulting from the interaction among multiple transgene copy number or an endogenous gene homology to the transgene leads to the homology dependent gene silencing (hdgs). the mechanism has been unclear, although some theoretical hypothesis have put forward that hdgs may be observed either through the transcription repression, called transcriptional gene silencing (tgs), or through mrna degradation, called posttranscriptional gene silencing (ptgs) 49. tgs is characterized as heavy methylation to be the mechanism of the silenced promoter and is meiotically heritable 50, it can be interpreted that the chromatin environment of the transgene is epigenetically modified in a way that can be heritable, most likely before or during gametogenesis. in fact, promoter regions in the repeated transgenes are often methylated, these repeated transgenes cause the methylation of homologous regions placed in trans; also transcription of aberrant promoters shows promoter methylation. heavy methylation can be seen 16 all res. j.biol, 2016, 7, 13-19     when prokaryotic vector sequences integrate into the plant genome, the excess vector sequences cannot be well tolerated by the eukaryotic genome and are often spontaneously resulted in heavy methylation that can extend into neighboring transgenes 49 it is possible these sequences have unusual compositions to bind the eukaryotic nuclear proteins, which subsequently are susceptible to plant methyltransferases 49 and to a conversion to different epigenetic states 50. ptgs is not meiotically inheritable and should be reinstituted in each gametogenesis. it leads to methylation of an increased amount of dna within the protein-coding region, resulting in the appearance of specific low molecular weight fragments of rna 49.   studies reported that the heritable instability could occur in the t2 to t4 generations, although travella et al (2005) reported that there was some evidence in which transgene instability occasionally could be appeared in the t1 population which contained the single-copy number of interested gene, as mentioned above the extra integrated vector sequence led to gene silencing, however it was recently reported that this extra vector sequence might influence transgene expression 51. sometimes the meiotic instability results in the loss of t-dna partially or completely, this instability frequently has been reported to be low for agrobacterium-mediated transformants with singlecopy inserts 17.   considering the aftermentioned reasons for the problem which we have met in the agrobacterium-mediated transformants in the t1 generation, according to scott et al (1998), it seems it is early to discuss the definite answer for this event now and more experiments require to be assayed. using scaffold attachment regions (sar) can be useful in nuclear stable transformation, as sars can be attached to the proteins of the nuclear scaffold and can lead to the organization of the chromatin into loop exposure to transcription machinery. so, flanking the transgenes with sar elements have been shown to contribute to the reduction of position effects and silencing of transgene expression 52– 54.     references     1 joshi, l., and lopez, l.c. 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(2002). matrix attachment regions (mars) enhance transformation frequencies and reduce variance of transgene expression in barley. plant mol. biol 49, 45– 58. 19 microsoft word corrected proofs prof van noorden 24 apr.docx publication bias in laboratory animal research by non-publication of “negative” results cornelis j.f. van noorden all res. j. biol., 2013, 4, 1 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com editorial                                                                                                                    issue 1, vol 4, 2013, 1 editorial: publication bias in laboratory animal research by nonpublication of “negative” results cornelis j.f. van noorden department of cell biology and histology, academic medical center, university of amsterdam, the netherlands.  e-mail: c.j.vannoorden@amc.uva.nl an interesting publication for the readership of the all results journal appeared recently, focused on publication bias in laboratory animal research. the article highlights the preference to publish results that show effects of treatment over publication of lack of effects of treatment. 1 the authors do not have hard evidence for the preference to publish effects rather than the lack of effects but show indirect evidence for this phenomenon by interviewing all known laboratory animal researchers in the netherlands in an internetbased survey. in the survey, a total of 454 researchers responded, which is 14-24% of the estimated number of laboratory animal researchers in the netherlands. researchers in for-profit organizations estimated that approximately 10% of all animal research is published, whereas researchers in non-for-profit organizations estimated that 80% was published. a major reason for non-publication was considered to be the lack of statistical significance between experimental animal groups. it is evident that such preference for publication of significantly-different or positive results creates publication bias. this bias, by non-publication of experiments that show lack of significant effects in treatment groups of laboratory animals, is unethical since it deprives researchers of the accurate data they need to estimate the potential of novel therapies in clinical trials, but also because the included animals are wasted as they do not contribute to accumulating knowledge. in addition, research that overstates effects may lead to further unnecessary animal experiments testing poorly founded hypotheses. 2 the authors recommend a number of measures to be taken to prevent non-publication of negative results, such as control by the institutional animal care and use committees or the submission of manuscripts for publication without any results as suggested previously.3 besides, the all results journals, the journal of negative results in biomedicine as well as specific sections for negative results publications in the journal of cerebral blood flow and metabolism, neurobiology of aging and gynaecological oncology were explicitly mentioned to minimize this type of publication bias. as section editor of the all results journals:biol, i am pleased with publications like ter riet et al.,1 of which i am a co-author, so i must warn the readership that i am not strictly objective. nevertheless, i am allowed to point at the publication as a stimulus to publish laboratory animal research studies that did not produce any statistically significant differences between treatment groups when the studies are properly designed. the all results journals are an excellent medium for the publication of these studies besides reducing publication bias. references 1. ter riet, g., korevaar d.a., leenaars, m., sterk, p.j., van noorden, c.j.f., bouter, l.m., lutter, r., oude elferink, r.p., hooft, l. (2012). publication bias in laboratory animal research: a survey on magnitude, drivers, consequences and potential solutions. plos one 7, e43404. 2. sena, e.s., van der worp, h.b., bath, p.m., howells, d.w., macleod, m.r. (2010). publication bias in reports of animal stroke studies leads to major overstatement of efficacy. plos biol. 8, e1000343. 10.1371/journal.pbio. 3. greenland, s. (2007). commentary: on ‘quality in epidemiological research: should we be submitting papers before we have the results and submitting more hypothesis generating research?’ int. j. epidemiol. 36, 944-945. 1 effects of thinning, burning, seeding, and slash arrangements on understory communities in pinyon-juniper woodlands of northern arizona maria r. irwin, alex j. finkral * and john d. bailey all res. j. biol., 2011, 2, 8-15 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol. 2, 2011, 8-15 8 effects of thinning, burning, seeding, and slash arrangements on understory communities in pinyon-juniper woodlands of northern arizona maria r. irwin 1 , alex j. finkral 2*, and john d. bailey 3 authors are 1 forest ecologist, ecosphere environmental services, durango, co 81301, usa; 2assistant professor, school of forestry, northern arizona university, flagstaff, az 86001 usa; 3associate professor, department of forest resources, oregon state university, corvallis, or 97331, usa. *correspondence: alex j. finkral, school of forestry, northern arizona university, flagstaff, az 86011, usa. email: alex.finkral@nau.edu. phone: (928)-523-1378 grafical abstract abstract: pinyon-juniper woodlands are a dominant ecosystem in the american southwest that have been increasing in density over the last century, generating concerns about the effects on wildlife habitat, livestock forage, and wildfire risk. we tested 16 treatment combinations designed to restore stands to historic conditions by examining the impact on understory plant richness and abundance. we thinned three sites comprised of different parent soil materials: limestone, sandstone, and basalt. each site had four slash arrangements: piled, broadcast, clustered, or no thinning. each of these arrangements received a different burning/seeding treatment: prescribed fire, seeding, prescribed fire and seeding, or none. this study corresponded with the driest period in the last 55 years, and plant species richness decreased by an average of 40% from the previous year in the control plots. richness was significantly different due to slash arrangement at the basalt site only. burning or seeding did not affect richness at any of the sites. plant species abundance was generally low and not influenced by treatment or site. this study demonstrates that extensive ecosystem manipulation in the pinyon-juniper woodlands of northern arizona did not affect understory richness or abundance the first year after treatment during a drought. keywords: piñon, restoration, prescribed fire, understory response, silviculture all res. j. biol, 2011, 2, 8-15 9 introduction pinyon-juniper woodlands occupy almost 30 million hectares in the western united states and are one of the largest ecosystems in the american southwest. 1 pinyon-juniper woodlands are usually found at elevations of 1,370 to 2,290 m, and are the most xeric forest type in the us with precipitation averaging 25-40 cm a year. 2 many studies have documented the expansion and contraction of pinyon-juniper woodlands over thousands of years, due to long term changes in climate. 3,4,5 in the last 150 years many pinyon-juniper areas have expanded their geographic extent and/or increased in density. 6,7,8,9,10 this period coincides with euro-american settlement in many areas, when livestock grazing, climatic changes, and fire suppression were introduced to pinyonjuniper forests. 11 the current expansion and densification of pinyon-juniper woodlands is generally considered an undesirable trend for land managers, who thin or remove pinyon-juniper woodlands for wildlife habitat improvements, increased forage for livestock, and fuels mitigation. many managers remove all pinyon and juniper trees by chaining (dragging a heavy chain between two bulldozers to knock over the trees) or mastication. 12 few studies have used historic reference conditions to guide thinning in pinyon-juniper woodlands. 13, 14 the inverse relationship between overstory and understory cover in pinyon-juniper woodlands has been documented. 15,16,17,18,19 increasing understory diversity and abundance has become a goal for many land managers. techniques for increasing understory health have included thinning, slash additions to bare soil, prescribed burning, and seeding. 13,20,21,22 leaving the slash created by thinning on the ground may create favorable microsites for understory establishment. slash amendments to the soil significantly increased residual woody and litter debris, reduced soil movement, and increased arbuscular mycorrhizal fungi and microbial carbon levels. 22 brockway et al. found that plant species richness and diversity increased most on sites where slash was either completely removed or scattered to serve as mulch and that understory biomass increased for all harvest treatments. 21 jacobs and gatewood determined that overstory reduction and slash mulching treatments produced two to sevenfold increases in herbaceous cover relative to controls. 13 it remains an open question whether slash additions to bare soil alone, without the confounding factors of thinning, increase understory diversity and cover. the use of prescribed fire has had limited applications in pinyon-juniper woodlands because of the difficulty of burning 23 and uncertainty about the historic fire regimes. 24 in many areas, only extremely dry and windy conditions will carry a fire through the canopy, resulting in a high severity, stand replacing fire. 25,26 prescribed fire success depends on stand structure, weather conditions, fuel availability, and fuel conditions. 27 some land managers have used prescribed fire followed by seeding to convert woodlands to grasslands, thus improving their rangeland for livestock. jacobs et al. used prescribed fire to maintain the mechanically created savanna structure by killing tree seedlings, but warn that excessive fuel loadings or less than optimal burning conditions can damage grass and forb communities. 13 prescribed fire has also been used to consume the slash created by thinning. understory abundance can increase when a site is burned several years after thinning. 13,20 the success or failure of seeding in pinyon-juniper woodlands is highly dependent on precipitation. water availability is critical for seedling establishment in arid ecosystems. 28,29 seeding is also affected by animal predation 30 and the availability of favorable microsites. 31 slash additions and minor soil disturbances can create favorable microsites for seed establishment. 22 seeding after wildfires is a common practice for the us forest service, and has been shown to effectively increase graminoid cover in degraded pinyon-juniper woodlands in northern arizona. 22 the objective of this study was to determine the effect of different silvicultural treatments in a pinyon-juniper woodland on understory richness and abundance. the treatments consisted of overstory thinning, different arrangements of slash, and burning and seeding in different combinations. our specific research questions were: (1) does burning and/or seeding after thinning influence resulting understory richness and abundance? (2) does slash arrangement influence resulting understory richness and abundance? to answer these questions, we measured posttreatment changes in forest structure, fuel creation and consumption, maximum soil temperature reached during the prescribed burn, and understory vegetation responses. we hypothesized that broadcasting slash followed by seeding would lead to the greatest understory abundance and richness and that burning would decrease both abundance and richness. the results from this study will assist land managers designing thinning prescriptions and in understanding the interactions of slash arrangements, burning, and seeding on resulting understory richness and abundance. methods study site this study was conducted in 2005 and 2006 on anderson mesa, located 150 km southeast of flagstaff, arizona. the climate of anderson mesa is semi-arid, receiving a mean annual precipitation of 470 mm. about half of the precipitation falls in july and august as rain, and the other half as snow in january, february, and march. the average high temperature in july is 29° c and the average low temperature in january is -9° c. 32 historically, there are few average years due to dramatic climatic fluctuations from year to year. 32 because of the effect of soil parent material on the developmental dynamics of vegetation in this region we selected three sites with different parental substrates. 10 these all res. j. biol, 2011, 2, 8-15 10 sites also have well documented historic forest structures (see 10 for detailed site descriptions). these sites were named after their soil parent material: limestone, sandstone, and basalt. all three sites were in the middle of the local pinyonjuniper elevational gradient. juniperus osteosperma (torr.) little and juniperus momosperma (engelm.) sarg. dominate the overstory and bouteloua gracilis (willd. ex kunth) lag. ex and gutierrezia sarothrae (pursh) britt. & rusby dominate the understory plant community at all sites. other common, yet less abundant grasses and forbs include elymus elymoides (raf.) swezey, chaetopappa ericoides (torr.) nesom, opuntia sp., descurainia sp., sphaeralcea parvifolia a. nels., lappula occidentalis (s. wats.) greene, lupinus kingii s. wats., lesquerella intermedia (s. wats.) heller, and arabis fendleri greene. the limestone and sandstone sites have had limited fall and spring livestock grazing since the 1950’s. the basalt site has not been grazed from 1920 to the present (jack metzger, flying m ranch, personal communication). other important grazers in the area include elk (cervus elaphus), mule deer (odocoileus hemionus), and pronghorn antelope (antilocarpa americana). all three sites have had very little modern human influence and have not been used as firewood gathering areas. the fire history of the area is unknown, although local anecdotal observations indicate that fires were limited to small (less than 1 ha), infrequent, high-severity fires. experimental design we created a split-plot design with one of four slash arrangements applied to the subplots and one of four seed/burn methods applied to the whole-plots (figure 1). at each site we created three 160 x 160 m units. each unit was divided into four 80 x 80 m whole plots, and the whole plots were divided into four 40 x 40 m subplots. each subplot was randomly assigned one of four slash arrangements: thin and pile, thin and cluster, thin and broadcast, or no thinning (control). then, we randomly chose a burn/seed method for each whole plot. there were four options: burn, burn and seed, seed, or no burn/seed method (control). therefore, each unit was composed of 16 subplots, and each subplot was a different slash arrangement and burn/seed method combination for a total of 16 treatments with three replications at each of the three sites. figure 1. split-plot experimental design replicated at each of the three sites. vegetation surveys a pre-treatment vegetation survey was conducted using a modification of the modified-whittaker plot in june of 2005. 33 we drew out a 50 m tape at a 45° angle from the southwest corner of each subplot, creating a 50 m line transect. a 1-m² frame (0.5 x 2 m) was placed along alternating sides of the transect at 14 meter intervals with a total of four samples per transect. in each frame, we visually estimated the aerial percent cover (abundance) of each plant species, bare soil, rock, coarse woody debris, litter, and moss. we averaged the cover estimates in these four frames to estimate plant species abundance for each subplot. to measure species richness in each subplot, we recorded all the species found within a five meter belt on either side of the line. a voucher specimen of each unknown species was collected and identified at the deaver herbarium at northern arizona university. posttreatment vegetation response was measured in the same way in june of 2006. thinning and slash arrangements thinning was conducted in the summer of 2005, following a bdq prescription for each site that was based on the 1860 stand structure at each site. 10 b stands for basal area in m 2 /ha, d stands for maximum diameter measured at root collar (drc) in cm, and q stands for the q-factor, a fixed ratio of trees in one diameter class to the next largest diameter class. 34 bdq thinning prescriptions are a silvicultural approach for controlling uneven-aged forest structure by setting targets of desired numbers of trees in each diameter class. 34 this method seeks to balance standing tree density all res. j. biol, 2011, 2, 8-15 11 with expectations for growth and mortality up to some maximum diameter. 35 these prescriptions did not consider p. edulis, which composed 1-10% of the woodland and much of which suffered from recent drought-induced mortality. in applying the prescription, we attempted to retain trees in a clumpy arrangement (3 trees or more together) when possible to mimic 1860 spacing patterns. 10 all of the thinning was done by hand with chainsaws. after each subplot was cut, we tallied the root collar diameter of all the stumps. these data, coupled with pre-treatment inventory data, allowed us to calculate forest density and diameter distribution at each plot before and after thinning. we arranged the slash as we were thinning the subplots. there were four possible slash arrangements: pile, cluster, broadcast, and no thinning (figure 2). we piled the slash for the pile arrangement. we felled the trees at the base and then left the limbs intact for the cluster arrangement. for the broadcast arrangement, we cut the slash into approximately one meter sections and then scattered it uniformly around the subplot. we left unthinned plots as controls. figure 2. four slash arrangements, clockwise from the upper left: pile, cluster, no thinning, and broadcast. fire measurements and the prescribed burn we measured surface fuel loading on each pile, cluster, broadcast, and no thinning sup-plot using planar intercept transects after the thinning. 36 we estimated the volume of slash piles according to hardy et al. 37 we used prescribed fire in the designated burn units in early november of 2005. even under windy conditions (gusts >24 km/hour) we had a difficult time getting the fire to carry because of a lack of continuous surface fuels. we placed 3 pyrometers at each subplot into areas of high, medium, and low slash accumulations. the pyrometers were composed of an “l” shaped strip of thin sheet metal, painted with 11 temperature-sensitive paints that detect temperatures ranging from 79°c to 760°c (tempilaq° g temperature indicating liquids, omega engineering, stamford, conn.). these pyrometers measured maximum soil temperature during the prescribed burn, and were also influenced by the duration of temperature, providing a somewhat integrated measure of intensity. 38,39 after the prescribed burn, we collected the pyrometers and recorded temperatures, and measured fuels to estimate fuel consumption. seeding we hand-seeded a custom native seed mix after the prescribed burn in november of 2005. we applied the seed mix directly to the ground in the whole-plots designated to be seeded. our seed mixture was composed of three shrubs, one forb and six grasses (artemisia tridentate, krascheninnikovia lanata, purshia tridentate, linum lewisii, achnatherum hymenoides, aristida purpurea, muhlenbergia wrightii, pleuraphis jamesii, elymus elymoides) and was applied at a rate of 62.9 kg/ha. all species in the seed mix were found on the sites in the 2005 vegetation survey. the seed and seeding rates were provided by granite seed in lehi, utah (www.graniteseed.com). to measure seed predation, we measured seedling emergence of pairs of protected and unprotected seeds. at every subplot in the limestone site and the sandstone site that was seeded, we placed 2 g of seed under a small cage (154.2 mm x 154.2 mm x 25.4 mm) made of hardware cloth. the cage was randomly placed within an area of the subplot that was not covered by slash. then 152 mm to the north of the cage, we placed 2 grams of seed mixture on the ground in the same sized area as the cage. data analysis since each of the three sites had different thinning prescriptions, they were treated as independent experiments and were analyzed separately. we used a split-plot design analysis of variance to test the influence of thinning, slash arrangements, seed/burn methods, and their interactions on understory richness and abundance. we used tukey-kramer honestly significant difference tests (hsd) to test for differences among means. we compared the differences in abundance and richness between years in the control plots at each site using paired t-tests. analyses were conducted using the statistical package jmp version 6 (sas institute, inc. 2004). all significances were found at the α=0.05 level. results thinning the prescriptions based on reference conditions resulted in basal area reductions across the three sites ranging from 28% to 61% (table 1). all res. j. biol, 2011, 2, 8-15 12 table 1. summary of the changes in forest structure after implementing the bdq thinning prescription at each of three sites. site bdq 1 prethinning density (trees/ha) postthinning density (trees/ha) density reduction (%) basal area reduction (%) limestone 301001.4 531 284 53 28 sandstone 201001.25 212 156 26 42 basalt 101001.5 441 138 69 61 1 b = basal area (m 2 ha -1 ), d = maximum diameter at root collar (cm), q = ratio of trees in one diameter class to the next largest diameter class. prescribed fire and fuels each of the four slash arrangements created a different fuel structure on the ground before and after the prescribed burn. the most consumption was seen in the pile arrangement, then the broadcast, then the cluster arrangement, and lastly in the no thinning subplots. the pyrometer readings showed that the pile slash arrangement burned hotter than all of the other slash arrangements, between 680 and 750 c. there was little difference between the maximum temperatures reached in the broadcast and the cluster slash arrangements; both ranged between 450 and 550 c. the plots that were not thinned reported the lowest maximum temperature readings, between 50 and 200 c. understory vegetation in 2005, we identified 115 species in the understory over all 3 sites. the basalt site had the greatest richness and abundance of the three sites. in 2006, we found 80 species over all 3 sites and few understory responses to treatments. understory species richness was not influenced by thinning, slash arrangement, or burn/seed method at the limestone site (table 2). at the basalt site, understory richness was influenced by thinning and slash arrangement, with the thinned plots and broadcast arrangement plots yielding the greatest richness (figure 3). at the sandstone site, we found a significant difference in richness only due to the slash arrangement by seed/burn method interaction, but the three treatments with the greatest species richness included the control (no thinning and no burn/seed method combination). understory abundance did not significantly differ by thinning, slash arrangements, or burn/seed method at any of the three sites (table 2). table 2. p-values for split-plot anova testing understory species richness differences due to the influences of thinning (thin vs no thin), slash arrangement (pile, cluster, broadcast, or no thinning), seed/burn method (burning, seeding, burning and seeding, or none), and the slash arrangement and seed/burn method interaction. all understory plant abundance results for the same variables were not significant (ns). variable limestone sandstone basalt thinning ns ns 0.02 slash arrangement ns ns 0.0004 seed/burn method ns ns ns slash arrangement x thin/burn method ns 0.003 ns figure 3. the basalt site understory species richness responses to different slash arrangements. data are expressed as means (n = 12) +/se. values indexed by different letters are significantly different at p ≤ 0.05 as determined by tukey’s hsd test. we compared understory plant richness and abundance for 2005 vs. 2006 in the 3 control (no thinning and no burn/seed method) sub-plots at each site. we found that species richness significantly decreased at all three sites (p=0.001 for the limestone site, p=0.001 for the sandstone site, and p=0.001 for the basalt site) (figure 4) by an average of 40%. plant abundance was not significantly different between years at the limestone site (p=0.7) or at the sandstone site (p=0.2), but was significantly reduced at the basalt site (p=0.02). seeding in june of 2006 we surveyed the seed cages and exposed seed plots and found no seedling emergence in either of the plots, at either of the sites. we found bare seeds lying on top of the soil inside of the cages. in the exposed plots, the seeds were no longer present, either consumed by herbivores or blown away by wind. there was no germination and therefore, no analysis was performed on the seed cage experiment. discussion although our experiment was not designed to test for the effect of moisture, we believe plant responses to our thinning, slash arrangements, and burning and seeding treatments were muted by the severe drought of the preceding winter and spring. pre-treatment vegetation measurements were conducted in a relatively wet period and post-treatment vegetation measurements were conducted in a very dry period. the seasonality of precipitation is very important in semiarid ecosystems. 40 our vegetation surveys were conducted in june, which is traditionally the peak of the understory plant abundance and richness at our research sites. 41 the growing season of 2005-2006 was the 3 rd driest growing season ever recorded. january to may of 2006 was the driest winter and spring in the last 55 years (western regional climate center). this same period in 2005 was relatively wet (85 th percentile), compared to the average precipitation year (figure 5). all res. j. biol, 2011, 2, 8-15 13 figure 4. differences in understory plant (a) richness and (b) abundance between 2005 and 2006 in the control plots at each site. an asterisk after site names indicate significant differences in the understory at the α=0.05 level. data are expressed as means (n = 16) +/se. species richness decreased 40% at the limestone site, 33% at the sandstone site, and 45% at the basalt site. figure 5. precipitation data from 1951 to 2006. (western regional climate center (http://www.wrcc.dri.edu)). the lines on the bars represent one standard deviation above and below the mean for the 1951-2006 precipitation data. we documented decreases in plant richness from 2005 to 2006 in the control plots; however, abundance levels did not significantly change in two of our three sites, probably because bouteloua gracilis accounted for a very high percent of total plant abundance at each site (90% at the limestone site, 63% at the sandstone site, and 67% at the basalt site). the above ground tufts of this hardy perennial grass persisted throughout the dry winter and spring of 2006, accounting for the majority of the abundance measurements. despite the dry growing conditions, we did see a significant plant richness response to the slash arrangement at one of the three sites. at the basalt site, broadcasting the slash resulted in the highest richness, followed by the cluster, then the pile and lastly the no thinning (figure 3). in other words, the more dispersed the slash was, the greater the resulting richness in the plant community. our findings on the basalt site support the idea that slash additions to bare soil can create favorable microsites for understory establishment. 10, 22 slash arrangement did not significantly influence resulting understory richness at the limestone and sandstone sites (table 2). the basalt site may have had a greater response because of increased moisture, a great reduction in overstory, soil type, or a combination of factors. the basalt site was 153 m higher than the other two sites and thus probably received more moisture and had the greatest overstory reduction (table 1). additionally, certain soil characteristics such as high calcium carbonate levels, high ph, and low phosphorous have been associated with no increase in perennial grass production. 42 burning did not influence understory richness or abundance at any of the sites after one year. other studies have burned slash created by thinning in pinyon-juniper woodlands and recorded an immediate decrease in plant abundance, followed by an increase in years following the burn. 20,43 burning heavy loads of juniper slash creates very hot soil temperatures and may have negative impacts on future understory regeneration. 43 our study showed that the maximum surface temperature exceeded 700°c under slash piles. the broadcast and the cluster slash arrangements also recorded high soil temperatures during the burn. soil heating can cause mortality to soil organisms, plant roots, alteration of physical soil properties, changes in nutrient cycling patterns, and nutrient volatilization. 44, 45 since seedling emergence often depends on soil water availability, 46,47 we attribute the total lack of germination in seeding cages to the dry growing season of 2006. seeding success in other studies had been mixed. stoddard (2006) found seeding increased biodiversity in degraded pinyonjuniper woodlands in northern arizona after the first two years of seeding. 22 judd and judd (1976) examined plant survival and found that none of the seeded species were present 30 years after seeding in pinyon-juniper woodlands of the tonto national forest in arizona. 29 a longer monitoring period is needed to determine the effects of treatments on understory response in pinyon-juniper woodlands. future studies on pinyon-juniper understory communities could be designed to control for moisture, reduce the numbers of influencing factors in the experimental all res. j. biol, 2011, 2, 8-15 14 design, and be remeasured to follow vegetation changes over many years and climatic patterns. management recommendations using an 1860 thinning prescription, as opposed to total tree removal, assures that structure of the pinyon-juniper woodland is maintained within the historical range of variability. 10 thinning represents a compromise between total tree removal which would maximize forage production and no management action. 48 broadcasting the slash created by thinning increased initial understory diversity on the basalt derived soil site, despite the dry year. burning slash did not affect initial grass and forb abundance and diversity, although it did produce exceedingly hot soil temperatures. land managers must weigh the tradeoffs of burning slash for wildlife and livestock mobility benefits, with the potential negative effects mentioned above. hand seeding was not found to be effective. variation in precipitation is the norm in the southwest. therefore, understanding temporal and spatial variability in the pinyon-juniper woodland understory plant community is vital to interpreting the influence of management actions. global climate change is expected to affect ecosystems worldwide 49 by raising temperatures and changing precipitation patterns. 50 given the central role that precipitation plays in semiarid ecosystems, changing precipitation regimes and inter-annual variability may have a stronger effect on pinyon-juniper understory biodiversity and abundance than land management decisions. acknowledgements this research was supported by usda forest service, rocky mountain research station under research joint venture rmrs-02-jv-11221615-135. we are extremely grateful to the diablo trust, particularly the flying m ranch, for allowing us to conduct research on private property. special thanks to bryan zebrowski, stephanie powers, and other members of the nau school of forestry silviculture lab, for making this project possible and to elizabeth kalies for thoughtful review comments. references 1. west, n.e. (1999). distribution, composition, and classification of current juniper-pinyon woodlands and savannas across western north america. in ecology and management of pinyon-juniper communities within the interior west, monsen, s.b., and r. stevens (comps.). usda for. serv. rmrs-p-9, ogden, ut. pp. 20-23 2. gottfried, g., swetnam, t.w., allen, c.d., betancourt, j.l., chung-magcoubrey, a. (1995). pinyon-juniper woodlands. in ecology, diversity, and sustainability of the middle rio grande basin. d.m. finch and j.a. tainter (tech. eds.). usda forest service general technical report rmgtr-268. fort collins, co. pp. 95-132. 3. van devender, t.r., betancourt, j.l., wimberly, m. (1984). biogeographical implications of a packrat midden sequence from the sacramento mountains, south-central new mexico. quaternary res. 22, 344-360. 4. mehringer, p.j., wigland, p.e. (1990). comparison of late holocene environments from woodrat middens and pollen: diamond craters, oregon. in packrat middens: the last 40,000 years of biotic change, betancourt, j.l., t.r. van devender, and p.s. martin (eds.). university of arizona press, tucson, az. pp. 13-16 5. woolfenden, w.b. (2003). a 180,000-year pollen record from owens lake, ca: terrestrial vegetation change on orbital scales. quaternary res. 59, 430-444. 6. cottam, w. p., stewart, g. (1940). plant succession as a result of grazing and meadow desiccation by erosion since settlement in 1862. j. forest. 38, 613-626. 7. burkhardt, j.w., tisdale, e.w. (1969). natural and successional status of western juniper vegetation in idaho. j. range manage. 22, 264-270. 8. blackburn, w. h., tueller, p. t. (1970). pinyon and juniper invasion in black sagebrush communities in eastcentral nevada. ecology 51, 841-848. 9. tausch, r.j., west, n.e., nabi, a.a. 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(1995). plant species composition patterns with differences in tree dominance on a southwestern utah pinyon-juniper site. in desired future condition for pinyon-juniper ecosystems, shaw, d.w., e.f. all res. j. biol, 2011, 2, 8-15 15 aldon, and c. losapio (eds.). usda for. serv. gen. tech. rep. rm-258, fort collins, co. pp. 16-23. 19. white, c.s., loftin, s.r., aguilar, r. (1997). application of biosolids to degraded semiarid rangeland: nine-year responses. j. environ. qual. 26, 1663-1671. 20. wood, k.m., javed, n. (2001). hydologic and vegetal responses to fuelwood harvest and slash disposal in a pinyon pine and juniper dominated grassland. water resources research institute rep. no. m27. new mexico state university, las cruces, nm. 21. brockway, d.g., gatewood, r.g., paris, r.b. (2002). restoring grassland savannas from degraded pinyon-juniper woodlands: effects of mechanical overstory reduction and slash treatment alternatives. j. environ. manage. 64, 179197. 22. stoddard, m.t. (2006). slash additions: a tool for restoring herbaceous communities in degraded pinyonjuniper woodlands. m.s. thesis, northern arizona univ. flagstaff, az, usa. 23. aro, r.s. (1971). evaluation of pinyon-juniper conversion to grassland. j. range manage. 24, 188-197. 24. baker, w.l., shinneman, d.j. (2004). fire and restoration of pinyon-juniper woodlands in the western united states: a review. forest ecol. manag. 189, 1-21. 25. arnold, j.f., jameson, d.a., reid, e.h. (1964). the pinyon-juniper type of arizona: effect of grazing, fire, and tree control. usda prod. res. rep. 84. 26. dwyer, d.d., pieper, r.p. (1967). fire effects on blue grama-pinyon-juniper rangeland in new mexico. j. range manage. 20, 359-362. 27. bruner, a.d., klebenow, d.a. (1979). predicting success of prescribed fires in pinyon-juniper woodland in nevada. usda for. serv. res. paper int-219. 28. griffiths, d. (1907). the reseeding of depleted range and native pastures. usda plant ind. bull. 117. 22 p. 29. judd, i.b., judd, l.w. (1976). plant survival in the arid southwest 30 years after seeding. j. range manage. 29, 248251. 30. archer, s., pyke, d.a. (1991). plant-animal interactions affecting plant establishment and persistence on revegetated rangeland. j. range manage. 44, 558-565. 31. harper, j.l., williams, j.t., sagar, g.r. (1965). the behavior of seeds in soil. i. the heterogeneity of soil surfaces and its role in determining the establishment of plants from seed. j. ecol. 53, 273-286. 32. western regional climate center. (2006). walnut canyon national monument, arizona. monthly total precipitation. available online at www.wrcc.dri.edu/cgibin/climontpre.pl?az9156; last accessed dec. 7, 2007. 33. korb, j. (2008). intraand inter annual vegetation change: implications for long-term research. restor. ecol. 16(1), 5-11. 34. smith, d.m., larson, b.c., kelty m.j., ashton, p.m.s. (1997). the practice of silviculture: applied forest ecology. wiley, new york, usa. 35. bailey, j.d., covington, w.w. (2002). evaluating ponderosa pine regeneration rates following ecological restoration treatments in northern arizona, usa. forest ecol. manag. 155, 271–278. 36. brown, j.k., (1974). handbook for inventorying downed woody fuels. usda for. serv. gen. tech. rep. int-16. 37. hardy, c.c. (1996). guidelines for estimating volume, biomass, and smoke production for piled slash. usda for. serv. gen. tech. rep. pnw-364. 38. odion, d.c., davis, f.w. (2000). fire, soil heating, and the formation of vegetation patterns in chaparral. ecol. monogr. 70, 149–169. 39. schwilk, d.w. (2003). flammability is a niche construction trait: canopy architecture affects fire intensity. am. nat. 162, 725–733. 40. schwinning, s., starr, b.i., ehleringer, j.r. (2005). summer and winter drought in a cold desert ecosystem (colorado plateau) part i: effects on soil water and plant water uptake. j. arid environ. 60, 547-566. 41. metzger, j., fying m. r. personal communication. 42. o’rourke, j.t., ogden, p.r. (1969). vegetative response following pinyon-juniper control in arizona. j. range manage. 22, 416-418. 43. jacobs, b.f., gatewood, r.g. (2002). reintroduction of fire maintains structure of mechanically restored pinyonjuniper savanna (new mexico). ecol. restor. 20, 207-208. 44. roberts, w.b. (1965). soil temperatures under a pile of burning logs. aus. for. res. 1, 21-25. 45. neary, d.g., klopatek, c.c., debano, l.f., ffolliot, p.f. (1999). fire effects on belowground sustainability: a review and synthesis. forest ecol. manag. 122, 51-71. 46. harper, j.l. (1977). population biology of plants. academic press, new york, new york, usa. 47. chambers, j.c. (2000). seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration. ecol. appl. 10, 1400-1413. 48. farichild, j.a. (1999). pinyon-juniper chaining guidelines for big game winter range enhancement projects. in proc. of ecology and management of pinyon-juniper communities within the interior west, monsen, s.b. and r. stevens (comps.). usda for. serv. rmrs-p-9, ogden, ut. pp. 278280 49. vitousek, p.m. (1994). beyond global warmingecology and global change. ecology 75, 1861-1876. 50. intergovernmental panel on climate change. (2001). climate change 2001: the scientific basis. contribution of working group i to the third assessment report of the intergovernmental panel on climate change, cambridge university press, cambridge, united kingdom. ebikeme_final layout the malaria problem: short communication. charles ebikeme, victoria valdivia giménez all res. j. biol, 2010, 1, 4-11 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com reviews issue 1, vol. 1, 2010, 4-11 4 the malaria problem: short communication charles ebikeme*a and victoria valdivia giménezb a) centre de résonance magnétique des systèmes biologiques (rmsb), umr5536 cnrs, université victor segalen bordeaux 2, 146 rue léo saignat, 33076 bordeaux, france ; b) instituto de investigaciones químicas, c.s.i.c-universidad de sevilla, c/américo vespucio, 49, isla de la cartuja, 41092 sevilla, spain corresponding author e-mail: charles@rmsb.u-bordeaux2.fr keywords: malaria, negative results, phase 1 drugs, drug resistance introduction over a decade ago the world decided to approach malaria in a new way. the goal is the eradication of malaria – and that, by 2015, will no longer be a major cause of mortality nor a barrier to social and economic development and growth anywhere in the world (www.rollbackmalaria.org). malaria is the world’s most prevalent infectious disease. almost 40% of the world’s population is at risk.1 in 2006, 247 million malaria cases caused around a million deaths,2 of which children and pregnant women were disproportionately affected. the african continent feels the greatest burden from this neglected disease, with 45 countries in the sub-sahara being endemic for malaria and accounting for 86% of cases worldwide.2 malaria is caused by protozoan parasites of the genus plasmodium. over 100 recognised species of plasmodium exist, infecting a wide range of vertebrate hosts including primates, rodents, birds and reptiles. the deadliest human malaria parasite is plasmodium falciparum, resulting in the most severe clinical symptoms of the disease and causing 90% of all malaria deaths. along with p. falciparum, p. vivax, p. ovale and p. malariae are the four main species that are known to infect humans. there are significant differences between the species; ranging from liver stage progression, parasite replication time within the host, and the timing in appearance of gametocytes in the bloodstream.3,4 each species causes a unique set of complications in terms of disease. p. falciparum infections are responsible for the most severe form of malaria and can result in cerebral malaria and pregnancy-related malaria. malaria caused by p. vivax is becoming an increasing problem,5 accounting for as much as 40% of the total disease burden.6 relapse can also be a problem with p. vivax infections. the development of dormant hypnozoite forms in the liver can last up to 20 years and cause subsequent reinfections in the blood.7 plasmodium parasites are transmitted by female mosquitoes of the genus anopheles, of which only 60 species are able to transmit the disease. vector control remains a central aspect of any malaria eradication strategy. so much so that in some highly endemic countries vector control measures has led to reductions in deaths from malaria.2 historically, emphasis was put on reducing the numbers of mosquitoes by combinations of environmental hygiene and insecticide spraying. none was more important than dichlorodiphenyl-trichloroethane (ddt). ddt use today is rare, highlighting the great importance that mosquito resistance has to any and future vector control strategies. the development of resistance to ddt was a primary cause of the collapse of previous malaria control programmes in the latter half of the late century.8 currently, there is a near-complete dependence on pyrethroids for vector control, and the development of new techniques for vector control need to be increased.9 this article gives a brief overview of the current state of antimalarial drug development, paying close attention to the history and current problems of drug treatment, and highlighting possible future drugs in phase i clinical trials. antimalarials and resistance antimalarials present the most important part of any integrated approach needed to combat the disease. antimalarials have direct benefits to patients and a general decrease in disability-adjusted life years (dalys) for the population in general. today, parasite resistance to all but one class of antimalarials exists in most endemic countries.10 resistance has prompted the wide-scale shift in first-line treatments against malaria, under recommendation by the world health organisation (who). however, many countries continued to use ineffective mono-therapy treatments, due to, in part, the disparity in costs between the more conventional chloroquine and sulfadoxinepyrimethamine based therapies and the recommended artemisinin combination drugs.11 however, the increase of international funding commitments,10,12 resulting in increased malaria control programmes has gone some way to rectify this problem. drug resistance is the major cause of malaria treatment failure. however, influencing the rapid rise of drug resistance are factors such as non-compliance or nonadherence to drug regimen, nutritional status of patients, incorrect drug usage, counterfeit drugs, and misdiagnosis of patients.13 further clouding the issue is the distinction all res. j. biol., 2010, 1, 4-11 5 between and outcome of drug resistance, treatment failure, and reinfection.14 the mechanisms, molecular and biochemical, underlying resistance of plasmodium species to the various antimalarial drugs has been greatly studied to date.15,16 the genetic events that confer antimalarial drug resistance are specific for each drug and consist of mutations or single gene copy number mutations in genes related to drug targets (table i). quinoline antimalarials have been widely used for the treatment of malaria. among these are mefloquine, quinine, pyronaridine, halofantrine, primaquine, and chloroquinine. the cheapest and more widely available antimalarial was quinine, the first known effective anti-malarial drug – an extract from the bark of the tree cinchona calisaya – was used as an antimalarial agent as early as 1632,17 and by the 19th century it was still the only known antimalarial agent. for decades first-line treatment of malaria involved chloroquine (cq), the first synthetic antimalarial compound introduced after the second world war18 following us government-sponsored clinical trials which showed that cq had prominent effects as an anti-malarial drug.19 today, cq is given as treatment for both uncomplicated malaria or severe malaria.20 parasite resistance to cq is widespread. cq-resistant p. falciparum (crpf) emerged from four independent foci. firstly, in southeast asia around the thai-cambodian border, where crpf infections were identified in 1957 and spread quickly to thailand.21 two other foci were identified in 1960 in south america, in venezuela and in the magdalena valley, colombia.22 in 1976, two confirmed cases of crpf infection were reported from port moresby in papua new guinea72 and probably represent the emergence of the fourth independent focus of crpf infection. in africa, crpf was first found in 1978.23 resistance spread from the african coastal areas inland and by 1983 had been observed in sudan, uganda, zambia, and malawi. in 1973, thailand was the first country to replace cq as a first-line drug. the spread of cq resistance was the main factor causing the increased child mortality rates observed in africa since the last decade of the last century.24 cq resistance is multigenetic and results in a reduced parasite accumulation of the drug,25,26 with reduced concentrations of the drug in the digestive vacuole of the parasite. cq enters the food vacuole and targets the polymerisation of toxic haem, binding and thus preventing its polymerisation to haemozoin.27,28 this results in an increase in toxic haem leading to enhanced oxidative stress, membrane damage and eventually parasite death.29 one mechanism of resistance is associated with polymorphisms in a 36 kb segment of the parasite's chromosome 7, which contains a polymorphic gene encoding a unique 330 kda protein, cg2.30 however, association of cg2 with chloroquine resistance in field isolates is incomplete.31 genetic crosses identified a role of the p. falciparum chloroquine-resistance transporter (pfcrt), a carrier protein located in the membrane of the digestive vacuole of the blood-stage parasite.32,33,34 multiple polymorphisms in the gene are associated with chloroquine resistance both in vitro and in vivo. however, pfcrt is not the sole molecular determinant of chloroquine resistance. mutations in the homolog of the major multidrugtransporter p. falciparum multidrug resistance gene (pfmdr) seems to modulate the extent of chloroquine resistance conferred by mutations in pfcrt.35 furthermore, the pfmdr1 gene has been shown to be involved in mefloquine resistance and cross-resistance to halofantrine.36,37 sulfadoxine-pyrimethanine (sp), a class of antifolates, is another drug used to treat uncomplicated malaria.20 its widespread use in many countries as a first-line antimalarial treatment was prompted by the emergence of cq resistance. however, resistance developed rapidly; sp was introduced in thailand in 1967 and resistance was reported within the same year.23,38 antifolates (including pyrimethaminesulfadoxine, chlorproguanil-dapsone, and proguanilatovaquonel) represent the more traditional second-line treatment option for malaria, but again, resistance is widespread.39 these classes of drugs have a mode of action through either inhibiting the formation of dihydropteroate catalyzed by dihydropteroate synthase (dhps) by competing for the active site of dhps;40 or by inhibition of dihydrofolate reductase (dhfr), thus preventing the nadph-dependent reduction of dihydrofolate to tetrahydrofolate by dhfr. mechanisms of resistance seem to be associated with several point mutations in the respective genes.41,42 a specific combination of these mutations is heavily associated with treatment failure.43 however, treatment outcomes become increasingly more difficult to predict as the level of mutation falls off .14 in 1972, chinese scientists discovered qing hao su – sesquiterpere lactone artemisinin – isolated from the leaves of the sweet wormwood artemisia annua. artemisininbased combination therapies (acts) are now generally considered as the best current treatment for uncomplicated falciparum malaria for a number of reasons.2 the most important being that no real mode of resistance has yet been implicated with artemisinin itself. most likely because a shorter half-life allows rapid clearance from the body, which avoids persistence of the drug at sub-lethal concentrations, and as a result will avoid the emergence of resistant parasites. currently, several treatment options are available – as mandated by the who2 – artemether-lumefantrine, artesunate + amodiaquine, artesunate + mefloquine, and artenusate + sulfadoxine-pyrimethamine. it is hoped that by combining antimalarial drugs with different modes of action parasite resistance can either be prevented or its onset delayed considerably, allowing completion of full dose regimens to end in high cure rates and an eventual decrease of disease transmission, benefiting patients and the larger community. today, more potent derivatives of its active chemical have been developed. these include artemether, artemotil, and artesunate. the problem of the appearance of resistance to artemisinin is one that health professionals, policy makers and research scientists are well aware of.44 to that effect, acts are now the recommended strategy both for clinical care and for the avoidance of drug resistance.2 to date, there has been no in vivo cases of resistance reported, however, in vitro susceptibility was found to vary with mutations in pfmdr1 and pfcrt (the two genes proposed to modulate sensitivity to cq).45 furthermore, cases of drug all res. j. biol., 2010, 1, 4-11 6 resistance induced experimentally are few and of a moderate level, and even then, have been proven to be transient.46 the mode of action of artemisinin and its derivatives is proving to be complicated and has not been completely elucidated, however, it seems to stem from alkylation of molecules by radicals produced from the reductive cleavage of the intact peroxide by ferroheme ferrous-protoporphyrin ix.31,47 acts represent the present best hope for treatment of malaria. if the history of the malaria parasite has taught us anything then it’s that onset of parasite resistance is a distinct inevitability, which brings up the question of where will the next generation of antimalarials come from? charles ledger “gave quinine to the world” even before the causative agent or the disease was known, the wars in europe and vietnam led to the development of chloroquine, mefloquine and halofantrine, and ancient china has brought the blue-green herb that is currently the first line of defence against the world’s most prevalent disease. the next generation of antimalarials are some way off from becoming a clinical reality. most antimalarial drugs developed thus far have been identified and developed using conventional drug discovery techniques.48 the future will bring many progressive leaps in the kind of techniques employed by researchers to increase the beneficial output of new compounds. pharmacogenetic-pharmacokinetic relations and parameters, pathogen and host genomic and proteomic information, as well as randomised trials and replication will all prove fruitful when applied to antimalarial drugs, not only in understanding the resistance factors at play but also in understanding the clinical success and failure of present and future antimalarial treatments.49 drugs in phase i clinical trials aminoquinoline antimalarial (aq-13) has been shown to be effective in vitro against p. falciparum malaria parasites resistant to cq and other antimalarials, as well as being active in a model of human infection with p. vivax, cqresistant p. falciparum in the squirrel monkey, a model of human infection with cq-resistant p. falciparum, and in two in vivo monkey models of human malaria (p. cynomolgi in the rhesus monkey macaca mulatta). its performance in human subjects is being investigated in phase 1 (safety/toxicity and pharmacokinetic) studies.50,51,52 furthermore, aq-13 has proven to be of similar safety to that of cq in preclinical studies performed by sri international (ind 55,670). the trial on aq-13 was appropriately designed as a randomized controlled phase i study, allowing the assessment of safety and physiological outcomes after treatment as compared to an existing and widely used drug, cq. a key limitation inherent to such studies is the small number of participants studied. this means that the study cannot prove that aq-13 is safe, or even as safe as cq, but rather simply that the findings do not raise immediate safety concerns.53 with malaria parasites often being resistant to cq and sp, chlorproguanil-dapsone is a potential alternative. the objective of the clinical trial was to compare chlorproguanildapsone with other antimalarial drugs for treating uncomplicated falciparum malaria.54 recent trials have shown that chlorproguanil-dapsone (with 1.2 mg chlorproguanil) as a single dose had fewer treatment failures than chloroquine (1 trial), but more treatment failures and people with parasitaemia at day 28 than sulfadoxinepyrimethamine (3 trials). two trials compared the threedose chlorproguanil-dapsone (with 2 mg chlorproguanil) regimen with sulfadoxine-pyrimethamine in new attendees. there were fewer treatment failures with chlorproguanildapsone by day 7 (1 trial). neither trial reported total failures by day 28. a further trial was carried out in participants selected because they had previously failed sulfadoxine-pyrimethamine. adverse event reporting was inconsistent between trials, but chlorproguanil-dapsone was associated with more adverse events leading to discontinuation of treatment compared with sulfadoxinepyrimethamine (1 trial). it was also associated with more red blood cell disorders. randomized controlled trials that follow up to day 28, record adverse events, and use an intention-to-treat analysis are required to inform any policy decisions. in all, it seems that there is insufficient data about the effects of the current standard chlorproguanil-dapsone regimen (three-dose, 2 mg chlorproguanil). however, chlorproguanil-dapsone has been withdrawn in 2008 following demonstration of post-treatment haemolytic anaemia in g6pd deficient patients in a phase iii trial of chlorproguanil-dapsone and chlorproguanil-dapsoneartesunate versus artemether-lumefantrine.55,56 significant reductions of haemoglobin levels in patients with g6pd deficiency have been observed with both cd and cda. mefloquine, a quinolinemethanol antimalarial, is effective as therapy and prophylaxis for all species of malaria infecting humans, including multi-drug resistant p.falciparum. mefloquine is a chiral molecule with two asymmetric carbon centres, which means it has four different stereoisomers. the drug is currently manufactured and sold as a racemate of the (r,s)and (s,r)-enantiomers by hoffman-laroche, a swiss pharmaceutical company. mefloquine's clinical utility has been impaired by its association with neuropsychiatric side effects.57 the pharmacological basis of these side effects are not known but two of the most reported hypotheses relate to its action on (i) the adenosine receptor58 and (ii) its effect on the cholinesterase enzyme.59 for both these mechanisms, there is a significant stereoselective activity of the two enantiomers.60 studies show that the (-) isomer is 50-100 fold more potent towards adenosine receptors compared with the (+) isomer.61 in addition, (-)-mefloquine has considerably more anti-cholinesterase activity.62 it has therefore been hypothesised that (+)-mefloquine may have a better central nervous system (cns) safety profile compared with either the racemate or (-)-mefloquine.63 the phase i clinical trial consisted of a randomized, ascending dose, double-blind, active and placebo-controlled, parallel group study in healthy male and female volunteers designed to investigate this hypothesis and to describe the comparative pharmacokinetics of the racemate and the single enantiomer.64 ferroquine (fq)+artesunate(as),65 a unique organometallic compound, is a novel antimalarial drug designed to overcome the chloroquine (cq) resistance problem currently in phase i clinical trials. fq has been all res. j. biol., 2010, 1, 4-11 7 revealed to be equally active on cq-sensitive and cqresistant p. falciparum laboratory strains and field isolates, and is also curative on rodent malaria parasites. as, a class of the artemisinin group of drugs that treat malaria, is a semi-synthetic derivative of artemisinin that is water-soluble and may therefore be given by injection.66 ferroquine and artesunate combination therapies have been the subject of phase i clinical trials.67 combinations of the two drugs were used to assess the safety of different doses of ferroquine with artesunate in adult african patients with uncomplicated malaria and to assess activity in reducing parasitemia and the pharmacokinetics of ferroquine and its metabolites. future prospects malaria is preventable and curable, however, in the absence of quick and effective treatment, symptoms of the disease progress and death results.68 accurate diagnosis is needed in all cases as well as accurate surveillance in all endemic areas.69 to curb the incidence of treatment failure, the spread of resistance, who recommends confirmation of malaria through parasite-based diagnosis in all patients prior to commencing treatment. prompt parasitologic confirmation by microscopy or alternatively by rapid diagnostic tests (rdts) are recommended in all patients suspected of malaria before treatment is started. thus, diminishing unnecessary use of acts and provide critical and accurate surveillance data to manage programmes and monitor impact. however, misdiagnosis and over-diagnosis of malaria still occurs.70 one reason is simply due to the fact that early symptoms of malaria are non-specific. bringing research agendas and control programmes together is one of the greatest challenges to fighting any infectious disease. for malaria endemic countries, strengthening the existing health systems is crucial. prevention, diagnosis and treatment needs to be followed up with accurate surveillance. the last few years have seen a great development in new treatments and new strategies, mainly because of the failure of old regimes due to parasite resistance. the unique biochemical aspects of the parasite continue to be exploited in the hope of drug design. parasite genome initiatives are ongoing efforts of full genomic sequencing to facilitate full understanding of how parasites develop, survive and reproduce in their respective hosts, of parasitehost and parasite-immune system interactions and of the factors that determine behaviour, pathogenicity, drug resistance and antigenic variation. parasite genome sequencing together with effective genetic manipulation (gene knock-out and gene silencing) provides a valuable means of mimicking loss of function attributable to therapeutic intervention (albeit with the caveat that pharmacological agents cannot mimic the zero activity state produced by conventional gene knock-out experiments). already, this year has seen significant advancements in the world of malaria research. glaxosmithkline has made available, in the public domain, thousands of compounds – confirmed-hit structures – to the general scientific community. the genome for artemisia annua – the herb producing the active ingredient in the most effective treatment for malaria – has been mapped.71 the further highlighting of genes and markers that could pave the way for higher yield varieties is another step in the road to complete eradication of this debilitating disease. all res. j. biol., 2010, 1, 4-11 8 table i: antimalarial compounds, targets, and resistance. structure molecule frequency of resistancea resistance gene ncl hn n chloroquine ++++b cg2/pfcrt/pfmdr1 n o ho n quinine ++ pfcrt /pfmdr1/ pfnhe n cf3 ho h n h cf3 h n oh n h f3c cf3 mefloquine +++ pfmdr1/other n n nh2 h2n cl pyrimethamine ++++ pfdhfr n n n h2n nh2cl cycloguanil ++ pfdhfr cl o o ho atovaquone + pfcytb cl cl ho n cl lumefantrine ++ pfcrt/pfmdr1 o o o o o h h h artemisinin + pfcrt/pfmdr1/ pfatpase6 all res. j. biol., 2010, 1, 4-11 9 n n och3 och3 h n s o o h2n sulfadoxine ++++ pfdhps a p.b. bloland. in: who/cds/csr/drs/2001.4, 2001 b 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(1976). chloroquine resistant malaria in papua new guinea. papua and new guinea medical journal, 19(3), 184-185. microsoft word 71-434-1-layoutediting.doc evaluation of growth, cell size and biomass of isochrysis aff. galbana (t-iso) with two led regimes miguel v. cordoba-matson*, bertha o. arredondovega and laura carreón-palau . all res. j. biol., 2013, 4, 7-15 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 1, vol 4, 2013, 7-15   evaluation of growth, cell size and biomass of isochrysis aff. galbana (tiso) with two led regimes miguel v. cordoba-matson a*, bertha o. arredondo-vega a and laura carreón-palau a a centro de investigaciones biológicas del noroeste, instituto politécnico nacional 195, col. playa palo de santa rita, la paz, baja california sur, 23096, mexico *corresponding author: celectronica@gmail.com abstract in contrast to crops, there are few studies using led-based light with microalgae and none cultivating the microalga isochrysis aff. galbana (t-iso) despite its importance to marine aquaculture. the objective was to evaluate white and red leds as an alternative source of light for the cultivation of i. aff. galbana (t-iso). in order to carry this out, white and red leds were used with a laboratory built erlenmeyer-type photobioreactor to determine productivity, cell number, size and biomass composition. results were compared with standard fluorescent lights of the same light intensity. the culture system consisted of 3 flasks for applying red leds, 3 flasks for white leds and 3 control group flasks illuminated with the normal fluorescent lighting at the similar light intensity of ~60 µm m–2 s–1. it was found that the population cell density did not significantly increase with either red leds or white leds (p > 0.05), if at all. standard fluorescent lighting (control group) showed significant increases in population cell number (p < 0.05). through microscopic observation cell size was found to be smaller for white leds, and even smaller for red leds compared to fluorescent lighting. the biochemical composition of proteins, carbohydrates and lipids was similar for all light regimes. the authors suggest that the unexpected non-growth of i. aff. galbana (t-iso), a haptophyte microalga with white and red leds, is possibly due to the fact that the cell growth initiation of this microalgae requires other wavelengths (possibly green) aside from red and blue, to allow auxiliary pigments, probably fucoxanthin, to capture light. keywords: microalgae, isochrysis galbana, leds introduction marine microalgae are recognized as a rich source of pigments: β-carotene, long-chain polyunsaturated fatty acids (pufas), polysaccharides and vitamins (1, 2). although microalgae cultivation is now over 40 years old, it is still a pertinent practice today. although it was established over 10 years ago, most of the microalgal species being grown commercially on a large scale are still spirulina spp. and chlorella spp. for health food (3), along with a handful of other species principally used in aquaculture as live food for farmed species (4). although microalgae biomass is still primarily commercially produced for human consumption and feedstuff in aquaculture, 25–30 years ago after the oil crises of the 1970s the united states department of energy (doe) started the aquatic species program from 1978 to 1996 looking into using microalgae as a biofuel (5). however, due to the low prices of oil at that time the program was discontinued in 1996. recently, escalating oil prices (> $100/barrel) due to depleting resources and high demand have revived interest in microalgae as a sustainable energy alternative (6, 7). microalgae are particularly attractive since they can lessen co2 emission, they are a nonfood crop, can produce oil with 7 all res. j. biol, 2013, 4, 7-15 low land usage (8) in saline or brackish or even wastewater (9) with high productivity and high yields since many strains contain up to 20-80% of their weight in triglycerides convertible into biodiesel (10, 11). for these reasons, there are currently many private companies actively attempting to commercialize fuel derived from microalgae—however, none as far as we are aware are in production. in addition, due to declining fisheries worldwide, aquaculture activity is on the rise thereby fueling demand for live microalgae (12, 13). this calls for the development of more sophisticated methods of microalgae cultivation, with higher productivity and lower contamination than that available by open ponds or raceways (14, 15). debates vary, but one general consensus is that ideally production should be in photobioreactors (pbrs) where growth can be better controlled; however, the cost of production has been a looming concern. one of the principal advantages of closed pbrs, as compared to open ponds or raceways, is that the light path length can be reduced leading to higher cell densities, which also diminishes the possibility of contamination thereby reducing harvesting costs. the development of the light emitting diode (led) technology over the years and its decrease in the cost of production has led to an increasing number of new applications of leds as a light source. leds offer a number of advantages over other light technologies such as low cost, longevity, low power consumption, low heat generation, and wavelength control. there are a limited number of studies utilizing high efficiency light-emitting diodes (leds) with microalgae, even though leds have been proposed as a primary light source in commercial crop cultures due to their lower energy costs compared to standard lighting (16). for example, matthijs et al., (17) reported that monochromatic exposure from red leds alone can support microalgae growth, whereas limited exposure to blue light failed to augment the biomass production of chlorella sp. however, in that study the effect of blue leds alone on the growth of chlorella sp. was not studied and the ratio of blue to red leds used may have possibly been too low to cause any effect. in another study, lee and palsson (18) reported also that in chlorella sp. red leds also had produced equivalent biomass growth in comparison to fluorescent light. oh et al. (19) studied the effects of irradiance with various wavelengths from light-emitting diodes on the growth of the harmful dinoflagellate heterocapsa circularisquama and the diatom skeletonema costatum; they reported light selectivity in stimulating the growth of one over the other. wang et al., (20) reported that red light-emitting diodes performed better than blue leds in the cultivation of spirulina platensis. isochrysis aff. galbana (t-iso) is one of the most commonly used microalgal species in aquaculture (14). isochrysis sp. are small (4–6 µm in diameter), motile with flagella, and since they a lack a cell wall (naked) they are readily digested by small (larval) invertebrates (21). they also have a high content of the essential fatty acid docosahexaenoic acid (dha, 22:6 (n-3) (22). isochrysis aff. galbana are considered an excellent candidate for mass cultivation (23). most of the previous studies with isochrysis sp. have been related to light intensity and microalgal growth and not with light quality (24, 25). for the first time, the present study applies both white and red leds in a small erlenmeyer-type bioreactor to determine the cell density, productivity, size and biomass composition of isochrysis aff. galbana (t-iso) and compares the results with standard fluorescent lights. the hypothesis being tested is whether this microalgae grows with red and white leds. materials and methods microalgae strain and culture conditions the microalga isochrysis aff. galbana (t-iso; utex lb 2307) from the culture collection of microalgae at the centro de investigaciones biológicas del noroeste s.c., la paz, b.c.s., mexico. the initial microalgae inoculums were grown in three 2-l erlenmeyer flasks in sterilized seawater (≈ 34 psu) with f/2 medium (26) with continuous air bubbling (1 vvm). full spectrum white light was supplied continuously at 60 µm m–2 s–1 on average with a warm-white fluorescent tube (mexico general electric co., mexico) positioned horizontally along the side of the flasks. ambient temperature was controlled at 23 ± 2 °c and the ph was 7.8. for cell synchronization each of the three 2-l flasks with culture were reinoculated every 3 days with 500 ml of cell suspension and 1500 ml of f/2 medium and this cycle was repeated twice prior to use with 12h:12h (light:dark). this was done in order to achieve synchronous growth and cell division of the microalgal cells. for experimental runs the doubly reinoculated suspension of microalgae was allowed to reach the fourth day and was used to prepare 9 different 1-l erlenmeyer flasks (kimble glass inc., usa) with 200 ml inoculum and 700 ml of f/2 medium for experimental runs. the experimental runs were repeated three times. remaining inoculums were resynchronized as indicated above. photobioreactor system using a 1-l erlenmeyer flask a laboratory built photobioreactor system was proposed and designed using red and white leds for illumination (fig. 1).   8 all res. j. biol, 2013, 4, 7-15   fig. 1. diagram of the proposed experimental system for production of microalgae with monochromatic light showing the 3 red leds (numbered 1,2 and 3) and 3 white leds (numbered 4, 5 and 6). the light generated from single leds applied to the base of an erlenmeyer flask allows for the generation of light intensity of 60 µm mol.m–2s–1 equal to that of flasks 7, 8 and 9 (control group) using light from a white fluorescent tube (general electric co., mexico) positioned horizontally to the side (not shown in diagram). the system built consisted of 3 flasks for applying red leds and three for white leds and 3 control group flasks illuminated with the normal fluorescent lighting typically used to grow i. galbana in the laboratory. the red leds were gaalas-based with a maximum of wavelength 680 nm and half the bandwidth of 20 nm while the white leds used were gan-based leds with a peak at about 465 nm (steren, mexico). since the intensity of light decreases inversely by the square of the distance the leds were mounted directly to the base of the erlenmeyer. each led illuminated flask base consisted of 4 components as shown in fig. 2a: (1) led, (2) parabolic mirror, (3) lens and (4) a circular base made of pvc. an assembled 1-l photobioreactor is shown in fig. 2b (right) covered with aluminum to exclude external light sources. fig. 2. photographic detail of the elements of the experimental system for the production of microalgae with leds that shows in the photo on the left: (a) led, lens, parabolic mirror and a circularring that provides the base support for an erlenmeyer flask; to the right: (b) is a flask in its base protected from external light with aluminum foil. this scheme allowed for an illumination intensity equal to that of fluorescent lighting of 60 µm m–2 s–1 as measured at the middle of the suspension culture (around the 300 ml level). leds fail to generate any significant heating and thus leds can be placed very close to bottom of flasks without any heat stress to the microalgae suspension, leds do not generate heat and the temperature was measured to be 23 ± 2 °c. a warm-white fluorescent tube (mexico general electric co., mexico) was positioned horizontally along the side of the three control group flasks and the distance of the flasks from the fluorescent lighting source was adjusted to match that of the leds light intensity of 60 µm m–2 s–1. the distance from the light source from the fluorescent (control) which was provided horizontally was adjusted so the horizontal lights provided were of the same intensity as the light from the leds from the bottom of the flask at mid level 200 ml. it is important to note that although the control light was provided horizontally and the experimental light was provided from the bottom of the flask, it is assumed that on average both microalgae cultures received similar light intensity doses due to fact that all flasks were air bubbled to provide mixing. the light intensity was measured using the 84022 sper scientific intensity meter (sper scientific ltd., scottsdale, az, usa) in lux and converted to µm m–2 s–1. in figure 3 is the actual system with leds assembled without flasks (left photograph), while on the right is the actual system assembled with flasks in place.     fig. 3. photographic detail of the elements of the experimental system shows : (a) the system with leds inside the base, and (b) the system with the 1-l flasks in light protected bases with external aluminum foil (rl = red led, wl = white led, fl = fluorescent light, i.e. the control group). the three flasks on the right are the control group and are illuminated on the side with a fluorescent light bulb. cell density at time zero and after each 5 days of light treatment the microalgae cell densities and cell size distribution were measured using a coulter counter 9 all res. j. biol, 2013, 4, 7-15 (multisizer ii; beckman instruments, inc., usa) following the procedure described by sheldon and parsons (27). the coulter counter not only provides total numbers but categorizes numbers of cells according to size. a 1:20 dilution was prepared by removing 1 ml of cell suspension per flask and adding of 19 ml of saline containing 10% buffered formol. the data of the coulter counter was converted by accucomp® software and exported to a spreadsheet to calculate cell densities. count numbers were multiplied by 20 (dilution factor). samples were measured in triplicate. size distributions due to light treatments were not obtained from this data since it has been documented that size numbers can be inaccurate with coulter counter when cells are nonspherical (28, 29, 30) or due to shrinkage caused by formaldehyde (31) to prevent cell growth. productivity (p) to obtain productivity (p) in units of mg l–1d–1, the total dry weight of lyophilized microalgal cells was calculated by dividing its harvested dry biomass by culture volume (l) concentration and by the 5 days of light treatment. determining specific growth rate (µe) the specific growth rate of microalgae was calculated using the equation:   (1) where µe = specific growth rate (d –1), n1 and n2 are the number of cells.ml–1 at the time t1 and t2, respectively. duplication time the time for the number of cells (cells.ml– 1) of microalgae to double in number was calculated using the equation:   (2) where tg (days) is the time for cell duplication and µe is the specific growth rate, and ln2 is the natural logarithm of 2 (approx 0.693). this equation is derived from equation 1 by setting n2 = 2n1 and tg = t2-t1. microscopic measurement of cell size live cell sizes after light treatment were measured with a mytek usb 2.0 digital microscope camera coupled to a microscope. a total of 9 cells were measured per each light treatment. protein quantification protein content after the 5 days of light treatment was carried for all samples out based on the procedure of lowry, et al. (32). the principle of this technique is based on the oxidation of the peptide bonds of the folin-ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid), after treatment with an alkaline solution of copper, which results in a blue color with intensity dependent on peptide content. after lyophilization dry weight samples weighing 5 mg were placed in test tubes, then 5 ml of 0.1n naoh was added to hydrolyze the proteins and then placed in thermal bath brand techne te-8j at 100 °c for 1 hour. the samples were allowed to cool to room temperature, and then the mixture was centrifuged at 3,000 rpm for 20 min at 10 °c. the extract was separated by pasteur pipette. two solutions were prepared: (1) na2co3 to 2% (w/v) in 0.1n naoh (solution a) and (2) 0.5% cuso4 in 1% k-tartrate and stirred (solution b). an aliquot of sample (alkaline extract) plus 0.1 n naoh to complete 1 ml was added in a test tube, and 5 ml of solution a and b in a 50:1 ratio was added, vortexed and was let to stand for 15 minutes. later the mixture was added to 500 µl of folin:h2o in a ratio of 1:1. the mixture was vortexed and allowed to stand in the dark for 40 minutes. the calibration curve was prepared from a standard solution of bovine albumin (bsa) with a concentration range of 0 150 µg/ml (0, 30, 60, 90, 120 and 150 µg/ml). absorbance of the extract was measured at 750 ηm in a thermo spectronic genesys 20 spectrophotometer. carbohydrate quantification the carbohydrate content was determined using the method of dubois et al. (33). the basis of this method is based on hydrolyzing glycosidic bonds forming polysaccharides and disaccharides to be converted to monosaccharides. the complexes vary in color from yellow to orange, depending on the amount of carbohydrates present in the sample. lyophilized samples of 5 mg were weighed and placed in test tubes, then 1 ml of sulfuric acid (h2so4) 1.0 n and the mixture was placed in an ultrasonic cleaner branson 200 for 5 min. then another 4 ml of sulfuric acid (h2so4) 1.0 n was added to the mixture and the test tubes were sealed with aluminum foil along with a screw cap. these test tubes were placed in a thermal bath brand techne te-8j at 100 °c for 1 hour. the test tubes with mixture were e l tg µ η2 = 10 all res. j. biol, 2013, 4, 7-15   regime response time light:dark period (hours) mean cell density (cells ml–1) days wl rl fl wl rl fl 0 12h:12h 12h:12h 12h:12h 2.66 ± 0.16x10 6 2.56 ±0.14x106 2.93 ± 0.19 x 106 5 12h:12h 12h:12h 12h:12h 2.94 ± 0.20x10 6 2.68 ±0.11x106 7.75 ± 1.01 x 106 removed and allowed to cool to room temperature. centrifuged at 3,000 rpm for 15 min at 10 °c and then separated the acid extract with a pasteur pipette. an aliquot of sample (acid extract) plus 1.0 n h2so4 to complete 1 ml was added into a test tube, and 1 ml of 5% phenol was added, mixed with a vortex and allowed to stand for 40 minutes. then 5 ml of concentrated sulfuric acid (h2so4) was slowly added in a fume hood, mixed with a vortex and cooled to room temperature. absorbance was measured at 485 ηm in a spectrophotometer thermo spectronic genesys 20 mark. the results were compared against a calibration absorbance curve prepared with 0, 24, 48, 72, 96, and 120 µg/ml, from a standard glucose solution of 120 µg/ml. lipid quantification lipids were extracted according to bligh and dyer (34) adapted to micro determinations. around 5 to 20 mg of freeze-dried sample was placed in individual test tubes, then 3 ml of chloroform/methanol/water (1:2:0.5) and 5 µl of antioxidant bht (1 mg ml-1) were added. the mixture was placed in an ultrasonic cleaner branson 200, then samples were incubated for 24 h at 4 °c protected from light. afterwards 2 ml of chloroform and 3 ml of water were added, mixed with a vortex and centrifuged at 3,000 rpm for 10 min at 10 °c. the bottom fraction (chloroform) was removed and placed in a test tube. an aliquot of sample (chloroform extract) was placed in a different tube and dried with n2. then 2 ml of concentrated sulfuric acid (h2so4) was added and the test tubes were sealed with aluminum foil along with a screw cap. this mixture was burned at a temperature of 200 °c for 15 minutes according to marsh and weinstein (35). then the fraction was allowed to cool to room temperature and placed in test tubes in an ice bath with 3 ml of distilled water and mixed with a vortex until the entire sample was homogeneous and no residual organic matter was visible. burned lipids have brown color directly related with quantity. for the calibration curve a known quantity of lipids of 0, 30, 60, 90, 120, 150 and 180 µg was used from microalgae. absorbance was measured at 375 ηm in a spectrophotometer thermo spectronic genesys 20 mark. statistics data was analyzed using statistica 6 (statsoft, tulsa, ok). the shapiro-wilk test was carried out and was found that all data had a normal error of distribution. subsequently, the bartlett´s box test was run to insure sample variance was equal across samples or homogenous. afterwards, analysis of variance (anova) was run in conjunction with tukey’s test (p <0.05) method for unplanned comparisons of means. for all measurements the number of samples consisted of three replicates per light treatment and was repeated three times (3x3, n=9). results cell density the cell density did not increase significantly in red or white leds after 5 days of treatment (p >0.05), however the cell density of the control group with fluorescent lighting did increase significantly based on tukey's test (p <0.05) after 5 days from 2.93 x 106 to 7.75 x 106 cells/ml (table 1) a distinct difference when compared to red and white leds. table 1. cellular density of isochrysis aff. galbana (t-iso) at time 0 and after 5 days of light treatment with white leds (wl), red leds (rl) and fluorescent lights (control) at 60 µm mol m–2s–1 (n=9). productivity and specific growth rates comparison of productivity (mg.l–1day–1) values of microalgae indicate that productivity was worse for red and white leds ~4 x compared to the control or fluorescent lighting (p < 0.05; table 2). this is also reflected in lower specific growth rates (µe) for white leds (4.8 x smaller) and red leds (4.2 x smaller) compared to the fluorescent lighting control group. growth or duplication time the best or lowest mean duplication time (tg) was 3.7 days with fluorescent light control group and was significantly different from both led light treatments (p < 0.05, table 3). the 3.7 day duplication time measured in i. galbana suggests at 5 days of light treatment the microalga had reached its maximum population growth with fluorescent lighting and had entered the saturation growth phase with delayed cell growth. we assume this because typical values of cell doubling time replication (tg) in the exponential phase for i. aff. galbana (t-iso) is less than a day, for example approximately 9.5 h in other studies (36). 11 all res. j. biol, 2013, 4, 7-15 the values of cell replication (tg) for white and red leds were very high, 38.4 and 18.7 days, respectively, indicating that with these light sources the microalgae are not growing and suggest that microalgae are in stasis or a photorespiration phase, probably waiting for better light conditions for reproduction. a simple ratio comparison of duplication times indicates that with both white and red leds duplication was 10.4 x and 5 x longer, respectively, than with fluorescent lighting (control). table 2. isochrysis aff. galbana (t-iso) productivity, p; specific growth rate growth, µe (d–1); and time in days for cellular duplication, tg after 5 days of light treatment with white and red leds and fluorescent light at 60 µm mol m–2s–1(n =9).   light source white led red led fluorescent parameter p µe tg p µe tg p µe tg (units) mg l–1d–1 (d–1) (d) mg l–1d–1 (d–1) (d) mg l–1d–1 (d–1) (d) mean 8.43 0.039 38.4 8.12 0.045 18.7 32.36 0.189 3.7 ± std dev 2.98 0.040 3.4 0.32 0.023 9.3 4.72 0.024 0.5   cell size obtained by microscopic photographs digital images were taken (fig. 4) after the light treatments (day 5) and showed that the size of the microalgae i. aff. galbana (tiso) is larger with fluorescent light (mean value of 49 µm2), followed in size with white led (mean value of 36.5 µm2, p < 0.05), and smallest with red leds (mean value of 22.6 µm2, p < 0.05), which corresponds to sizes that are 1.3 x and 2.2 x smaller, respectively, compared to fluorescent light or control group (n=9). fig. 4. microscopic photographs of isochrysis aff. galbana (t-iso) showing typical differences in sizes with white led (wl) , red led (rl) and fluorescent light (fl = control group) after 5 days of light treatment in the experimental system at 1000x magnification (n =9). biochemical content mean values of protein, carbohydrates, and lipids of isochrysis aff. galbana at the end of 5 days of culture were very similar with red and white led and fluorescent light (table 3). table 3. protein, carbohydrate and lipid biochemical composition of isochrysis aff. galbana (t-iso) at the end of 5 days of culture with red and white leds and fluorescent light (n =9).                     *fl=  fluorescent  light  (control) discussion according to the present findings the cell density growth rate did not significantly differ from zero after 5 days of light treatment in red and white leds. this result differs from the limited studies with leds with other microalgae, dinoflagellates or cyanobacteria where growth is observed irrespective of whether treated with either red or blue leds (17,18,19,20). however, this is significant for as far as the authors are aware this is the first study that isochrysis aff. galbana (t-iso) has been cultivated, or attempted to be cultivated with led-derived light sources, herein monochromatic red and multi-chromatic white leds. also, all previous reported studies with leds and microalgae have used green microalgae, while i. aff. galbana (t-iso) is a golden-brown microalga. the result is surprising since it is well known that in i. aff. galbana the main pigments are chlorophyll a, c1, c2 and fucoxanthin (37). chlorophyll a is considered generally to be sufficient to drive photosynthesis and it strongly absorbs in the red and blue region. however, the importance of antenna   biochemical content light source proteins carbohydrates lipids mean ± std mean ± std mean ± std (mg/g) (mg/g) (mg/g) white led 301 ± 28 75 ± 19 388 ± 68 red led 322 ± 57 87 ± 37 356 ± 77 *fl 276 ± 47 80 ± 10 378 18   12 all res. j. biol, 2013, 4, 7-15 pigments photon capture is well recognized at least in cyanophytes. the transfer of energy trapped by phycobilisomes (pbs) to the chlorophyll a of photosystem i (ps i) or photosystem ii (ps ii) is regulated either at the transfer point from the pbs to the two photosystems or at a transfer point between the chlorophyll a of ps ii and ps i (38). it is important to note, that white light leds used in this study are actually mainly a high power blue emitter led with some phosphor in conjunction, the current technical solution and as such is not true white light. one possibility is that there was not enough energy at the specific wavelength to activate antenna pigments like fucoxanthin. it is hypothesized that although cell sizes were very different, the percentages or ratio of biochemical content was maintained irrespective of biomass loss. this may be a fitness strategy that the microalgae have maintained during inadequate light intensity or wavelength in order to survive while waiting for better light resources for reproduction. herein, we also report that red and white led-derived light leads to a reduction in cell size compared with fluorescent lighting in this non-green microalga, isochrysis aff. galbana (t-iso). studies of microalgal cell size changes induced by light quality are few. however, we found at least one study in the literature that reports a similar effect, but with a green freshwater microalga. lee and palsson (18) reported that with chlorella vulgaris a narrow-spectrum monochromatic red light reduced average cell volume from 60 to 30 µm3 cell–1, and also found by switching light sources to full spectrum that the two dissimilar cell population states were interchangeable. another possibility of the size reduction observed with red and white leds is that the light fluence level applied is below the light intensity compensation point (38) and the microalga is smaller simply because its respiration rate is higher than its photosynthetic rate and it utilized its energy stored. we suggest that this indeed may be a possibility, but not because the fluence rate is not high enough, 60 µm m–2 s– 1 is more than sufficient, but possibly because isochrysis aff. galbana (t-iso) needs another specific wavelength to grow. in summary, the data in this study suggest that isochrysis aff. galbana (t-iso) needs a wavelength possibly a green wavelength to grow and divide other than the red and white leds provided for in this study for the following reasons: (i) it is generally accepted that the minimum photosynthesis level for unicellular photoautotrophic growth, although at low growth rates, is at 2 µm m–2 s–1 (40) our fluence rate was well above this at 60 µm m–2 s–1, (ii) lee and palsson (18) observed growth in chlorella (a green microalgae) with narrow-bandwidth red light with leds, but their microalgae was green with obviously different pigments or ratio of pigments, (iii) isochrysis aff. galbana (t-iso) is a yellowbrown microalgae haptophyte possibly indicating it has pigments like fucoxanthin as part of the main auxiliary pigments (38) which absorbs between 480 and 530 nm (41) mostly corresponding to green light, and (iv) many earlier landmark studies with photosynthesis and with microalgae have shown that a combination of different wavelengths affects photosynthesis rates (39) and this is due to accessory pigments (42); however, most of these studies were conducted with green or red microalgae. conclusion hence, the results suggest that the non-growth of isochrysis aff. galbana, a golden-brown colored haptophyte, with red or white (mainly blue) leds is because the microalgae needs another wavelength to activate auxiliary pigments. this conclusion is supported by two facts: (1) the microalgae did not divide with leds (numbers were essentially the same before and after light treatment with leds) and (2) the size of the microalgae with leds was significantly smaller compared to fluorescent (control) light treatments, suggesting that with leds the microalgae was only in a respiration phase and was using its energy stores. it is also important to note that this microalga is a different pigmented microalga than that was used in the earlier pioneering studies of photosynthesis which used green and red microalgae, a possibly reason why this response has not been previously reported. moreover, it is still unclear whether a single wavelength or combinations of wavelengths are necessary to influence photosynthesis or photomorphological responses in this microalga studied, i.e. isochrysis aff. galbana. further light studies will be required to confirm and disentangle the light processes in this microalga. acknowledgments we thank centro investigaciones biológicas del noroeste (cibnor) for supporting this research with the project ac0.2. the authors especially thank juan francisco villa medina, jorge cobos anaya, alfonso alvarez casillas, marte félix virgen, julián alfonso garzón favela, adriana greene yee, cynthia elizabeth aldana avilés, eleonora puente carreón and rosa linda salgado garcía whose technical assistance at cibnor was indispensable in carrying out the work. mvc thanks conacyt for fellowship grant no. 211773.   13 all res. j. biol, 2013, 4, 7-15 references 1. cohen z [ed.] chemicals from microalgae. taylor & francis inc. usa. 1999; 419 pp. 2. arad s, richmond a. industrial production of microalgal cell-mass and secondary products–species of high potential: porphyridium sp. in: richmond, a. 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properties of algal pigments. in: spinrad rw (ed.) ocean optics. x proc soc. photo-opt. instrum eng 1990; 1302: 90-302. 42. haxo ft. the wavelength dependence of photosynthesis andthe role of accessory pigments. in: allen mb (ed) comparative biochemistry of photoreactive pigments. academic press, new york, 1960; pp 339–361. 15 microsoft word 121-713-3-le.doc in vitro study of modulatory effects of strobilanthes crispus extracts on human cdna-expressed cytochrome p450 2a6 (cyp2a6) and cyp3a4 pan yan*, hsu chia jie, koh rhun yian, ong chin eng, and chieng jin yu all res. j. biol., 2016, 7, 1-12 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 1, vol 7, 2016, 1-12 in vitro study of modulatory effects of strobilanthes crispus extracts on human cdna-expressed cytochrome p450 2a6 (cyp2a6) and cyp3a4 pan yan1*, hsu chia jie2, koh rhun yian2, ong chin eng3, and chieng jin yu4 1) department of biomedical science, the university of nottingham malaysia campus, jalan broga, 43500 semenyih, selangor darul ehsan, malaysia. 2) school of medical sciences, international medical university, 126, jalan 19/155b, bukit jalil, 57000 kuala lumpur, malaysia. 3) jeffrey cheah school of pharmacy and health sciences, monash university sunway campus, jalan lagoon selatan, 46150 bandar sunway, selangor darul ehsan, malaysia. 4) faculty of medicine and health sciences, universiti putra malaysia, 43400 upm serdang, selangor darul ehasan, malaysia graphical abstract abstract: aim: cytochrome p450 2a6 (cyp2a6) and cyp3a4 play important roles in biotransformation of endogenous substances as well as xenobiotics. strobilanthes crispus (l.) blume (s. crispus) has been found to have anticancer activities and it has been suggested that this is due to inhibition of enzymes involved in metabolic activation of procarcinogens. the purpose of this study was to look into the potential inhibitory effects of various extracts (aqueous, hexane, chloroform, ethyl acetate, and methanol) of s. crispus from leaf and stem on human cdna-expressed cyp2a6 and cyp3a4 activities. methods: the activity of cyp2a6 was examined via a fluorescence-based 7-hydroxylase coumarin assay. meanwhile, a high performance liquid chromatography (hplc)-based testosterone 6β-hydroxylase assay was performed to assess cyp3a4 activity. results: it was shown that none of the extracts from either leaf or stem potently inhibited cyp2a6 or cyp3a4 activities, with 50% inhibitory concentration (ic50) values above 100µg/ml. conclusion: s. crispus is unlikely to display anti-cancer properties through the modulation of cyp2a6 or cyp3a4 activities, but other mechanisms might be involved and merit further investigation. in addition, potential drug-herb interaction occurring between cyp2a6/cyp3a4 substrates and s. crispus preparations would probably be limited in effect, but this requires further investigations via in vivo animal as well as clinical studies. keywords: cyp2a6; cyp3a4; drug-herb interaction; procarcinogen-activation; strobilanthes crispus 1 all res. j.biol, 2016, 7, 1-12     introduction cytochrome p450s (cyps) are essential enzymes widely distributed within almost all living organisms. in mammals, they are responsible for biotransformation of a wide spectrum of endogenous compounds including fatty acids, cholesterol, steroids, retinoids, vitamin d derivatives, and bile acids. these enzymes are also involved in the metabolism of numerous xenobiotics such as environmental chemicals, pollutants, natural plant products and medicinal drugs.1 among human cyp families, the cyp1, cyp2, and cyp3 families are mainly involved in metabolism of exogenous substances while the cyp4 family contributes to a smaller extent.2 reactive oxygenated intermediates can be formed from parent compounds catalysed by cyps, and are able to covalently bind to dna and proteins, leading to carcinogenesis. the damage to dna or protein by reactive oxygenated intermediates may directly affect various stresssignalling and/or checkpoint-signalling pathways.3-4 if this continues for a matter of decades, human carcinogenesis processes are set in motion. in addition to initiating carcinogenesis, cyps can also be involved in cancer promotion or cancer progression by influencing various signal transduction pathways. consequently, the cell cycle may be altered and apoptosis or aberrant cell growth may be take place. cyp2a6 accounts for approximately 10% of human microsomal cyp proteins, contributing to the biotransformation of nicotine and cotinine as well as some pharmaceuticals (such as fadrozole, tegafur etc.) and a number of coumarin-type alkaloids.5-6 attention has been focused on cyp2a6 after nicotine and some tobacco-specific nitrosamines were discovered to be high-affinity substrates for this enzyme. 4-(methyl-nitrosamino)-1-(3-pyridyl)-11butanone (nnk), one of the most potent and abundant procarcinogens contained in tobacco smoke, is mainly activated to a mutagenic reactive metabolite via the cyp2a6 metabolic pathway.7-8 moreover, cyp2a6 genotypes associated with reduced metabolic activity are associated with a lower risk of developing lung cancer.9 it has been postulated that employment of a cyp2a6 inhibitor such as methoxsalen could lower cancer risk.10 cyp3a4, the most abundant cyp isoform in the human liver, is responsible for metabolizing more than 50% of drugs used today, including anti-cancer agents (eg. gefitinib and imatinib). coadministration of gefitinib with cyp3a4 inhibitor itraconazole resulted in increased exposure to gefitinib.11 serious adverse reactions to anti-cancer drugs are often reported, and concomitant administration of natural products with cyp3a4-metabolised drugs is one of the major causes. for example, grapefruit juice reduces dehydrofelodipine/ felodipine auc ratio and increases absolute dehydrofelodinpine auc by inhibiting cyp3a412. moreover, cyp3a4 is able to activate certain procarcinogens, such as aflatoxin b1.13-15 strobilanthes crispus (l.) blume (s. crispus) is a traditional medicinal plant in malaysia and indonesia known locally as ‘pecah kaca’ or ‘jin batu’ in malaysia and ‘pecah beling’ or ‘kecibeling’ in indonesia. its leaves have been used to treat various conditions including breast and uterine cancers and gastrointestinal and kidney diseases.16-17 crude extracts of this plant have been found to be cytotoxic to human cancer cell lines and protective against chemically-induced hepatocarcinogenesis in rats.18-19 administration of s. crispus extract also reduced the severity of hepatic necrosis in rats with diethylnitrosamineand acetylaminofluorene-induced hepatocellular carcinoma and this was suggested to be due to the inhibition of enzymes involved in metabolic activation of the carcinogens.20 this in vitro study was designed specifically to look into the modulatory effects of various s. crispus extracts on cyp2a6 and cyp3a4 activities, which may serve as one of the mechanisms involved in s. crispus’s anti-cancer properties. additionally, this study was able to provide information to that suggests potential drug-herb interactions. methods reagents isopropyl β-d-1-thiogalactopyranoside (iptg), tris-base, ethylenediaminetetraacetic acid (edta), dithiothreitol (dtt), and glycerol were purchased from promega (madison, wi, usa). terrific broth media were purchased from invitrogen corporation (carlsbad, ca, usa). organic solvents were purchased from merck (darmstadt, germany). all other chemicals were purchased from sigma-aldrich (st. louis, mi, usa). preparation of s. crispus extracts fresh leaves and stems of s. crispus were collected from paka, terengganu, malaysia. a sample of the plant was deposited at forest research institute malaysia (voucher specimen number: pid 040114-04). the plant materials were oven dried at 60 °c and then separated into leaves and stems, and subsequently blended into smaller pieces. the fine 2 all res. j.biol, 2016, 7, 1-12     pieces of leaves and stems of s. crispus were prepared separately in 10 different extracts (5 from leaves and 5 from stems). water extraction was prepared by mixing the plant materials (50 g) with distilled water (500 ml). the mixture was macerated overnight at room temperature and then maintained at 60 °c for 3 hours. the water extract was then filtered through filter paper. the filtrate was freeze-dried to obtain powder form of the water extract. approximately 12 g of leaf extract and 9 g of stem extract were obtained by these procedures. organic solvent extractions were prepared by mixing the plant materials (100 g) with hexane, ethyl acetate, chloroform or methanol (500 ml) and macerating for 3 days at room temperature. the resulting suspensions were filtered and the organic solvents evaporated in a rotary evaporator. the procedures yielded approximately 2 g of leaf extract using hexane as the solvent, 7 g using ethyl acetate, 12 g using chloroform and 10 g using methanol. extraction from the stems yielded approximately 1.5 g using hexane, 5 g using ethyl acetate, 9 g using chloroform and 5 g using methanol. co-expression of cyp2a6 and cyp3a4 with nadph-cyp reductase in e. coli human cyp2a6 and cyp3a4 cdna were cloned into the bacterial expression vector pcwori+, as detailed in previous articles [21-22 respectively]. pcw-2a6/pcw-3a4 were subsequently co-transformed with pacyc plasmid containing nadph-cyp reductase cdna into escherichia coli dh5α competent cells to achieve optimum activity. three-hundred and fifty milliliters terrific broth (tb) medium culture was inoculated with 3.5 ml overnight luria– bertani (lb) culture of co-transformed bacteria. induction of cyp expression was initiated by the addition of 1.0 mm iptg and 0.5 mm-aminolevulinic acid (∆-ala) when the optical density of the culture at 600 nm was approximately 0.7. the incubation proceeded for 24 h at 30 °c and 125 rpm in a shaker incubator, after which bacterial cells were harvested and fractionated.23 cells were pelleted in tris– edta–sucrose (tes) buffer (15 ml per gram of wet cells). following lysozyme treatment of the cells (3 mg/ml in tris buffer; 100 µl of lysozyme per gram of wet cells) and centrifugation, the spheroplasts were resuspended in a buffer (20 mm potassium phosphate, ph 7.4, containing 500 mm potassium chloride and 20% glycerol, v/v) and stored at −80°c. spheroplast suspensions were thawed on ice and sonicated in the presence of protease inhibitors (250 µl per gram of wet cells) and phenylemethanesulfonyl fluoride (pmsf) (1 mm) using an ultrasonicator (labsonic®p, sartorius ag, goettingen, germany). the membranes containing the expressed cyp2a6/cyp3a4 were isolated by ultracentrifugation with a beckman nvt65 rotor at 37,000 × g for 75 minutes at 4 °c. the membrane pellets were subsequently resuspended in 50/50 tes/water and stored at −80 °c until use. a control protein was expressed following the same procedures described above, employing authentic pcwori+ instead of pcw-2a6/3a4. coumarin 7-hydroxylase assay full cyp2a6 enzyme kinetic activity was assessed by a fluorescence-based coumarin 7-hydroxylase assay following a published protocol.21 0.31-40 µm of coumarin was incubated and the metabolite 7-hydroxycourmarin was quantified using a tecan infinite® 200 microplate reader (tecan, männerdorf, switzerland) at the excitation wavelength of 365 nm and emission wavelength of 450 nm, which was used to establish a michaelis-menten plot. absorbance data were exported to microsoft excel to acquire pharmacokinetic parameters (km and vmax). testosterone 6β-hydroxylation assay the evaluation of the cyp3a4 activity was performed via testosterone 6β-hydroxylation assay using hplc according to our published method.24 1 to 500 µm testosterone was incubated and the metabolite 6-hydroxytestosterone was measured by an hplc system equipped with a gilson 307 pump, a gilson uv/vis-152 detector, and an ods hypersil column (thermo, 4.6 x 250 mm, 5 µm). the mobile phase used was 70% methanol/water and was pumped at a flow rate of 1 ml/min at a wavelength of 242 nm. quantification of metabolites was achieved by comparing the peak area ratio of the metabolite with that of an internal standard, corticosterone. inhibition studies with s. crispus extracts aqueous extracts were dissolved in water and organic solvent extracts were dissolved in dmso. the percentage of dmso was kept as 1% (to retain enzyme activity) in all reactions except those involving aqueous extracts. ic50 values were determined to estimate the inhibitory potencies for various s. crispus extracts (aqueous, chloroform, ethyl acetate, methanol and hexane) from stem and leaf separately. this was achieved by incubating co-expressed cyp2a6 or cyp3a4 and nadph-cyp reductase proteins (50µg per reaction) with coumarin (5 µm, close to km) or testosterone (100 µm, close to km) in the absence (set as control) and presence of various concentrations of s. crispus aqueous extract (up to 500 µg/ml) or organic solvent extracts (up to 3 all res. j.biol, 2016, 7, 1-12     100 µg/ml), and subsequently the formation of 7-hydroxy coumarin and 6-hydroxytestosterone were quantified by the assay described in the previous sections. the cyp2a6/cyp3a4 enzyme activities for various concentration of each extract was calculated on the basis of its activity relative to the control reaction (set as 100%). data analysis enzyme kinetics (km,and vmax values) were ascertained via non-linear regression analysis with prism version 6.00 for windows (graphpad software, la jolla california usa, www.graphpad.com). ic50 values were estimated by nonlinear regression analysis with the help of sigmaplot™ (version 12.0, systat software inc, usa). results kinetics of cyp2a6 and cyp3a4 enzyme assays the plots shown in figure 1 are consistent with and typical of thel michaelis-menten kinetic model {v=vmax[s]/(km+[s])}, in which, by increasing coumarin concentration, the reaction velocity (v) follows a hyperbolic pattern and reaches the maximum velocity vmax. km, known as the michaelis dissociation constant, reflects how well the enzyme and substrate interact. it is the substrate concentration at which the reaction rate is half of its maximum. the km and vmax values derived from figures 1a and b were 3.9±1.4 µm (mean± std. error) and (1261±135.1pmol/min/mg protein (mean± std. error) for cyp2a6; 86.2±16.3 µm (mean± std. error) and (4176.7±200.4 pmol/min/mg protein (mean± std. error) for cyp3a4. b c o n c e n t r a t i o n o f t e s t o s t e r o n e (µ m ) v e lo c it y ( p m o l/ m in /m g p ro te in ) 0 2 0 0 4 0 0 6 0 0 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 figure 1: representative michaelis–menten plot of cyp2a6 (a) and cyp3a4 (b) activities versus coumarin and testosterone as substrates. points are experimentally determined values while solid lines are computer-generated curves of best fit. each data point was found by taking the mean of 3 experimentally determined values. inhibition studies with s. crispus extracts the ic50 values determined for the effects of s. crispus leaf and stem aqueous extracts on cyp2a6 were 399.1 and 390.9µg/ml respectively (figures 2a and 3a). ic50 values of all solvent extracts from both leaf (figure 2b, c, d, and e) and stem (figure 3b, c, d, and e) samples were more than 100µg/ml. in the case of cyp3a4, ic50 values of leaf and stem extracts were more than 500µg/ml (aqueous extracts) and 100µg/ml (solvent extracts) (figures 4a, b, c, d, and e; figures 5a, b, c, d, and e). table 1 shows the percent control activities of various s. crispus solvent extracts at a concentration of 100µg/ml. figure 2a s. crispus leaf aqueous extract (mg/ml) 0 100 200 300 400 500 600 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 concentration  of  coumarin  (μm)   4 all res. j.biol, 2016, 7, 1-12     figure 2b s. crispus leaf hexane extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120 figure 2c s. crispus leaf chloroform extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120 figure 2d s. crispus leaf ethyl acetate extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120 figure 2e s. crispus leaf methanol extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120 figure 2. effects of s. crispus leaf extracts on cyp2a6-mediated coumarin 7-hydroxylase activity: aqueous extract (2a), hexane (2b), chloroform (2c), ethyl acetate (2d), and methanol (2e). points are experimentally determined values, while solid lines are best-fit curves generated by sigmaplot for windows version 12.0. the x axis represents the s. crispus extract concentrations (µg/ml), while the y axis represents the percentages of control activity. data points are the average values of duplicate determinations. 5 all res. j.biol, 2016, 7, 1-12     figure 3a s.crispus stem aqueous extract concentration (mg/ml) 0 200 400 600 p er ce nt c on tro l a ct iv ity (% ) 30 40 50 60 70 80 90 100 figure 3b s. crispus stem hexane extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120     figure 3c s. crispus stem chloroform extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120       figure 3d s. crispus stem ethyl acetate extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120 figure 3e s.c rispus stem methanol extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120 figure 3. effects of s. crispus leaf extracts on cyp2a6-mediated coumarin 7-hydroxylase activity: aqueous extract (2a), hexane (2b), chloroform (2c), ethyl acetate (2d), and methanol (2e). data points are experimentally determined values, while solid lines are best-fit curves generated by sigmaplot for windows version 12.0. the x axis represents the s. crispus extract concentrations (µg/ml), while the y axis represents the percentages of the control activities. each data point is the average value of duplicate determinations.   6 all res. j.biol, 2016, 7, 1-12     figure 4a s. crispus leaf aqueous extract concentration (mg/ml) 0 100 200 300 400 500 600 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120     figure 4b s. crispus leaf hexane extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120   figure 4c s. crispus leaf chloroform extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120       figure 4d s. crispus leaf ethyl acetate extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120   figure 4e s. crispus leaf methanol extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120       figure 4. effects of s. crispus leaf extracts on cyp3a4-mediated testosterone 6β-hydroxylase activity: aqueous extract (2a), hexane (2b), chloroform (2c), ethyl acetate (2d), and methanol (2e). data points are experimentally determined value, while solid lines are best-fit curves generated by sigmaplot for windows version 12.0. the x axis represents the s. crispus extract concentrations (µg/ml), while the y axis represents the percentages of the control activities. data points are average values of duplicate determinations.   7 all res. j.biol, 2016, 7, 1-12     figure 5a s. crispus stem aqueous extract concentration (mg/ml) 0 100 200 300 400 500 600 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120   figure 5b s. crispus stem hexane extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120   figure 5c s. crispus stem chloroform extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120         figure 5d s. crispus stem ethyl acetate extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120     figure 5e s. crispus stem methanol extract concentration (mg/ml) 0 20 40 60 80 100 120 p er ce nt c on tro l a ct iv ity (% ) 0 20 40 60 80 100 120   figure 5. effects of s. crispus stem extracts on cyp3a4-mediated testosterone 6β-hydroxylase activity: aqueous extract (2a), hexane (2b), chloroform (2c), ethyl acetate (2d), and methanol (2e). data points are experimentally determined values while solid lines are best-fit curves generated by sigmaplot for windows version 12.0. the x axis represents the s. crispus extract concentrations (µg/ml), while the y axis represents the percentages of the control activities. data are the average values of duplicate determinations. 8 all res. j.biol, 2016, 7, 1-12     table 1. percent control activity of s. crispus solvent extracts at a concentration of 100 µg/ml: cyp isoform solvent percent control activity (%) cyp2a6 leaf hexane 90.6 chloroform 74.5 ethyl acetate 81.5 methanol 61.2 stem hexane 74.3 chloroform 64.8 ethyl acetate 84.9 methanol 72.5 cyp3a4 leaf hexane 93.5 chloroform 89.0 ethyl acetate 96.7 methanol 73.3 stem hexane 65.5 chloroform 55.1 ethyl acetate 54.2 methanol 88.1 discussion km is thought to be relatively consistent from one study to another. the km value acquired for cyp2a6-mediated coumarin 7-hydroxylase assay derived from figure 1a was 3.9±1.4µm (mean± std. error), which fell within the reported range (1.7-10.6 µm).25-28 similarly, the km value determined for cyp3a4-mediated testosterone 6β-hydroxylase assay was 86.2±16.3µm (mean± std. error), which was compared with previously reported values (see figure 1b).22, 29-31 the major factors contributing to the slight deviation from values established in previous studies were considered to be different sources of cyp enzymes, incubation conditions, and co-factor variables. in comparison to these small deviations, vmax values (1261±135.1 pmol/min/mg cyp2a6 protein; 4176.7±200.4 pmol/min/mg cyp3a4 protein, mean± std. error) were shown to have a bigger difference from values established in previous other studies. this is not unusual since the vmax value is a function of the expression level of enzyme, which varies based on enzyme concentration and incubation conditions. because of this, the in vitro cyp2a6 and cyp3a4 enzyme systems served as an activity marker for the following inhibitory screening assays. it has been suggested that one of the anti-cancer mechanisms of s. crispus may be the inhibition of cyp isoforms, which are able to bio-activate procarcinogens.32 the initial intention of this study was to evaluate the inhibitory potencies of various s. crispus extracts prepared from both leaf and stem on cyp2a6-coumarin 7-hydroxylation as well as cyp3a4testosterone 6β-hydroxylation, which might account for the reported anti-cancer properties of this plant, and potentially enable it to be used in cancer prevention. aqueous and organic solvents ranging from polar to nonpolar (methanol, chloroform, ethyl acetate and hexane) were used to do extractions, to provide a complete screening outcome covering a wide spectrum of active constituents. leaf and stem samples were collected and processed separately since they are the most commonly consumed portion of s. crispus. nevertheless, none of the extracts demonstrated potent inhibition towards cyp2a6 and cyp3a4 activities, with ic50 values more than 100µg/ml (figure 2-5). it was suggested that the anti-cancer potential of s. crispus was unlikely to result from modulation of cyp2a6 or cyp3a4 activity. hence, alternative mechanisms and pathways must be assumed to play more important roles. for example, s. crispus extract has been shown to induce apoptosis in breast cancer cells via mitochondria the dependent p53 pathway.33 alternatively, the anti-cancer effect of the plant extract might be mediated by downregulation of c-myc, an oncogene, in hepatocellular carcinoma.34 in addition, s. crispus extract has been found to possess anti-angiogenic properties and hence inhibit cancer cell growth in that way.35 it is advised that other cyp isoforms also be investigated, such as cyp2e1, which is involved in the metabolism of nitrosamine nnitrosodiethylamine (ndea) in rats’ livers,8 or cyp1a2 and cyp1b1, which play roles in colon cancer carcinogenesiss. crispus extract’s cytotoxicity to this cancer has been demonstrated.36 independence of cyp2a6 and cyp3a4 activities from action of s. crispus extracts was an encouraging finding in terms of drug-herb interaction assessment. known to metabolise the chemotherapy drug tegafur to active component 5-fluorouracil, cyp2a6 enzyme is crucial for patients with malignant cancer. this could be gastric cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, breast cancer, pancreatic cancer or biliary tract cancer.37 the majority of currently prescribed anti-cancer drugs are biotransformed via cyp3a4 pathways, including docetaxel, erlotinib, imatinib, irinotecan, paclitaxel and vinicristine.38 the use of herbal products is popular among cancer patients.39 although herbal products are claimed to be natural and health-improving, concomitant use together with anti-cancer drugs potentially leads to drug-herb interaction, which has become one of the safety issues among cancer patients. in patients who wish to consume s. crispus herbal infusion for anti-cancer or other health-promoting purposes, it is important that it does not affect anti-cancer agents’ 9 all res. j.biol, 2016, 7, 1-12     metabolism rate in the liver or the chemotherapy outcome. found not to affect cyp2a6 or cyp3a4 drug-metabolising activity, s. crispus is believed to be safe to take together with drugs metabolized by cyp2a6 and cyp3a4. however, this finding has to be supported with more clinical data. caution is urged for those taking alternative medicine or herbal remedies. to prevent the possibility of serious adverse reactions due to drug-herb interactions, it is suggested that other cyp isoforms be tested with s. crispus extracts including cyp2c9, cyp2d6, and cyp2c19. these cyp enzymes altogether contribute to the metabolism of approximately 40-50% clinically used drugs. conclusions in vitro enzyme systems have been constructed successfully to be used to evaluate cyp2a6 and cyp3a4 activities. no significant inhibitory effects for aqueous or organic solvent extracts of s. crispus plant were discovered in the current study, suggesting that the anti-cancer properties of this plant involve mechanisms other than the inhibition of the cyp2a6 or cyp3a4 pathways. moreover, co-administration of s. crispus products with drugs metabolised by cyp2a6 or cyp3a4 is unlikely to result in drug-herb interactions, which merits more clinical assessments. acknowledgements the authors would like to thank international medical university for funding (bmsc i01/09 (05)2011) this project as well as biomedical science final year students yew mei yeng, chow pui yee, ten yi yang, teo siew yong and wong jhia yi for assisting with this study. conflict of interest the authors declare no conflict of interest associated with this paper. references 1. gotoh, o. 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(2004). herbal remedies in the united states: potential adverse interactions with anti-cancer agents. j clin oncol. 22(12):2489-503.     12 microsoft word layoutediting.docx evaluation of the toxic potential of aspartame in third instar larvae of transgenic drosophila melanogaster (hsp70lacz) bg9 simmi anjum, rahul, smita jyotiand, yasir hasan siddique* all res. j. biol., 2017, 8, 16-24 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol. 8, 2017, 16-24 evaluation of the toxic potential of aspartame in third instar larvae of transgenic drosophila melanogaster (hsp70-lacz) bg9 simmi anjum, rahul, smita jyotiand, yasir hasan siddique* drosophila transgenic laboratory, section of genetics, department of zoology, faculty of life sciences, aligarh muslim university, aligarh, uttar pradesh202002, india. *corresponding author, email: yasir_hasansiddique@rediffmail.com grafical abstract abstract: aspartame is commonly used as a sweetener in prepared foods, beveragesand recipes. in the present study, the possible toxic effects of aspartame were studied in third instar larvae of transgenic drosophila melanogaster (hsp70-lacz) bg9. the third instar larvae were allowed to feed on the diet having different doses of aspartame. the results obtained in the present study suggest that the exposure of third instar larvae to the diet having 20, 40, 80, 160 and 320 mm of aspartame did not show anysignificant the toxic effects after24hours of the exposure compared to control (p<0.05). the results for pupae formation and theemergence of flies also did not showany significant difference compared to control (p<0.05). keywords: aspartame;drosophila melanogaster;toxicity introduction aspartame is widely used as an artificial sweetener in many soft beverages, cakes and variety of other foods(simintzi et al. 2007).it is a low-calorie artificial sweetener consumed by 200 million people worldwide. it was discovered by james schlatter in 1965 of g.d. searle company and was approved for the use by fda in the 1970s(ashok and sheeladevi 2014).it is a dipeptide, composed of the aminoacids phenylalanine and aspartic acid plus a small quantity of 16 all res. j.biol, 2017, 8, 16-24 methanol(butchko et al. 2002).the acute toxicity of aspartame, tested in rats, mice, rabbits and dogs was very low (marinovich et al. 2013). for more than 30 yearsaspartame has been widely used as a food additive because of its very strong, sweet taste(soffritti et al. 2006). it has 200 times more sweet taste than sucrose(mazur 1984). although fda has approved it after a series oftests, after an experimental demonstrationof it as a multipotential carcinogenic agent at a dose of 20mg/kg body weight,has raised the issue regarding its use in more than 6000 products worldwide(soffritti et al. 2006).the use of animals in toxicological research and testing has become an issue of concern for both science and ethics. as a result, the emphasis has been given on the use of analternative to mammals in research and testing (mukhopadhyay et al. 2003). the fruit fly (drosophila) has been developed as an invivo model organism for the toxicological evaluations due to short life cycle with distinct developmental stages(ging et al. 2014; ong et al. 2015). in conserved domains, the overall identity between fly and mammal is about 80-90%(celotto and palladino 2005). heat shock proteins (hsps) were initially reported to be expressed in response to heat, but their expression is also triggered in response to adiverse range of stress(gupta et al. 2005). in the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals(mukhopadhyay et al. 2003). since there are contrary reports on the toxic potential of aspartame and most of them are reported at very high doses (saleh 2014; reshman et al. 2015; martins et al. 2007; holder and yirmiya 1989; tilson et al. 1991; fisher 1989). hence, we decided to investigate the toxic potential of aspartame on the third instar larvae of transgenicdrosophila melanogaster (hsp70-lacz) bg9. materials and methods fly strain. a transgenic d. melanogaster line expressing bacterial β-galactosidase in response to stress was used in the present study (lis et al. 1983). in the present strain, the transformation vector is inserted with a p-element i.e. the line contains wild-typehsp70 sequence upto lacz fusion point. the flies and larvae were cultured on food (without sugar) containing agar, corn, yeast and propionic acid at 24 ± 1°c (nazir et al. 2003; siddique 2012). experimental design. the aspartame was dissolved in distilled water, and the final concentrations of 20, 40, 80, 160, and 320 mm were established in the diet. the third instar larvae were allowed to feed for 24 hours. to study the effect of aspartame at same selected doses on pupation and emergence of flies separate experiments were set. the larvae are voracious feeders and are routinely used to study developmental and physiological processes. the future adult structures of the fly are contained within the larvae, and for the primary high throughput screening, third instar larvae are widely accepted. hence, we also decided to study the effect of aspartame on the pupation and emergence of flies. soluble o-nitrophenyl-β-d-galactopyranoside (onpg assay).the expression of hsp70was quantified by performing soluble o-nitrophenyl-β-d-galactopyranoside (onpg) assay asdescribed by nazir et al. (2003). after giving a wash of phosphate buffer, the larvae were placed in microcentrifuge tubes (30 larvae/tube, five replicates/group), permeabilized for 10 min by acetone and incubated overnight at 37°c in 600µl of onpg buffer. after incubation for the desired duration, the reaction was stopped by adding 300µl of na2co3, and the extent of the reaction was quantified by measuring absorbance at 420 nm (chowdhuri et al. 1999; 2001). in situ histochemical β-galactosidase activity. the larvae (30 larvae/treatment; 5 replicates/group) were dissected out in pole’s salt solution (pss), and x-gal staining was performed using the method as described by chowdhuri et al. (1999). the larvae explants were fixed in 2.5% glutaraldehyde, washed in 50mm sodium phosphate buffer (ph 8.0) and stained overnight in x-gal staining solution at 37°c in the dark. trypan blue exclusion test. for studying the extent of tissue damage in larvae caused by the exposure to different dosages of aspartame dye exclusion test was performed (nazir et al. 2003; krebs and feder 1997). briefly, the internal tissues of larvae were explanted in a drop of pss, washed in phosphate buffer saline (pbs), stained with trypan blue (0.2 mg/ml in pbs) for 30 min, washed thoroughly with pbs and scored immediately for dark blue staining. about 30 larvae per treatment (10 larvae per dose; 5 replicates/group) were scored for the trypan blue staining on an average composite index per larvae: no colour =0; any blue = 1; darkly stained = 2; large patches of darkly stained cells = 3; or complete staining of most cells in the tissue = 4 (krebs and feder 1997). preparation of larval homogenate.the larvae (30 larvae/dose; 5 replicates/group) were homogenised in 1ml of cold homogenising buffer (0.1m phosphate buffer containing 0.15m kcl; ph 7.4). after centrifugation at 9000g, the supernatant was used for estimating lipid peroxidation, glutathione content, glutathione-s-transferase activity and protein carbonyl content. lipid peroxidation assay.lipid peroxidation was measured according to the method described by ohkawa et al. (1978). the reaction mixture consisted of 5µl of 10mm butylhydroxytoluene (bht), 200µl of 0.67% thiobarbituric acid, 600µl of 1% o-phosphoric acid, 105µl of distilled water and 90µl of the supernatant. the resultant mixture was incubated at 90ºc for 45 min, and the od was measured at 535nm. the results were expressed as µmol of tbars formed/h/g tissue. estimation of glutathione (gsh) content. the glutathione (gsh) content was estimated colorimetrically using ellman’s reagent (dtnb) according to the procedure described by (jollow et al. 1974). the supernatant was precipitated with 4% sulphosalicyclic acid (4%) in the ratio of 1:1. the samples were kept at 4ºc for 1 hour and then subjected to centrifugation at 5000 rpm for 10 min at 4ºc. the assay mixture consisted of 550 µl of 0.1m phosphate buffer, 100 µl of supernatant and 100 µl of dtnb. the od was read at 412 17 all res. j.biol, 2017, 8, 16-24 nm, and the results were expressed as µ moles of gsh/gramme tissue. estimation of glutathione-s-transferase (gst) activity. the glutathione-s-transferase activity was determined by the method ofhabig et al. (1974). the reaction mixture consists of 500 µl of 0.1 m phosphate buffer, 150 µl of 10mm cdnb, 200 µl of 10mm reduced glutathione and 50 µl ofsupernatant. the od was taken at 340 nm, and the enzyme activity was expressed as µ moles of cdnb conjugates/min/mg protein. estimation of protein carbonyl content. the protein carbonyl content was estimated according to the protocol described by hawkins et al. (2009). the larvae homogenate was diluted to a protein concentration of approx 1mg/ml. about 250µl of each diluted homogenate was taken in eppendorf centrifuge tubes separately. to it, 250µl of 10mm 2,4-dinitrophenyl hydrazine (dissolved in 2.5m hcl) was added, vortexed and kept in the dark for 20 min. about 125µl of 50% (w/v) trichloroacetic acid (tca) was added, mixed thoroughly and incubated at -20°c for 15min. the tubes were then centrifuged at 4°c for 10 min at 9000 rpm. the supernatant was discarded and the pellet obtained was washed twice withcold ethanol: ethyl acetate (1:1). finally, the pellets were re-dissolved in 1ml of 6m guanidine hydrochloride, and the absorbance was read at 370nm. effect of pupation and emergence of flies. to study the effect of aspartame on the pupation and emergence of flies the aspartame was mixed in the diet at a final concentration of 20, 40, 80, 160 and 320mm. to these doses, 50 first instar larvae were introduced. triplicate sets of each treatment group (including the untreated and positive control) were employed in the study. each day the numbers of larvae pupate followed by flies emergence were recorded separately, and the data was expressed as the mean of three replicates (podder and roy 2015). statistical analysis. the statistical analysis was done using statica soft inc. the student t-test was applied to observe the significant difference between treated and untreated groups. results the results obtained for β-galactosidase activity are presented in fig. 1. the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not show any significant increase in the activity of β-galactosidase (fig. 1). however, the exposure of mms (as a positive control) showed a significant 1.71 fold increase in the β-galactosidase activity compared to the control (p<0.05; fig.1). the results obtained for x-gal staining are shown in figs. 2 (a-g). the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not show any β-galactosidase expression (figs. 2 a-e). however, the exposure of mms to third instar larvae showed an intense staining in the midgut (fig. 2g) compared to control (fig. 2f). the results obtained for trypan blue staining are shown in figs. 3 (a-g). the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not show any tissue damage compared to control (figs. 3a-f). the results obtained for lipid peroxidation are shown in fig. 4. the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not show any significant increase in the lipid peroxidation compared to control (p<0.05; fig. 4). however, the exposure of larvae to mms showed a 2.56 fold significant increase in the lipid peroxidation compared to control (p<0.05; fig. 4). figure 2(a-g) x-gal staining in the third instar larvae of transgenic drosophila melanogaster (hsp70-lac z) bg9 after the exposed to * 0 0.1 0.2 0.3 0.4 0.5 a1 a2 a3 a4 a5 c pc o d a t 4 20 n m ( m ea n ± se ) figure 1 β-galactosidase activity measured in the transgenic drosophila melanogaster (hsp70-lacz)bg9 third instar larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20mm aspartame; a2-40 mm aspartame; a3-80 mm aspartame; a4-160 mm aspartame; a5-320 mm aspartame; ccontrol; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant at p<0.05 with respect to control. 18 all res. j.biol, 2017, 8, 16-24 different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 mm aspartame; a5-320 mm aspartame; c-control; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate. [bgbrain ganglia, sgsalivary gland, pvproventriculus, fgforegut, mgmidgut, hghindgut, mtmalpighian tubule, gcgastric caeca]. figure 3. (a-g) trypan blue staining in the third instar larvae of transgenic drosophila melanogaster (hsp70-lacz) bg9after the exposed to different doses of aspartame for 24 hrs of duration. {a120µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4160 µm aspartame; a5-320 µm aspartame;c-control; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate [bgbrain ganglia, sgsalivary gland, pvproventriculus, fgforegut, mgmidgut, hghindgut, mtmalpighian tubule, gcgastric caeca]. the exposure of mms to larvae showed the tissue damage in the brain ganglia, salivary gland, proventriculus, foregut, midgut, hindgut, malpighian tubule, gastric caeca. the results obtained for the gsh content is shown in fig. 5. the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not shownany significant increase or decrease compared to control (fig. 5). however, the exposure of mms to larvae showed a 1.51 fold decrease compared to control (p<0.05; fig. 5). the results obtained for the gst activity are shown in fig. 6. the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not show any significant increase in gst activity compared to control (fig. 6). the exposure of mms to larvae showed significant 1.55 fold increase in gst activity compared to control (p<0.05; fig. 6). * 0 100 200 300 400 500 a1 a2 a3 a4 a5 c pc n m ol es o f t b a r s fo rm ed / ho ur / gr am ti ss ue ( m ea n ± s e ) figure 3. lipid peroxidation measured in the transgenic drosophila melanogaster (hsp70-lacz)bg9 third instar larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 µm aspartame; a5-320 µm aspartame;ccontrol; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant with respect to control (p<0.05). * 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 a1 a2 a3 a4 a5 c pc t ot al g sh (µ m ol es o f g sh / g ra m ti ss ue ) (m ea n ± se ) figure 5. glutathione content measured in the transgenic drosophila melanogaster (hsp70-lacz)bg9 third instar larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 µm aspartame; a5-320 µm aspartame;ccontrol; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant at p<0.05 with respect to control (p<0.05). * 0 10 20 30 40 50 60 a1 a2 a3 a4 a5 c pc t ot al g st (µ m ol es o f c d n b co ju ga te s/ m in / m g pr ot ei n) (m ea n ± se ) figure 6. glutathione-s-transferase activity measured in the transgenic drosophila melanogaster (hsp70-lacz)bg9 third instar larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 µm aspartame; a5-320 µm aspartame;c-control; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant at p<0.05 with respect to control. 19 all res. j.biol, 2017, 8, 16-24 the results obtained for protein carbonyl content are shown in fig. 7. the exposure of larvae to 20, 40, 80, 160 and 320mm of aspartame did not show a significant increase in the pc content compared to control (p<0.05; fig. 7). however, the exposure of mms to larvae showed a 2.66 fold significant increase in the pc content compared to control (p<0.05; fig. 7). the results obtained for pupae formation for the larvae exposed to 20, 40, 80, 160 and 320mm of aspartame are shown in fig. 8. almost similar number of larvae pupates in each of the exposed groups, and complete pupation was observed for eight (8) days in aspartame as well as control groups. no significant difference in the number of pupation/day was observed in aspartame exposed as well as a control group (p<0.05; fig. 8). however, the larvae exposed to mms showed a significant difference in the number of pupae formation for 5th, 6th, 7th and 8th day compared to control as well as aspartame exposed groups. the process of pupation was observed till 12th day for the larvae exposed to mms (p<0.05; fig. 8). the results obtained for the emergence of flies are shown in fig. 9. in each group, the number of flies emerged was counted each day. the first emergence was observed at the 12th day and no significant difference in the mean number of flies emergence at 12th, 13th,14th, 15th ad 16th day was observed in the aspartame-treated as well as control groups (p<0.05; fig. 9). however, in mms exposed group a significant decrease in the mean number of flies emerged at 13th,14th, 15th and 16th day was observed compared to control as well as aspartame treated groups (fig. 9; p<0.05). the emergence was observed till the 19th day in the mmstreated group (fig. 9; p<0.05). discussion the results obtained in the present study suggest that the aspartame is not toxic at 20, 40, 80, 160 and 320mm. the selected doses in our present study are very high, and the plasma concentration of the aspartame in humans hardly reaches 320mm(ranney and oppermann 1978).in humans and other mammalian species, aspartame is metabolised in the gastrointestinal tract by esterases and peptidases into three components i.e. aspartic acid, phenylalanine and methanol(stegink 1987; butchko et al. 2002). it may completely hydrolyze in the gastrointestinal line and absorbed or may be hydrolyzed to methanol and aspartylphenylalanine dipeptide. the dipeptide may be absorbed into the gastrointestinal mucosa cells and then cleaved into amino acids(stegink 1987). in some cases, aspartame may be absorbed into mucosal cells before hydrolysis, and be cleaved within the cell into three components(matthews 1984). there is very much similarity in both structure and function of drosophila gut and human gastrointestinal tract. the midgut is the main site of digestion and nutrient absorption(buchon et al. 2013). drosophila genome encodes a vast array of putative digestive enzymes (probably 349) involved in the processing of carbohydrate, proteins and lipids(lemaitre and miguel-aliaga 2013). esterases and peptidases are predominantly present in the fly/larval midgut hence the possibility of the metabolism of * 0 0.1 0.2 0.3 0.4 a1 a2 a3 a4 a5 c pc o d a t 3 70 n m (m ea n ± s e ) figure 7. protein carbonyl content measured in the transgenic drosophila melanogaster (hsp70-lacz)bg9 third instar larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 µm aspartame; a5-320 µm aspartame;ccontrol; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant at p<0.05 with respect to control. * * * * 0 5 10 15 20 25 5th 6th 7th 8th 9th 10th 11th 12th n um be r of p up ae (m ea n ± s e) day a1 a2 a3 a4 a5 c pc figure 8. pupation of larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 µm aspartame; a5-320 µm aspartame;c-control; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant at p<0.05 with respect to control. * * * * * * * * 0 2 4 6 8 10 12 14 16 18 20 12th 13th 14th 15th 16th 17th 18th 19th n um be r of il ie s em er ge d (m ea n ± s e) day a1 a2 a3 a4 a5 c pc figure 9. emergence of flies from the larvae exposed to different doses of aspartame for 24 hrs of duration. {a1-20µm aspartame; a2-40 µm aspartame; a3-80 µm aspartame; a4-160 µm aspartame; a5-320 µm aspartame;c-control; p-positive control; p-mms(0.5µl/ml methylmethane sulphonate)}*significant at p<0.05 with respect to control. 20 all res. j.biol, 2017, 8, 16-24 aspartame cannot be ruled out in the larval tissue(lemaitre and miguel-aliaga 2013). although the global organisation of mammalian and insect guts is not conserved, similar functional sequences can be clearly observed(buchon et al. 2013). drugs are chemical substances that exhibit some side effect (toxicity) in the organism due to overdose, accumulation or inability to eliminate it. the expression of hsp70 has been correlated with early cytotoxic events and cellular damage due to its conservation and inducibility by a wide variety of environmental agents and also being a part of the cellular defence (steinmetz and resing 1997; ait-aissa et al. 2000). in our present study, the β-galactosidase activity was not significantly higher as compared to control at all of the selected doses. but in mms exposed group the activity was significantly higher as compared to untreated, and aspartame exposed groups. this demonstrates the sensitivity of larvae against the chemical agents. the β-galactosidase activity as an indicator of hsp70 expression has been used to monitor the environmental stress, heat and cadmium toxicity in transgenic caenorhabditislegans(guven et al. 1999; stringham and candido 1994).trypan blue staining performed on the larvae exposed to various doses of aspartame as well as control larvae showed no tissue damage compared to the larvae exposed to mms. the trypan blue staining is widely accepted and used to evaluate the damaging potential of a particular compound on a specific organ or tissue (mukhopadhyay et al. 2003; siddique et al. 2013). the midguts of insects have been reported to have high microsomal and cytochrome activity(wilkinson and brattsten 1972). the results obtained for the gst activity showed no increase or decrease, compared to the control. the midguts of the insects express glutathione-s-transferase (gsts) homologous to mammals and are associated with oxidative stress(perrimon and owusu-ansah 2014). it has been suggested that a vast majority of harmful chemicals are taken care off with the help of p450s present in the midgut. the larvae exposed to mms showed an increase in the activity of gst. the results obtained for the gsh content also show no increase or decrease in the level compared to the control. the larvae exposed to mms showed a significant decrease in the gsh content compared to control as well as aspartame exposed groups. the results obtained for lipid peroxidation and protein carbonyl content also did not show any significant increase compared to control groups. lipid peroxidation is a measure of oxidative stress resulting from the production of reactive oxygen species that damage the cell membranes, proteins and dna(ryter et al. 2007). however, there are reports of high tbars and low gsh content in the kidney tissues of the rats exposed to 500mg/kg body weight for 42 days (saleh 2014). the protective effect of pomegranate juice against the aspartame induce an oxidative injury i.e. reduction in superoxide dismutase, catalase and reduced glutathione has also been reported(darwish et al. 2009). here, the rats were given 250mg/kg/day by gavage for 28 days (darwish et al. 2009). in another study, the rosemary extract was reported to attenuate the increase in lipid peroxidation and enhanced the levels of reduced glutathione and antioxidant enzymes activities in kidney and testis in male rats exposed to 1000mg/kg body weight of aspartame (hozayen et al. 2014). the low concentrations of aspartame metabolites did not affect the membrane enzyme activity, but the higher concentrations partially decreased the membrane acetylcholinesteraseactivity in human erythrocytes(tsakiris et al. 2006). the long term consumptions of aspartame lead to the imbalance in the antioxidant/prooxidant status in the brain of rats exposed to 1000 mg/kg body weight(abhilash et al. 2013). the chronic ingestion of abuse doses of aspartame produced no significant chemical changes in brain capable of altering behavioural parameters believed to be controlled by monoamines in rats(torii et al. 1986). the exposure of aspartame to 1000mg/kg body weight has been reported not only a hepatotoxic but also a potent carcinogenic agent(alkafafy et al. 2015). aspartame has been reported to increase protein carbonyl, lipid peroxidation levels, superoxide dismutase, glutathione-s-transferase, glutathione peroxidase and catalase activity in the rats exposed to 40mg/kg body weight of aspartame along with 0.2mg/kg/day of methotrexate(ashok and sheeladevi 2014). these results were obtained when methotrexate was also given with aspartame. but in normal humans or rats, the aspartame is quickly metabolised in the gastrointestinal tract(magnuson et al. 2007). the inhibition of acetylcholinesterase activity in vivo conditions was reported by the metabolites of aspartame in the frontal cortex(simintzi et al. 2007). the researchers have also focussed on the toxicity of its metabolites such as methanol, formaldehyde and formate(oyama et al. 2002). mean peak blood methanol concentration exceed 2mg/dl in subjects administered abuse doses of aspartame(stegink 1987). but these methanol concentrations are still lower than those reported in methanol intoxication(nolla-salas et al. 1995; meyer et al. 2000). in our study on the third instar larvae, no tissue damage was observed even at 360mm of aspartame. the direct and indirect cellular effects of aspartame on the brain have also been reported as the aspartic acid is also thought to play a role as an excitatory neurotransmitter in central nervous system(humphries et al. 2008). according to the epidemiologic data intake of aspartame is not associated with hematopoietic neoplasms, brain cancer, digestive sites, breast, prostate and several other neoplasms(marinovich et al. 2013). however, there are reports on the vestibular toxicity in a pair of siblings, suggesting a genetic susceptibility to aspartame toxicity(pisarik and kai 2009). aspartame has also been reported to cause neurobehavioral changes and activation of neurodegenerative apoptosis following the long-term consumption of it in folate deficient rat brain(ashok and sheeladevi 2015). to study the effect on development by aspartame, the third instar larvae were also allowed to pupate, and the emergence of the flies was also followed. the durations were noted, and there was no significant difference in the number of larvae pupated and the emergence of flies in control and larvae exposed to various doses of aspartame. however, the delay and significant difference in the number of larvae pupated and the emergence of flies was observed in the mms exposed group. hence it is concluded that if aspartame possesses any toxic effect, it would have also induced the delay in the pupation or emergence of flies. in our study, no effect of aspartame was observed in the pupation and emergence of flies. further, the emerged flies were also observed for any morphological differences; they exhibit no morphological differences compared to control 21 all res. j.biol, 2017, 8, 16-24 group. however, one study has reported the toxic effect of aspartame in drosophila melanogaster and danio rerio. in this study, the same concentration was used for both invertebrate as well as avertebrate model, and the concentrations were exceptionally high (highest tested dose 500 mg/ml)(reshman et al. 2015). the group has reported marked phenotypic changes and dna damage in both flies and fishes at the same doses(reshman et al. 2015). the genotoxicity studies carried out on aspartame in vitro as well as in vivo have been extensively reviewed by magnuson et al.(2007). there are no reports of its genotoxicity in vitro as well as in vivo models. it did not induce micronucleus and chromatid exchanges in cultured human peripheral blood lymphocytes(rencuzogullari et al. 2004). there was also no evidence of dna damaging activity by aspartame in primary rat hepatocyte culture at 5 and10 mm of aspartame(jeffrey and williams 2000). the in vivo cytogenetic assay performed on rats for aspartame at a dose of 500, 1000, 2000, and 4000 mg/kg/body weight/ day for 5 days did not induce chromosomal aberrations in bone marrow cells from all groups(magnusan et al. 2007). the contradictory reports on the aspartame are mainly due to the dose differences, the route of administration and the duration of exposure. here in our present study, we have studied various doses of aspartame on larvae for 24 hours of duration and also the larvae were allowed to pupate on the same doses of exposure and the emergence of the flies were also noted. it is concluded from our study that aspartame does not exhibit any toxic effects at the doses selected in our study, and these doses are higher than the plasma concentrations in human even after a high dose intake (stegink et al. 1983; finkelstein et al. 1983). aspartame is completely digested by the gastrointestinal tract, into amino acids and methanol, which is subsequently metabolised into carbon dioxide and water (magnuson et al. 2007). conclusions the contradictory reports on the aspartame are mainly due to the dose difference, the route of administration and the duration of exposure. here in our present study, we have studied various doses of aspartame on larvae for 24 hrs of duration and also the larvae were allowed to pupate on the same doses of exposure and the emergence of the flies were also noted. it is concluded from our study that aspartame does not exhibit any toxic effects at the doses selected in our study, and these doses are still higher than the plasma concentrations in human even after a high dose intake (stegink et al. 1983). aspartame is completely digested by the gastrointestinal tract, into amino acids and methanol, which is subsequently metabolised into carbon dioxide and water (oyama et al. 2002). acknowledgements: we are thankful to the chairman, department of zoology, amu, aligarh for providing the laboratory facility. we also thankful to the laboratory colleagues mr. fahad ali, mrs. ambreen fatima, mrs. saba khanam, mrs. barkha shakya and ms. falaq naz for their kind help and support in conducting the experiments. references alkafafym.e.s., ibrahim z.s., ahmed m.m., and el-shazly s.a.(2015). impact of aspartame and saccharin on the rat liver: biochemical, molecular, and histological approach. int. j. immunopath. pharmacol.28, 247-255. abhilash m., sauganth paul m.v., varghese m.v., and nair r.h.(2013). long-term consumption of aspartame and brain antioxidant defense status. drug chem. toxicol. 36,135-140. ashok i., and sheeladevi r. 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(1986). dietary aspartame with protein on plasma and brain amino acids, brain monoamines and behaviour in rats. physiol. behav.36,765-771. wilkinson c.f., and brattsten l.b.(1972). microsomal drug metabolizing enzymes in insects. drug metab. rev. 1,153-227. 24 microsoft word 73-479-1-layoutefinal.docx low effects on cytotoxicity against neuroblastoma cancer cell line (sk-n-sh) and free-radical scavenging activity of stemona alkaloids sumet kongkiatpaiboon, primchanien moongkarndi, wandee gritsanapan all res. j. biol., 2013, 4, 19-23 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 3, vol 4, 2013, 19-23 low effects on cytotoxicity against neuroblastoma cancer cell line (sk-nsh) and free-radical scavenging activity of stemona alkaloids sumet kongkiatpaiboona, primchanien moongkarndib, and wandee gritsanapana,* a) department of pharmacognosy b) department of microbiology, faculty of pharmacy, mahidol university, bangkok 10400, thailand *corresponding author email: wandee.gri@mahidol.ac.th; wandeegrit@yahoo.co.th abstract: stemona plants contain alkaloids with different skeletal types. they are well-characterized and are commonly used for insecticide, killing head lice, treating skin diseases, antitussive, and anticancer treatment. eleven stemona alkaloids of three skeletal types, protestemonine-, croomine-, and stichoneurine-type, i.e. protostemonine-type (didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, oxystemokerrine), croomine-type (croomine), and stichoneurine-type (tuberostemonine, tuberostemonine a, tuberostemonine n, neotuberostemonine) were isolated from various stemona roots and investigated for their cytotoxicity and free radical scavenging activity, which have not yet been reported. cytotoxicity on neuroblastoma cancer cell (sk-n-sh) was determined by mtt assay, while free radical scavenging activity was investigated using the dpph radical scavenging method. protostemonine type alkaloids possessed insignificant effect on the cytotoxicity and free radical scavenging activity, while croomine and stichoneurine type alkaloids showed weak to moderate effects. keywords: anticancer; antioxidant; mtt assay; stemofoline; stemona; tuberostemonine introduction stemona spp. of the family stemonaceae are commonly known in thailand under the name of “non tai yak.” these plants have been used as a natural insecticide against head lice, scabicide, anticancer agent, and for treatment of skin and respiratory diseases1-3. stemona alkaloids represent a unique chemical character of the family stemonaceae4,5. stemona plants contain various amounts of these alkaloids6,7, which can be divided into 3 main skeletal types4, i.e. protostemonine-type, croomine-type, and stichoneurine-type (fig. 1). the major alkaloids of the first skeletal type includes didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, oxystemokerrine, and stemokerrine, while the second type includes croomine, and the last type includes tuberostemonine, tuberostemonine a, tuberostemonine n, and neotuberostemonine. the reported biological activities of these stemona alkaloids are insect toxicity8,9, antitussive10,11, oxytocin antagonist12, nitric oxide inhibition13, and increasing chemosensitivity via p-glycoprotein-mediated multidrug resistance14-16. cytotoxicity of stemona extracts or their isolated alkaloids has been reported against several human cancer cell lines such as medullary thyroid carcinoma17,18, human fibroblast18, leukemia carcinoma19, nasopharyngeal19, gastric carcinoma19, breast adenocarcinoma20. however, free radical scavenging activity and cytotoxicity of these alkaloids on neuroblastoma cancer cells has not been reported. oxidative stress can lead to numerous pathophysiological conditions, including cancer. antioxidants can terminate or retard the oxidation process by scavenging and sequestering the free radicals that are implicated in many chronic diseases. in the present study, eleven stemona alkaloids (fig. 1), which were isolated from underground parts of various stemona plants in our previous works6, were investigated for their free radical scavenging activity and cytotoxicity against neuroblastoma cell line (sk-n-sh). materials and methods chemical and reagents rpmi 1640 medium and fetal calf serum (fcs) were obtained from biochrom (berlin, germany). 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) and 1,1-diphenyl-2-picrylhydrazyl radical (dpph) were purchased from sigma (st. louis, mo). eleven stemona alkaloids of three skeletal types, i.e. didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, oxystemokerrine of protostemonine-type; croomine of croomine-type; and tuberostemonine, tuberostemonine a, tuberostemonine n, neotuberostemonine of stichoneurine-type were isolated from the roots of various stemona plants and identified in our previous work6. 19 all res. j.biol, 2013, 4, 19-23     cell culture sk-n-sh cell line was obtained from the american type culture collection (rockville, md) was cultured in rpmi 1640 medium supplemented with 5% (v/v) fsc, 100 mg/l of streptomycin and 100,000 u/l of penicillin g, at 37°c in 5% co2 incubator. cell viability evaluation by mtt assay mtt assay was a conventional method to assess the number of viable cells growing in microtiter plate after treatment with various substances. serial dilutions of samples (50 µl) were added into each of 96-well plates. the cells were plated at a density of 1 × 104 cells/well and incubated for 48 hrs. after incubation, the medium was removed and the cells in each well were incubated with phosphate buffered saline (pbs) containing 1 mg/ml mtt for 2 hrs at 37°c in 5% co2 incubator. mtt solution was then discarded and 50 µl of isopropanol was added into each well to dissolve insoluble formazan crystals. plates were then agitated for 5 min at room temperature to complete the solubilization. the color of formazan derivative was analyzed on a microplate reader (molecular devices, ca) at a wavelength of 590 nm. each concentration was done in triplicate. the percentage of cell viability was calculated according to the following equation: cell viability (%) = (od of treated cells / od of control cells) × 100%. determination of free radical scavenging activity the free radical scavenging activity of stemona alkaloids was investigated using dpph radical scavenging assay. the solution of dpph• was prepared at concentration of 60 mg/l (152 µm) in methanol, while stock solution of the sample (1 mg/ml) was serially diluted between 12.5 and 800 µg/ml. the dpph• scavenging reaction was performed by adding dpph• solution (100 µl) to the sample solution of the same volume. the mixture was incubated for 30 min at room temperature and measured for absorbance at 517 nm by a microplate reader. the corresponding blank sample was also taken and percent inhibition was then calculated as follows: scavenging activity (%) = [1 (a1 a2) / a0] × 100% where a0 was the absorbance of control (dpph• solution without sample), a1 was the absorbance of dpph• solution in the presence of the sample and a2 was the absorbance of the sample without dpph• solution. statistical analysis the experiments were repeated three times and the results were expressed as mean + s.d. statistical analysis was done using two-tailed student’s t test and p values at a level of 95% confidence limit. results cytotoxicity on neuroblastoma cell line sk-n-sh the cytotoxic effect of stemona alkaloids on neuroblastoma cell line sk-n-sh was investigated by mtt assay. cells were treated with stemona alkaloids at concentrations ranging from 0 to 200 µg/ml for 48 hours and the percentage of cell viability was analysed. the ec50 was then calculated as shown in table 1. croomine and stichoneurine derivatives, i.e. tuberostemonine, tuberostemonine a, tuberostemonine n, and neotuberostemonine inhibited the proliferation of sk-nsh cells in a dose-dependent manner. a similar result was observed when quercetin was used as a positive control. free radical scavenging activity the free radical scavenging activity of stemona alkaloids was determined by the dpph• free radical scavenging assay. at a concentration of 400 µg/ml, protostemonine alkaloid derivatives didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, and oxystemokerrine increased free radical scavenging activity by only 8.81%, 9.92%, 45.03%, 8.55%, 8.52%, and 59.27%, respectively, whereas croomine and the stichoneurine derivatives tuberostemonine, tuberostemonine a, tuberostemonine n, and neotuberostemonine increased scavenging activity by 88.84%, 88.76%, 79.98%, 84.81%, and 84.73%, respectively. serial dilutions of each sample were also measured for their free radical scavenging activity. the ec50 of each alkaloid was then calculated (table 1). croomine and stichoneurine derivatives possessed free radical scavenging activity in a dose-dependent manner. similar results were observed when quercetin and trolox were used as positive control. discussion stemona alkaloids could be classified, based on biosynthetic hypothesis, into three skeletal types: protostemonine-, stichoneurine-, and croomine-type. protostemonine type alkaloids, which include didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, and oxystemokerrine, possessed insignificant cytotoxicity on skn-sh cells and low free radical scavenging activity, while croomine and the stichoneurine-type alkaloids, which are tuberostemonine, tuberostemonine a, tuberostemonine n, and neotuberostemonine, showed weak to moderate activities compared to the positive control (table 1). although stemona roots have been traditionally used as an ingredient in several anti-cancer preparations, only a few scientific studies on their anti-cancer properties were reported. only medullary thyroid carcinoma cell lines gave positive results when treated with stemona extracts17,18 whereas other cell lines showed weak responses or no cytotoxicity activity15,19,20. although those compounds do not seem to exhibit a strong cytotoxicity, they might still be acting to potentiate the effect of anti-neoplastics. for example, stemofoline, stemocurtisine, and stemocurtisinol were reported to sensitize the multidrug resistant cancer cell to putative chemotherapeutic agents including vinblastine, paclitaxel and colchicine via p-glycoprotein mediated pathway14-16. 20 all res. j.biol, 2013, 4, 19-23 figure 1 structure of stemona alkaloids used in this study. 21 all res. j.biol, 2013, 4, 19-23     table 1 cytotoxicity and free radical scavenging activity of stemona alkaloids typea compounds ec50 (µg/ml) cytotoxicityb free radical scavenging activityb i didehydrostemofoline > 200 >> 400 stemofoline > 200 >> 400 stemocurtisine > 200 > 400 stemocurtisinol > 200 >> 400 stemokerrine > 200 > 400 oxystemokerrine > 200 156.68 + 2.72 ii tuberostemonine 140.49 + 7.92 34.88 + 1.92 tuberostemonine a 144.58 + 10.39 218.33 + 2.00 tuberostemonine n 193.44 + 10.57 57.03 + 0.68 neotuberostemonine 71.27 + 2.52 66.74 + 1.56 iii croomine 198.42 + 5.48 107.74 + 0.33 postive control trolox 2.62 + 0.05 quercetin 36.67 + 4.39 2.29 + 0.02 a i = protostemonine-type alkaloid, ii = stichoneurine-type alkaloid, iii = croomine-type alkaloid b results expressed as mean + sd (n = 3) conclusion protostemonine alkaloid derivatives showed an insignificant effect on both cytotoxicity of sk-n-sh cells and free radical scavenging activity, while stichoneurine alkaloid derivatives and croomine alkaloid demonstrated these activities at low to moderate levels. the highest cytotoxic effect was observed in neotuberostemonine with an ec50 of 71.27 µg/ml, which was 2-fold lower activity than that of quercetin (ec50 36.67 µg/ml). the highest free radical scavenging activity was observed in tuberostemonine with ec50 of 34.88 µg/ml, which is about 15 times lower than those observed with trolox and quercetin (ec50 2.62 and 2.29 µg/ml, respectively). these results demonstrate that stemona alkaloids, the major contents in the roots of stemona species, may not directly possess cytotoxicity on cancer cells. when included in traditional anti-cancer preparations, stemona possibly synergizes and enhances the activity of other active compounds through increasing chemosensitivity. acknowledgements :    this study is a part of a ph.d. thesis at mahidol university, financially supported by thailand research fund and mahidol university (royal golden jubilee ph.d. program grant no. phd/0139/2550). the project is also supported by the office of the higher education commission and mahidol university under the national research universities initiative. we thank mr. panupon 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(2005). antiviral and anticancer activities of stemona collinsae. thai j. pharm. sci. 29, 125-136. 23 microsoft word layoutfinal-corregido.docx novel plant regeneration and transient gene expression in catharanthus roseus abdullah makhzoum*, anica bjelica, genevieve petitpaly, mark a. bernards all res. j. biol., 2015, 6, 1-9 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol 6, 2015, 1-9 novel plant regeneration and transient gene expression in catharanthus roseus abdullah makhzoum*1, anica bjelica1, genevieve petit-paly2, mark a. bernards1 1department of biology & thebiotron, the university of western ontario, london, on, canada, n6a 5b7. 2biomolécules et biotechnologies végétales, université françois-rabelais, 37200, tours, france abstract: catharanthus roseus genetic transformation represents a real challenge due, in part, to the lack of regeneration capability and to the recalcitrant nature of this species to genetic transformation. in the present work, we demonstrate the regeneration of c. roseus plants from hypocotyls and cotyledons, using specific growth regulator conditions. plants derived from hypocotyls and cotyledons were successfully acclimated and grown in the greenhouse. furthermore, c. roseus meristem tissues were shown to have high shoot regeneration potential under conditions optimised for cotyledonand hypocotyl-derived shooting. meristem tissues were, therefore, investigated as a genetic transformation target using both agrobacterium tumefaciens and a. rhizogenes. although meristem-derived shoots transformed with a. tumefaciens harbouring a p35s gusplus construct revealed transient gus expression and protein accumulation, they were not amenable to selection even after two months on selection medium. transformation of c. roseus meristem tissues with a. rhizogenes resulted in a typical hairy root phenotype, in which the adventitious root tissue strongly expressed the p35s gusplus construct, as revealed by intense gus staining. keywords: catharanthusroseus, regeneration, meristem, transient genetic transformation introduction medicinal plants represent important sources of drugs and the use of genetic transformation systems to increase the production of these molecules requires efficient plant regeneration and genetic transformation systems. catharanthusroseus(l.) g. don (apocynaceae), native to madagascar, is known to produce many indole alkaloid compounds. of these, vinblastine and vincristine are effective anti-cancer agents against leukaemia and hodgkin’s diseases1. tissue-based compartmentalization of the last steps of indole alkaloid biosynthesis means that specific cell types, but especially idioblasts and laticifers in leaves and young buds, are required for vinblastine and vincristine biosynthesis in vivo 2 ,3 . these differentiated tissues do not exist in cell cultures (e.g., callus, cell suspension) and the vindoline pathway is blocked in these cultures because the final reactions catalyzed by desacetoxyvindoline 4-hydroxylase (d4h) 4 and acetyl coenzyme a (coa): deacetylvindoline-4o-acetyltransferase (dat) are absent 2 ,5. there is one report of vindoline production in callus culture transformed by different agrobacterium tumefaciens6; however, this has not been further substantiated. thus, it is necessary to be able to genetically transform and subsequently regenerate c. roseus plants to be able to modify and/or analyse the last steps of the indole alkaloid pathway in this species. however, c. roseus has proven to be difficult to transform using standard genetic transformation systems, particularly in the steps for plant regeneration. hairy roots system has been extensively exploited in plant genetic engineering, gene and promoter and plant molecular pharming studies2 ,7-9. with the exception of one report of the regeneration of c. roseus plants from genetically transformed hairy roots under very special conditions 10 and another from hypocotyles11 ,12, no regeneration of genetically transformed plants has been reported for this species from apex or primordia tissues; however, these two systems are not reproducible based on our experiments on most organs and tissues explants. limited success in c. roseus plant regeneration has been had with zygotic embryos 11and petiole explants 13, the latter of which gave rise to shoots with a 40% regeneration efficiency. a few groups have reported the successful regeneration of c. roseus using seedlings as explants 14 ,15. more recently, an alternative method to generate c. roseus shoots in vitro through somatic embryos derived from embryogenic calli was described 16. yet, another approach made use of nodal explants 17 containing 1 all res. j. biol, 2015, 6, 1-9     two auxiliary buds as a target tissue source for microparticle bombardment and plant regeneration 18, albeit with very low transformation rates and the generation of numerous chimeras. although meristem shoot regeneration systems have been described for many plant species 19,   this system has not been exploited for c. roseus. in general, plants are characterized by their post-embryonic organogenesis and have sets of stem cells, localized in shoot apical and root meristems that remain in an undifferentiated state 20. shoot apical meristem stem cells are characterized by quick regeneration and competence for genetic transformation and have been successfully used to obtain transgenic plants in maize, wheat, rice, oat, barley, sorghum, and millet 21. furthermore, meristematic tissues have been used for shoot apical meristem genetic transformation of several plants such as rice 22, maize 23 ,24 and yellow lupin25. similarly, axillary shoot meristems have been used for the transformation of pear cultivars recalcitrant to regeneration 26. bulk meristematic tissue obtained by removing the apical dome 27has been used to transform cotton 28or castor mature seed embryo axes 29. in this paper, we describe a highly efficient shoot regeneration system using hypocotyl-and cotyledon-derived callus, as well as shoot apical meristems and their surrounding cells. this degree of shoot formation provides an optimal amount of explants for plant regeneration and potentially genetic engineering of this species. we further regenerated whole plants from shoots obtained from hypocotyl-and cotyledon-derived callus, including their acclimation and transfer to the greenhouse. lastly, genetic transformation using both a. tumefaciens and a. rhizogenesis was demonstrated. results plant regeneration in a preliminary set of experiments, we applied varying amounts and ratios of naa and bap to different organs (e.g., roots, hypocotyls and cotyledons) from cv. pacifica pink to determine the optimal conditions for shoot formation. shoot formation was highest (ca. 70%) from callus derived from hypocotyl explants after 35 days of culture on media supplemented with naa (1 mg/l) and bap (1 mg/l) (figure 1, 2a). calli from roots and cotyledons did not produce many shoots. next we tested the shooting ability of hypocotyl explants from six c. roseus cultivars using 1 mg/l each of naa and bap. we readily obtained shoots from callus derived from the upper part of hypocotyls of all six cultivars, but less so from the lower part (table1). in this experiment, the highest amount of shoot regeneration was obtained from the cv. cooler coconut (16.6 %). although this was much lower than that obtained with cv. pacifica pink in our preliminary experiments, we were able to subsequently obtain whole plants from these shoots (see below). table 1. yield of shoots from hypocotyl-derived calli segments from six cultivars of catharanthusroseus. hypocotyl segments from in vitro grown seedlings were divided into upper and lower parts prior to plating on ms medium supplemented with 1 mg/l naa and bap. the number of explant-derived calli pieces on which shoots were evident was counted after 2 months in culture, and expressed as a fraction of the total number of calli plated. cultivar name abbreviations: bp = blue pearl, cc = cooler coconut, cmi = cooler mix improved, ml = mediterranean lilas, pp = pacifica pnk, sr = stardust rose. cultivar number of calli with shoots upper hypocotyls lower hypocotyls bp 4/40 (10%) 0/40 (0%) cc 10/60 (16.6%) 0/60 (0%) cmi 5/80 (6.3%) 0/50 (0%) ml 2/40 (5%) 1/50 (2%) pp 9/100 (9%) 0/80 (0%) sr 9/70 (12.9%) 0/80 (0%) figure 1. shoot formation from catharanthus roseus explants. various explant tissues (cotyledons, hypocotyls, roots) from in vitro grown c. roseus plantlets (cv. pacifica pink) were cultured on ms medium supplemented with different concentrations of naa and bap. categorical values in the x-axis are expressed as ratios of naa/bap (both in mg/l). the percentage of explant-derived calli with regenerated shoots (y-axis) was recorded after 35 days in culture. data are pooled from a minimum of 120-150 explants plated on 4 to 5 independent plates (minimum of 30 explants per plate) for each naa/bap combination. root formation: shoots obtained from hypocotyl and cotyledon explants were transferred to ms medium containing various growth regulators, and sub-cultured on a 4-week cycle. after four to five subculture cycles on media supplemented with either iaa (10 mg/l) and bap (0.1 mg/l) or iba (10 mg/l) and bap (0.1 mg/l), roots began to appear (figure 2b, table 2). prior to acclimation, these were transferred to ms solidified with gelrite instead of agar to improve rooting and allow plantlet recovery with minimal damage to the roots. plant acclimation: plants were successfully acclimated and transferred to the greenhouse (figure 2c). out of 40 shoots taken through the rooting steps, only 14 survived through to full plants in the greenhouse (table 2). the overall process, from initial invitro shoot formation from hypocotyls or cotyledons through 2 all res. j. biol, 2015, 6, 1-9     to the potting of regenerated plants in the greenhouse took approximately 1 year. figure 2. regeneration of catharanthus roseus plants from callus. (a) shoots were obtained from hypocotyl-derived callus after several transfers on ms medium supplemented with 1 mg/ml each of naa and bap. (b) shoots were transferred to ms medium supplemented with either iaa (10 mg/l) and bap (0.1 mg/l) or iba (10 mg/l) and bap (0.1 mg/l). (c) mature plants of c. roseus were obtained from in vitro plantlets, after a period of acclimation. data for cv. pacifica pink are shown. table 2. yield of rooted hypocotyl-and cotyledon-derived shoots from catharanthusroseus. shoots derived from calli obtained from either cotyledons or hypocotyls were rooted on ms media supplemented with either iaa 10 mg/l and bap 0.1 mg/l or iba 10 mg/l and bap 0.1 mg/l and later on the same medium solidified with gelrite instead of agar. after hardening and acclimation, plants placed into pots of vermiculite under 25°c ± 1°c, 18 µe/cm2/s, (12h/12h) conditions for one month before transferring them to the greenhouse (20°c ± 1°c) under natural light and day length. original explant number of explants number of acclimated plants time to acclimation (months) cotyledon 16 7 (43%) 12-15 upper hypocotyl 9 3 (33%) 14-15 lower hypocotyl 15 4 (26%) 15 meristem culture meristem explants obtained from all six cultivars were able to form shoots with similar efficiency (table 3) in as little as 30 days, when cultured with naa (1 mg/l) and bap (1 mg/l). the highest efficiency achieved (97%) was with the cultivar mediterranean lilas. adding high concentrations (e.g., higher than 5 mg/l of bap) caused shoot malformation (data not shown). table 3. yield of shoots from meristem explants from six cultivars of catharanthusroseus. meristem segments from 9-to 10-day old in vitro grown seedlings were plated on ms medium supplemented with 1 mg/ml naa and bap. the number of explants on which shoots were evident was counted after 35 days in culture, and expressed as a fraction of the total number of explants plated. cultivar number of meristems with shoots bp 35/50 (70%) cc 130/138 (94.2%) cmi 122/127 (96.1%) ml 77/79 (97.5%) pp 122/129 (94.5%) sr 136/151 (90.1%) transformation of meristems with agrobacterium tumefaciens amplification of a band of the correct size (750 bp) using an exon-exon junction primer confirmed the presence of the βglucuronidase (gusplus) transgene in meristem cells (figure 3a). further proof of the incorporation of gusplus into meristem tissues, as well as its expression, was obtained using a dot-blot assay to detect his-tagged proteins. thus, proteins extracted from meristems co-cultivated with a. tumefaciens were blotted onto nitrocellulose and probed with anti-his antibody 30. only those extracts obtained from transformed meristems revealed the presence of his-tagged protein (i.e., gus); untransformed meristems did not contain any his-tagged proteins (figure 3b). transient reporter gene expression was further observed in c. roseus apices co-cultured with a. tumefaciens harbouring the pcambia gusplus construct using a colourimetric assay for gus enzyme activity (figure 4a-f). in an attempt to optimize gus expression levels, we tested a variety of parameters, including the age of apices at the time of inoculation, the number of days of co-cultivation, the optical density of the cultures used, and wounding the tissue prior to agrobacterium treatment. the duration of the co-cultivation period had an effect on gene expression since those cocultured for 4 days showed a higher level of gus activity than for those co-cultivated for only 2 days (figure 4a,b). even though a wounding treatment resulted in an enhanced level of (transient) gus activity, there were no significant 3 all res. j. biol, 2015, 6, 1-9     differences in gus activity observed between apices that were wounded with either a scalpel or needle, regardless of whether the wounding was longitudinal or transversal (figure 4c-f). no difference in transient gene expression levels were noted for apices with or without first leaves or old (9 or 15 days) apices (data not shown). by contrast, bacterial colony density had an impact on gene expression, with cultures adjusted to an od600 = 0.5 resulting in higher gus activity than with cultures used at a higher optical density (data not shown). figure 3.gus gene expression and protein accumulation in agrobacterium tumefaciens-transformed catharanthus roseus meristem tissue. (a) rt-pcr using an exon-exon bridging primer was used to detect c. roseus derived gus transcripts (see materials and methods for details) from mrna isolated from a. tumefaciens transformed meristems. (b) his-tagged gus protein was detected in crude protein extracts obtained from a. tumefaciens transformed meristems (gus). the negative control (-ve) is a crude protein extract from non-transformed c. roseus meristem cultures. the positive control (+ve) is a sample of his-tagged recombinant potato fatty acid ω-hydroxylase expressed in e. coli. crude protein extract (10 µl) was spotted onto nitrocellulose and probed with anti-his antibody conjugated with horseradish peroxidase and detected using the chemiluminescence (ecl) assay system (ge healthcare). transformation with a. rhizogenes normally, plant tissue infected with a. rhizogenes generates a hairy root phenotype, and this is what we observed (figure 4g). that the tissue harboured the gus gene was evident from intense blue staining in the root tissue when assayed for glucuronidase activity. at this stage, we have not been successful in regenerating whole plants from hairy roots obtained from meristem cultures transformed with a. rhizogenes. nevertheless, it is clear that c. roseus is readily transformed with a. rhizogenes, and this may be a good alternative to a. tumefaciens. figure 4. transient expression of gus in apical meristem and hairy root cultures of catharanthusroseus. (a-f) apical meristems (20 to 30 per plate) were subjected to different conditions to optimize gus transformation using agrobacterium tumefaciens, including (a) 2-or (b) 4-day of co-culture, (c) transverse or (d) longitudinal wounding (with 2-day co-culture), and wounding by (e) needle or (f) scalpel (with 2-day co-culture). in situ gus activity is evident by dark staining, especially at the meristem ends of the explants. two replicate plates were analysed; a representative plate form each treatment is shown. (g) in vitro cultured c. roseus meristems were infected with a. rhizogenes harbouring a p35s::gusplus construct. after approx. 1 month on non-selective media, the tissue was subjected to gus staining (see materials and methods). the intense dark staining of the adventitious (hairy) roots is indicative of active gus protein. discussion catharanthus roseus produces medicinally valuable indole alkaloids, including vinblastine and vincristine, which are used in the treatment of leukemia and hodgkin’s disease. since the amounts of indole alkaloids produced in this species is relatively low (< 1% of dry weight), and over 500 kg of c. roseus leaves is required to produce 1 g of vincristine, 31 it is desirable to apply biotechnological approaches to enhance production. while there have been many attempts to genetically engineer c. rosues to enhance indole alkaloid production, most have been unsuccessful. this is in part due to (1) compartmentation of indole alkaloid biosynthesis in planta, (which largely rules out the use of tissue cultures for indole alkaloid production), (2) difficulty with regenerating c. roseus plants from in vitro cultures (i.e., as part of an agrobacterium-mediated gene transfer system), and (3) the lack of a suitable, stable genetic transformation protocol for this species. therefore, the development of a genetic transformation protocol for c. roseus requires progress in both plant regeneration and plant genetic transformation. herein we report that c. roseus cv. pacifica pink plants have been regenerated from hypocotyland cotyledon-derived 4 all res. j. biol, 2015, 6, 1-9     callus tissue. shoots were obtained after one to two months on ms medium supplemented with naa and bap (both at 1.0 mg/l). shoots were subsequently rooted on medium supplemented with either iaa (10 mg/l) and bap (0.1 mg/l) or iba (10 mg/l) and bap (0.1 mg/l), acclimated and transferred to the greenhouse, where they developed and produced flowers. thus, we were able to generate whole, mature plants starting with hypocotyls and cotyledons obtained from in vitro germinated seeds. this process required up to one year, with an overall efficiency of 35%. in addition, shoots were readily obtained in high yield from apical meristems cultured on ms medium supplemented with naa and bap (both at 1.0 mg/l). consequently, meristem explants represent an excellent means whereby shoots can be readily obtained, and are superior to somatic embryos, which require more than nine months, many subculture cycles and a few combinatorial hormones 16. the formation of shoots from meristems is important because we were also able to demonstrate transient gene expression in agrobacteriuminfected meristems (see below) using both a. tumefaciens and a. rhizogenes. previously, transient gene expression systems for c. roseus have been attempted using protoplasts 32, biolistic bombardment 33agroinfiltration34 or vacuum infiltration 35. we recently expressed a promoter-gus construct using the c. roseus dat gene (which encodes the enzyme acetylcoa:deacetylvindoline-4-o-acetyltransferase involved in the last step of vindoline biosynthesis) promoter in agroinfiltrated c. roseus leaves and a. rhizogenes-derived c. roseus hairy roots, albeit only transiently 2. successful transformation of c. roseus by a. tumefaciens was performed on suspension cultures 36. however, given the difficulty in regenerating plants from suspension culture and the need for intact plant tissue to study some biochemical processes, an alternative transformation system is necessary. for this we proceeded with agrobacterium-mediated transformation of meristem cultures, with the goal of ultimately re-generating transformed plants from them. as proof of concept, we initially worked with a p35s::gusplus construct. we tested many factors to find the best transformation conditions, including the agrobacterium strain, preculture period, bacterial suspension density, methods of co-cultivation (i.e., with or without wounding and type of wounding), and the duration of the co-cultivation period (2 and 4 days). while wounding may facilitate attachment or the release of virulence gene inducers 37, we did not observe a significant enhancement of transformation (inferred using in situ gus enzyme activity as a proxy for gene expression) when meristems were wounded. instead, the highest levels of gus enzyme activity were observed when suspension cultures of a. tumefaciens (e.g., od600 = 0.5) were used, and co-cultivation was allowed to continue for four days. other conditions resulted in less apparent transformation, as evident by less intense in situ gus activity. to ensure that meristems treated with a. tumefaciens were indeed transformed and contained the gus gene, exonexon junction primers were used to avoid the amplification of bacterial dna and to only amplify cdna generated from properly spliced mrna. while we have only demonstrated transient gus gene expression (i.e., the a. tumefaciens transformed tissue was not subjected to selection), the process provides a potential means to obtain stable genetic transformation of c. roseus. even though meristems have been shown to overcome agrobacterium infections, giving rise to healthy plants 38, we have now demonstrated, through the transient expression of gus, that these explants can be an agrobacterium-mediated transformation target for c. roseus. the use of meristem cultures allows the regeneration of c. roseus shoots in vitro without a callus stage. such an approach increases genotype fidelity and avoids the complications of somaclonal variation 39. agrobacterium rhizogenes can also be a useful tool to generate transgenic plants with high genetic stability and growth rate 8 ,40 , even for species that are difficult to transform using a. tumefaciens41 ,42. however, plant regeneration from hairy roots, derived from a. rhizogenes transformation, is difficult, and has only been reported once for c. roseus7. nevertheless, we obtained a typical hairy root phenotype when we treated c. roseus meristems with a. rhizogenes harbouring the same p35s::gus construct as with a. tumefaciens. adventitious roots obtained in this manner demonstrated intense in situ gus activity, especially when these were derived from tissue below the main apical meristem. conclusions the establishment of a c. roseus genetic transformation system is a challenge in part because of the difficulty in regenerating shoots from primary transformed tissues; however, it is also a challenge because of difficulty in generating primary transformants. in the present paper, we address two of these issues and demonstrate the regeneration of plantlets from hypocotyl-and cotyledon-derived tissue as well as agrobacterium-mediated (both a. tumefaciens and a. rhizogenes) transformation of c. roseus meristems. we are still not at a stage where we can combine these two results into a routine genetic transformation system because it remains technically challenging to generate genetically modified c. roseus tissue that is also amenable to plant regeneration. it is beyond the scope of the parameters considered herein to recommend a strategy for c. roseus transformation and plant regeneration. nevertheless, our data provide the basis for further studies on shoot apical meristem genetic transformation as a tool to overcome the intrinsic difficulties in c. roseus genetic transformation, as well as the basis for plantlet regeneration. material and methods plant material explants were obtained from seedlings grown in sterile culture from seed. for whole plant regeneration from various organs (hypocotyls, cotyledons and roots) and for genetic transformation assays, only the pacifica pink cultivar was employed. to estimate shooting efficiency and for meristem experiments, six different cultivars (blue pearl, cooler coconut, cooler mix improved, mediterranean lilas, pacifica pink and stardust rose) were examined. 5 all res. j. biol, 2015, 6, 1-9     bacterial strains and plasmid construct agrobacterium tumefaciens agl1 and a. rhizogenes 15834 were transformed, using electroporation, with the binary vector pcambia1305.1 in which the t-dna region harboured the reporter gene gusplustm encoding a 6 x histagged β-glucuronidase (gus) and hygromycin phosphotransferase (hptii). both genes are driven by separate 35s promoters. sterilization and germination of seeds sterilization of c. roseus seeds was carried out by immersion in ethanol for 2 minutes followed by na-hypochlorite (3%) for 20 minutes, and finally washing three times in sterile distilled water. later, seeds were soaked in sterile distilled water in the dark for one day, then sown on petri dishes containing hormone-free ms medium 43  (0.8% agar), covered by aluminium foil and left in the dark to germinate. after three days, the foil was removed and the seedlings were grown at 24°c with a 16 hour light, 8 hour dark, photoperiod (18µe/cm2/s). meristems from nineand ten-day old plantlets were used for agrobacterium-mediated transformation. plant culture explants were excised from 9-or 10-day old seedlings and sub-cultured on ms medium supplemented with different naa and bap ratios (see below), under the same conditions noted previously. to study the influence of organs and hormones on regeneration, various organs (cotyledons, roots and hypocotyls) from c.v. pacifica pink were used with different naa/bap combinations (values in mg/l) including: 1.0/5.0; 1.0/2.0; 1.0/1.0; 1.0/0.5; 1.0/0.1; 0.5/2.0; 0.5/1.0; 0.5/0.5; 0.5/0.1. calli arising from explants were subcultured onto new medium every 3 weeks. for regeneration of whole plants and acclimation, shoots derived from callus were first rooted on ms media supplemented with either iaa 10 mg/l and bap 0.1 mg/l or iba 10 mg/l and bap 0.1 mg/l for several sub-culture cycles. once roots appeared, plantlets were subcultured on the same medium solidified with gelrite instead of agar. for hardening and acclimation, plants were removed from gelrite cultures and the roots washed with water. the plants were then placed into pots of vermiculite wetted with knop solution 44, under 25 °c ± 1°c, 18 µe/cm2/s, (12h/12h) conditions for one month. later, plants were transferred to the greenhouse (20 °c ± 1°c) under natural light and day length. the appearance of shoots on hypocotyls was established separately for the upper and lower segments of hypocotyls from 6 c. roseus cultivars on ms supplemented with naa 1 mg/l and bap 1 mg/l. for meristem shoot regeneration, meristems from all 6 cultivars were used on ms containing naa 1 mg/l and bap 1 mg/l. meristems of the pacifica pink cultivar were used to optimize growth regulator concentrations by placing them on ms media supplemented with different concentrations of bap (1 mg/l, 2 mg/l, 5 mg/l, 10 mg/l, 15 mg/l and 20 mg/l) in combination with 1 mg/l naa. meristem genetic transformation assays agrobacterium tumefaciens transformation: meristems from 9-to 10-day old plantlets were isolated and plated on ms basal medium as above. agrobacterium tumefaciens suspensions (strain agl1), harbouring the binary vector pcambia 1305.1, containing the gusplustm gene (modified with a 6 x his c-terminal tag) encoding βglucuronidase were prepared in lb medium supplemented with kanamycin monosulphate (50 mg/l). the optical density of overnight cultures was measured and adjusted (to as low as 0.5; 45 and meristems, with or without preinoculation wounding (stabbing or cutting) were immersed in bacterial suspension for 10 minutes prior to transfer to petri dishes containing hormone free ms medium. after 2 or 4 days of co-culture, the meristem explants were washed twice with sterile, distilled water and once with ms (hormone free) containing 500 mg/l cefotaxime, and subjected to gus assay (see below). agrobacterium rhizogenes transformation: transformation of apical meristems by a. rhizogenes was carried out with a needle dipped in solid colonies, using a dissecting microscope to target tissue (e.g., meristem or apical meristem zones, or the main veins of young leaves from in vitro-grown plantlets). finally, explants were placed in petri dishes on ms medium with bap (1 mg/ml) and naa (1 mg/ml) supplemented with 500 mg/l cefotaxime and incubated at room temperature with 16 hours light and 8 hours dark. rt-pcr to demonstrate transient transformation of meristems, we amplified gus cdna using an exonexon junction-based primer 46 ,47. total rna was extracted from 100 mg infected and washed meristems using the rneasy plant mini kit (qiagen), according to manufacturer’s instructions. firststrand cdna was synthesized from 5 µg of total rna using superscript ii reverse transcriptase (invitrogen) primed by oligo(dt). pcr was performed using first-strand cdna as the transformed meristems template and the over-intron (e.g., exon-exon primer) pintr3f primer (gpint3f tggtagatctgaggaaccga) as forward primer with gp3r (aatctccacgttaccgctca as the reverse primer. the pcr conditions were as follows: initial conditions: 94°c for 4 min, followed by 35 cycles at 94°c for 30 seconds, 54°c for 45 seconds, 72°c for 2 min and a final cycle at 72°c for 7 min. the pcr reactions were carried out on a techne thermal cycler (tc-3000g). dot blot assay in order to test for gus accumulation in meristems transformed with a. tumefaciens, total protein was isolated from 500 mg of transformed or untransformed (negative control) meristems, and probed for his-tagged protein. briefly, total protein was extracted using qb extraction buffer (100 mm kpo4 (ph 7.8), 1 mm edta, 1% triton x100, 10% glycerol, dh2o and 1 mm dtt) 48 (2 ml of qb buffer/g of ground tissue) and subsequently centrifuged for 15 minutes at 4˚c at 13,000 rpm. the concentration of the total protein in the supernatant was determined using a bca kit, according to manufacturer’s instructions (pierce). sixty micrograms of total protein (in 10 µl) isolated from transformed and untransformed meristems was spotted onto nitrocellulose membrane. after blocking for 1h in 5% skim milk, the membrane was probed with anti-his hrp 6 all res. j. biol, 2015, 6, 1-9     conjugated antibody (1:5000) (invitrogen). protein presence was revealed using ecl (ge healthcare). histochemical gus staining transient and stable genetic transformation of explants was assessed by assay for gus enzyme activity in situ as described 49.  explants were immersed in staining solution (50 mmna-phosphate, (ph 7.0), 0.5 mm potassium ferricyanide, 0.5 mm potassium ferrocyanide, 1% triton x-100, 1 mg/ml 5-bromo-4-chloro-3-indolylglucuronide) in the dark at 37°c for 2 or 3 days. next, chlorophyll was removed from explants by immersion in 70% ethanol. gus staining was observed under a microscope (olympus bx51) and photographed. acknowledgements the authors gratefully acknowledge financial support from the department of plant physiology, tours university (gp) and the natural sciences and engineering research council (nserc) of canada (mab). additional financial support to abm, from the ministry of higher education in syria is gratefully acknowledged. references 1. svoboda, g. h., and blake, d. a. 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(2013) hairy roots: an ideal platform for transgenic plant production and other promising applications, in phytochemicals, plant growth, and the environment gang, d. r., ed.(springer new york), pp. 95-142. 9. makhzoum, a., tahir, s., locke, m. e. o., trémouillaux-guiller, j., and hefferon, k. (2014). an in silico overview on the usefulness of tags and linkers in plant molecular pharming plant science today1, 201-212. 10. choi, p. s., kim, y. d., choi, k. m., chung, h. j., choi, d. w., and liu, j. r. (2004). plant regeneration from hairy-root cultures transformed by infection with agrobacterium rhizogenes in catharanthusroseus. plant cell rep22, 828-831. 11. kim, s., in, d., choi, p., and liu, j. (2004). plant regeneration from immature zygotic embryoderived embryogenic calluses and cell suspension cultures of catharanthusroseus. plant cell, tissue and organ culture76, 131-135. 12. wang, q., xing, s., pan, q., yuan, f., zhao, j., tian, y., chen, y., wang, g., and tang, k. (2012). development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants. bmc biotechnology12, 34. 13. lee, s. y., choi, p. s., chung, h. j., in, d. s., choi, d. w., and liu, j. r. (2003 ). comparision of adventitious shoot formationin petiole explant cultures of 20 cultivars of catharanthusroseus. j plant biotechnol 5, 59-61. 14. constabel, f., gaudet-laprairie, p., kurz, w. g., and kutney, j. p. (1982). alkaloid production in catharanthus roseus cell cultures : xii. biosynthetic capacity of callus from original explants and regenerated shoots. plant cell rep1, 139-142. 15. yuan, y. j., hu, t. t., and yang, y. m. (1994). effects of auxins and cytokinins on formation of catharanthus roseus g. don multiple shoots. plant cell, tissue and organ culture37, 193-196. 16. piovan, a., filippini, r., caniato, r., dallavecchia, e., innocenti, g., cappelletti, e. m., and puricelli, l. ( 2000 ). somatic embryogenesis and indole alkaloid production in catharanthusroseusplant biosystems 134, 179-184. 17. morard, p., and henry, m. (1998). optimization of the mineral composition of in vitro culture media. journal of plant nutrition21, 1565-1576. 18. zárate, r., memelink, j., van der heijden, r., and verpoorte, r. (1999). genetic transformation via particle bombardment of catharanthus roseus plants through adventitious organogenesis of buds. biotechnology letters21, 997-1002. 19. sai kishore, n., visarada, k. b., aravinda lakshmi, y., pashupatinath, e., rao, s. v., and seetharama, n. (2006). in vitro culture methods in sorghum with 7 all res. j. biol, 2015, 6, 1-9     shoot tip as the explant material. plant cell rep25, 174-182. 20. baurle, i., and laux, t. (2003). apical meristems: the plant's fountain of youth. bioessays : news and reviews in molecular, cellular and developmental biology25, 961-970. 21. sticklen, m., and oraby, h. (2005). shoot apical meristem: a sustainable explant for genetic transformation of cereal crops. in vitro cell.dev.biol.-plant41, 187-200. 22. park, s. h., pinson, s. r., and smith, r. h. (1996). t-dna integration into genomic dna of rice following agrobacterium inoculation of isolated shoot apices. plant molecular biology32, 11351148. 23. zhang, s., williams-carrier, r., and lemaux, p. (2002). transformation of recalcitrant maize elite inbreds using in vitro shoot meristematic cultures induced from germinated seedlings. plant cell rep21, 263-270. 24. zhong, h., sun, b., warkentin, d., zhang, s., wu, r., wu, t., and sticklen, m. b. (1996). the competence of maize shoot meristems for integrative transformation and inherited expression of transgenes. plant physiology110, 1097-1107. 25. li, h., wylie, s. j., and jones, m. g. k. (2000). transgenic yellow lupin (lupinusluteus). plant cell rep19, 634-637. 26. matsuda, n., gao, m., isuzugawa, k., takashina, t., and nishimura, k. (2005). development of an agrobacterium-mediated transformation method for pear (pyruscommunis l.) with leaf-section and axillary shoot-meristem explants. plant cell rep24, 45-51. 27. mezzetti, b., pandolfini, t., navacchi, o., and landi, l. (2002). genetic transformation of vitisvinifera via organogenesis. bmc biotechnology2, 18. 28. gould, j., and magallanes-cedeno, m. (1998). adaptation of cotton shoot apex culture to agrobacterium-mediated transformation. plant molbiol rep16, 283-283. 29. sujatha, m., and sailaja, m. (2005). stable genetic transformation of castor (ricinuscommunis l.) via agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. plant cell rep23, 803-810. 30. koo, s. c., choi, m. s., chun, h. j., park, h. c., kang, c. h., shim, s. i., chung, j. i., cheong, y. h., lee, s. y., yun, d. j., chung, w. s., cho, m. j., and kim, m. c. (2009). identification and characterization of alternative promoters of the rice map kinase gene osbwmk1. molecules and cells27, 467-473. 31. dewick, p. m. (1997 ). medicinal natural products: a biosynthetic approach vol., ed.(new york ny john wiley & sons ). 32. galun, e. (1981). plant protoplasts as physiological tools. annual review of plant physiology32, 237266. 33. ferrer, e., linares, c., and gonzalez, j. m. ( 2000 ). efficient transient expression of the b-glucuronidase reporter gene in garlic (allium sativum l.) agronomie 20, 869-874. 34. batra, s., and kumar, s. (2003). agrobacteriummediated transient gus gene expression in buffel grass (cenchrusciliaris l.). journal of applied genetics44, 449-458. 35. di fiore, s., hoppmann, v., fischer, r., and schillberg, s. (2004). transient gene expression of recombinant terpenoid indole alkaloid enzymes incatharanthus roseus leaves. plant molbiol rep22, 15-22. 36. garnier, f., label, p., halard, d., and chénieux, j.c. (1996 ). transgenic periwinkle tissues overexproducingcytokinins do not accumulate enhanced levels of indole alkaloids plant cell, tiss organ cult 45, 223-230. 37. saini, r., and jaiwal, p. k. (2005). transformation of a recalcitrant grain legume, vignamungo l. hepper, using agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures. plant cell rep24, 164-171. 38. stahl, y., and simon, r. (2005). plant stem cell niches. the international journal of developmental biology49, 479-489. 39. giri, c. c., shyamkumar, b., and anjaneyulu, c. (2004). progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. trees18, 115-135. 40. giri, a., and narasu, m. l. (2000). transgenic hairy roots. recent trends and applications. biotechnology advances18, 1-22. 41. guillon, s., tremouillaux-guiller, j., pati, p. k., rideau, m., and gantet, p. (2006). hairy root research: recent scenario and exciting prospects. current opinion in plant biology9, 341-346. 42. guillon, s., tremouillaux-guiller, j., pati, p. k., rideau, m., and gantet, p. (2006). harnessing the potential of hairy roots: dawn of a new era. trends in biotechnology24, 403-409. 43. murashige, t., and skoog, f. (1962). a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiologiaplantarum15, 473-497. 44. gautheret, r. j. (1959 ). la culture de tissusvégétaux. techniques et réalisations vol., ed.( paris: masson and cie (eds) ). 45. cheng, m., lowe, b., spencer, t. m., ye, x., and armstrong, c. (2004). factors influencing agrobacterium-mediated transformation of monocotyledonous species. in vitro cell.dev.biol.plant40, 31-45. 46. le hir, h., gatfield, d., izaurralde, e., and moore, m. j. (2001). the exon-exon junction complex provides a binding platform for factors involved in mrna export and nonsense-mediated mrna decay. the embo journal20, 4987-4997. 47. vancanneyt, g., schmidt, r., o'connor-sanchez, a., willmitzer, l., and rocha-sosa, m. (1990). construction of an intron-containing marker gene: 8 all res. j. biol, 2015, 6, 1-9     splicing of the intron in transgenic plants and its use in monitoring early events in agrobacteriummediated plant transformation. molecular & general genetics : mgg220, 245-250. 48. ni, m., dehesh, k., tepperman, j. m., and quail, p. h. (1996). gt-2: in vivo transcriptional activation activity and definition of novel twin dna binding domains with reciprocal target sequence selectivity. the plant cell8, 1041-1059. 49. jefferson, r. (1987). assaying chimeric genes in plants: the gus gene fusion system. plant molbiol rep5, 387-405.   9 microsoft word 80-539-2-le.docx modeling temperature and light acclimation of photosynthetic capacity in seedlings and mature trees of pinus ponderosa bahram momen* and paul .d. anderson all res. j. biol., 2014, 5, 14-17 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                      issue 2, vol 5, 2014, 14-17 modeling temperature and light acclimation of photosynthetic capacity in seedlings and mature trees of pinus ponderosa bahram momen*1 and paul .d. anderson2 1environmental science and technology department, university of maryland, college park, md 20742, usa 2pacific northwest research station, usda forest service, corvallis, or 97331-8550, usa *corresponding author e-mail: bmomen@umd.edu abstract a preliminary step to understand the impact of possible rise in temperature on carbon dynamics of forests is to examine the temperature elasticity of key processes involved in carbon fixation in forest trees. for seedling and mature ponderosa pines of three genotypes, we used a response-surface methodology and anova to evaluate changes in maximum net photosynthesis (an max), and its corresponding light (lan max) and temperature (tan max) to diurnal and seasonal changes in ambient temperature during summer and autumn. as seasonal ambient temperature decreased: (1) an max did not change in seedlings or mature trees, (2) lan max did not change in mature trees, but it decreased for current-year foliage of seedlings from 964 to 872 µmol photons m-2 s-1, and (3) tan max did not change in seedlings but it decreased in mature trees for both currentand one-year-old foliage, from 26.8 to 22.2, and 24.6 to 21.7 °c, respectively. keywords light acclimation, temperature acclimation, photosynthetic capacity, ponderosa pine introduction the global mean temperature in the next 100 years may increase by 1-4 °c 1. temperature is an important factor affecting plant biochemical and physiological processes. plants have been evolved under warming and cooling periods. however, plants’ ecological stability and reproductive success in their current habitat may depend on their potential to cope with projected increase in temperature. photosynthesis is a key process that involves many temperature-dependent biochemical reactions 2. temperate forest plants experience diurnal and seasonal changes in temperature that may affect photosynthetic adaptation/acclimation to longand short-term temperature fluctuations. in this study, we investigated maximum photosynthetic capacity (an max) and its associated temperature (tan max) and light (lan max) levels as related to diurnal and seasonal changes in ambient temperature in ponderosa pine (pinus ponderosa dougl. ex laws) in the field. we examined currentand one-year-old foliage in both seedlings and mature trees of three ponderosa pine genotypes. these genotypes have been selected by the usda forest service for future plantations in california, and differ in their phenotypic vigor. the unique aspects of this research are: 1) both mature trees and seedlings are used, 2) plants acclimated naturally to seasonal conditions in the field and then their photosynthetic response was measured under rapid changes in temperature and light levels (resembling rapid, diurnal changes), and 3) no previous study of this nature has caught our attention for the three important ponderosa pine genotypes we used. materials and methods plant material consisted of mature clones of three ponderosa pine genotypes (usda forest service designations 3087, 3088, 3399) and their related half-sib seedlings. the clonal trees were part of a grafted seed orchard growing at the usda-forest service chico tree improvement center (ctic) in chico, california. grafts of clones 3088, 3087, and 3399 displayed low, moderate, and high vigor, respectively, in terms of growth, foliage color, and foliage retention characteristics when growing at ctic. we deliberately included this clonal variation to explore whether 14 all res. j. biol, 2014, 5, 14-17     photosynthetic elasticity would be differentially expressed among genotypes differing in degree of local adaptation. the clonal trees were produced from bud scion grafted onto three-year-old rootstocks. the grafts were planted at the ctic in the mid to late 1970’s. although these grafts were 12to 15-yr old at the time of the study, they expressed mature characteristics in terms of branch and needle morphology, and cone production. half-sib seedlings of the three clones were grown from seed of the three clonal source trees. seeds were sown in containers in the spring of 1989, and seedlings were grown in a greenhouse for one season, and then transplanted to the field in the spring of 1990. the ctic is located in the sacramento valley at the base of the west slope of the sierra nevada (n 39.8º latitude, w 121.9º longitude; elevation 75 m), at the lower elevation limit of ponderosa pine habitat. the climate is mediterranean with hot, dry summers and mild, wet winters. mean temperature is greater and mean precipitation is lower at the ctic site compared with the natural habitat of the parent trees. soil is a vina silt loam, alluvium, about 120-cm deep. soil fertility was maintained by annual application of soil sulfur (220 kg ha-1, pure elemental s), ammonium sulfate (1100 kg ha-1, 24% s and 21% n), manganese sulfate (140 kg ha-1, 50% mn), and n:p:k (1200 kg ha-1, 16:20:0). plant material was watered biweekly from may to october. competing herbaceous species and rodents were removed regularly. these cultural treatments minimized potential confounding effects of resource limitations with that of experimental treatments (temperature and light). six mature trees, two from each genotype, were planted in the field in a completely randomized manner. therefore the experimental design for mature trees was a crd with two mature trees per treatment. for seedlings, the field layout was a randomized complete block design (rcbd), in which the three genotypes were randomized within two sites (blocks) resulting in having two seedlings per treatment. we used three temperature and three light levels (explained below). the three temperature levels were sub-plots within each genotype, and the three light levels were sub-plots within each temperature level. measurements for the two foliage ages and two seasons were analyzed separately. net photosynthesis (an) was measured on one replicate in the mornings and on the second replicate in the afternoons (between 0800 to 1900 h) in late august (period i) and mid november (period ii). midday mean (se) temperatures in august and november were 37 (0.38) and 24 (0.27) °c, respectively. an was measured under controlled temperature, light, vapor pressure deficit (vpd), and external co2 conditions using an infrared gas analyzer and data acquisition console (li-6200, licor, inc., lincoln, ne) interfaced with a 0.95-liter temperature-controlling cuvette (ddg-9920, data design group, la jolla, ca) and temperature controller (cn9111, omega engineering, stamford, ct). light was supplied by two 27-watt, electronic compact warm fluorescent lamps (2700k chromaticity, 82% color rendering index, general electric co.). ambient light was excluded. within the cuvette, vpd was held at 1.5 ± 0.5 kpa using a dew point generator (li-610, licor, inc., lincoln, ne), and the co2 concentration was maintained at 340 ± 5 µmol mol-1. for each replicate, 8 to 12 needles were placed flat inside the temperature-controlling cuvette and exposed to a series of target temperatures that followed the diurnal progression of ambient temperature. the sequence of target temperatures was 18, 25, and 32 °c for the morning measurements, and 32, 25, and 18 °c for the afternoon measurements. the measurement protocol for a given sample consisted of achieving a target temperature followed by sequential measurements at light intensities of 250, 500, and 1000 µmol photons m-2 s-1. with each change in temperature/light condition, the cuvette environment was allowed to equilibrate with respect to co2 concentration, vpd, and temperature. this equilibration was maintained for an additional 15 minutes and then an was measured and calculated per unit area and time. in mature trees, an was measured on currentand one-yearold foliage in august and november. in seedlings, an was measured on both foliage ages in august, but not all seedlings/genotypes retained healthy one-year-old foliage in november, and thus, for seedlings seasonal comparisons would be made on current-year foliage only. response-surface regression analysis was performed on each set of photosynthetic responses to the nine combinations of three temperature-by-three light levels for each sample to derive estimates of maximum photosynthesis (an max) and corresponding light (lan max) and temperature (tan max) values. this resulted in a total of 24 response surface models [3 genotypes × 2 plant forms (mature, seedling) × 2 diurnal measurements (morning, afternoon) × 2 seasonal measurements]. response surface analysis was performed using the resreg procedure of the sas system. detailed explanation of our response surface analysis is given elsewhere [3]. analysis of variance was then performed on estimates of an max, lan max, and tan max, which were obtained from the individual response surface models, to detect the main or interactive effects of genotype, plant form, diurnal, and seasonal differences. 15 all res. j. biol, 2014, 5, 14-17   results and discussion we acknowledge the limitations of having three light and three temperature levels for the construction of the response surface models, as well as having two replications per each of the six light-by-temperature treatment combinations for seedlings or mature trees. however, such limitations were unavoidable due to scarcity of desired plant material. time was also a limiting factor considering the amount of time needed to complete each photosynthetic measurement after equilibration to the six light-by-temperature treatment combinations. all 24 response surface models demonstrated excellent fits as indicated by the residual plots (not shown) and r2 > 0.99. all models had well-defined, stationary points of an max. no significant threeor two-way interactive effects, genotype, or diurnal main effect were detected for any of the three responses (an max, lan max, tan max ) examined. both seedlings and mature trees maintained similar an max in august and november (table 1). in mature trees, lan max did not change significantly from august to november, but tan max decreased from 26.8 to 22.2 °c (p < 0.01) in current-year foliage, and from 24.6 to 21.7 °c (p < 0.07) in one-year-old foliage. these results suggest that the photosynthetic capacity of experimental plants during active shoot growth was maintained under cool and short days in november, and that the optimal temperature for photosynthesis shifted in the direction of seasonal changes in the mean ambient temperature. similar photosynthetic acclimation to temperature has been demonstrated in loblolly pine (pinus taeda l.) seedlings grown under controlled environment 4 and in scots pine (pinus sylvestris l.) seedlings grown in open-top chambers 5. in current-year foliage of seedlings lan max decreased significantly (p < .03) from 964 to 872 µmol photon m-2 s-1, but it did not change in mature trees (table 1). table 1: mean (standard error) of maximum photosynthesis (an max) and its corresponding temperature (tan max) and light (lan max) values for ponderosa pine seedlings and mature trees. main-effect means are shown because of non-significant threeand two-way interactive effects of diurnal, seasonal, and genotype. temperature: °c, light and an max: µmol m -2 s-1. sample size is 2. plant foliage component mean (se) p august november seedling current-year an max 7.87 (.5) 6.94 (.5) 0.24 lan max 964 (25) 872 (25) 0.03 tan max 26 (1) 24 (1) 0.16 mature tree current-year an max 7.59 (.5) 6.82 (.5) 0.48 lan max 953 (23) 944 (23) 0.78 tan max 26.8 (.8) 22.2 (.8) 0.01 1-year-old an max 4.79 (.4) 5.49 (.4) 0.22 lan max 929 (24) 875 (24) 0.14 tan max 24.6 (1) 21.7 (1) 0.07 lan max of current-year foliage of seedlings decreased significantly from august to november, from 964 to 872 µmol photon m-2 s-1. this decrease may be due to seedlings’ adaptation to changes in understory conditions as light availability decreases with shortening day length. although seedlings were grown in the open in this study, it seems that they did not acclimate to higher light availability in the open and responded to the site of their long-term adaptation under canopy with low light. mature ponderosa pines, occupying co-dominant and dominant canopy positions, may not experience a wide range of seasonal light limitations but they may be exposed to a wider temperature range compared with seedlings, which are evolved under canopy with greater insulation from light fluctuations and subsequent effects on soil and above-soil temperature levels. if so, seasonal adjustment of tan max may be more important to mature tree fitness than seasonal adjustment of lan max. previous research on the same plant material indicated genotype differences in mid-day photosynthesis for both mature trees and seedlings when averaged across measurements made from february to october 6. in the current study, however, no genotype difference in the 16 all res. j. biol, 2014, 5, 14-17     maximum photosynthesis is detected regardless of the seasonal change in the ambient temperature. this may be explained by a genotype-by-midday temperature interaction, as suggested elsewhere 6, because mid-day measurements under ambient temperature do not reflect potential maximum photosynthesis. although our current results do not indicate genotype differences in photosynthetic capacity, the fact that genotype 3088 retained one cohort of foliage, as opposed to genotypes 3087 and 3399 that retained two cohorts, suggests that at the ctic, clone 3088 is farther displaced from its optimal environment than the other two genotypes. the retention of fewer foliage cohorts may indicate shifts in biomass allocation as a result of environmental stress. alterations of biomass allocation have been reported for ponderosa pine growing under contrasting mountain and desert thermal regimes 7. based on the result that mature ponderosa pines can sustain maximum photosynthesis under almost 4.5 °c change in optimum temperature, it may be concluded that predicted increases in mean temperature between 1 and 4 °c would not have a serious effect on ponderosa pine photosynthetic capacity. additionally, our result may be valid under current range of temperature and may not hold under an elevated range of temperature due to global warming. moreover, the effects of global warming should be considered in broader physiological and ecological terms. the effects of elevated range of temperature on biomass allocation 8 and respiration 9 must be considered with respect to the carbon budget at individual tree 4 and broader scales. interspecific differences in photosynthetic performance and competitive ability are expressed at the ecosystem levels, affecting species composition 10. under a most optimistic scenario of no major direct effect of a 2-4 °c increase in mean temperature on ponderosa pine in monoculture, increased temperature may differentially affect the competitive ability of components of ponderosa pine ecosystems and result in unpredicted outcomes. furthermore, having two replications limited the power of our analysis. references 1. united nations’ intergovernmental panel on climate change: http://www.grida.no/publications/vg/climate/page/307 6.aspx. 2. long s. p. and woodward fi. eds. (1988) plants and temperature. symposia of the society for experimental biol., 42. 3. momen b., anderson p. d., and helms j. a. (2001) application of response surface methodology and anova to detect pollution effects on photosynthetic response under varying temperature and light regimes. forest ecol. mgmt. 152, 331-337. 4. teskey r. o., and will r. e. (1999) acclimation of loblolly pine (pinus taeda) seedlings to high temperatures. tree physiol. 19, 519-525. 5. kellomäki s., and wang k. (1996) photosynthetic response to needle water potentials in scots pine after a four-year exposure to elevated co2 and temperature. tree physiol. 16, 765-772. 6. anderson p.d., houpis j.l.j., helms j.a., and momen b. (1997) seasonal variation of gas-exchange and pigmentation in branches of three grafted clones of mature ponderosa pine exposed to ozone and acidic rain. environ. pollut. 97, 253-263. 7. carey e. v., delucia e. h., and ball j. t. 1996) stem maintenance and construction respiration in pinus ponderosa grown in different concentrations of atmospheric co2. tree physiol. 16, 125-130. 8. callaway r. m., delucia e. h., and schlesinger w. h. (1994) biomass allocation of mountain and desert ponderosa pine: an analog for response to climate change. ecology. 75, 1474-1481. 9. bassow s. l., and bazzaz f. a. (1998) how environmental conditions affect canopy leaf-level photosynthesis in four deciduous tree species. ecology. 79, 2660-2675. 10. stroch m., vrabl d., podolinska j., kalina j., urban o., and spunda v. (2010) acclimation of norway spruce photosynthetic apparatus to the combined effect of high irradiance and temperature. j. plant physiol. 167, 597–605. 17 microsoft word 87-512-1-layoutediting final.docx anxiogenic like activity of sarcocephalus latifolius fruit extract in mice david arome*, chinedu enegide, solomon fidelis ameh all res. j. biol., 2014, 5, 1-6 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 1, vol 5, 2014, 1-6 anxiogenic like activity of sarcocephalus latifolius fruit extract in mice david arome*, chinedu enegide, solomon fidelis ameh 1department of science laboratory technology (physiology and pharmacology technology), university of jos, nigeria. corresponding author email: davearome@gmail.com abstract: the use of pharmacological agents in the treatment of anxiety disorders have fallen out of favour as their unwanted side effects have become evident. the present challenges call for an inward look into harnessing the full potential of medicinal plants that abound around us. the present study evaluates the anxiogenic activity of ethanolic fruit extract of sarcocephalus latifolius in mice. the prepared extract at 200, 400, and 600 mg/kg as well as 2.5 mg/kg of diazepam, the reference standard was administered orally. the anxiogenic activity of the extract was evaluated using elevated plus maze and open field models. in the elevated plus maze, the extract showed an anxiogenic effect in all the experimental dosage levels by decreasing the time spent and number within the open arms. animals in the extract and physiological saline groups spent more time in the enclosed arms to avoid the open arms, probably to avoid falling off. the reference standard showed a significant (p<0.001) anxiolytic effect, evidenced by the increased number of entries into the open arms and prolonged time spent in the open arms and centre axis. in the open field model, decreased horizontal locomotor activity was observed in the extract groups. the degree of horizontal locomotion was less in the extract groups compared to the reference standard which had the highest horizontal locomotor activity. also, there were reductions in the number of rearings at extract doses of 400 and 600 mg/kg. in conclusion, data from the present study suggested that the ethanolic fruit extract of sarcocephalus latifolius is not only devoid of anxiolytic activity, but it appears to exert anxiogenic like effects. keywords: sarcocephalus latifolius, diazepam, anxiogenic activity, elevated plus maze, and open field models 1. introduction: anxiety disorders represent the most common forms of psychiatric illnesses,1 prevalent among children and adults. its prevalence changes during childhood and adolescence,2 affecting about one-eighth of the total world population.3 anxiety disorders may lead to severe distress over a period of time,4-5 and disrupt the lives of individuals suffering from them.4-5 the intensity and frequency of anxiety involved in these disorders is often debilitating.6 the causes of anxiety disorders still remain unclear, but studies have implicated genetic, environmental factors, psycho-stimulating drugs,7 mental illnesses and brain injury as possible causes. anxiety disorders are suspected whenever features like uncontrollable worry, tension, fear, phobias, limited abnormalities of thought, and previous traumatic flashbacks present themselves often.8 anxiety disorders are managed with psychotherapy and medication. psychotherapy techniques are generally considered as a first line of treatment especially in 1 all res. j.biol, 2014, 5, 1-6   children and adolescents,9 which may be used alone or in conjunction with medications such as antidepressants and sedative anti-anxiety agents in severe anxiety disorders.9 high relapse rates have been reported with the use of pharmacological agents in the treatment of anxiety disorders.10 the use of pharmacological agents in the treatment of anxiety disorders has fallen out of favour as their unwanted side effects have become evident.11 these challenges call for an inward look into harnessing the full potential of medicinal plants that abound around us. sarcocephalus latifolius commonly called african peach is a multi-stemmed shrub with irregular and dense foliage that grows up to 12m. it is predominantly found in africa and some parts of asia. sarcocephalus latifolius have a wide range of medicinal applications which include cough remedy, diabetes, malaria treatment,12 diarrhoea and central nervous system diseases such as epilepsy.13-14 decoction root extract of sarcocephalus latifolius has been reported to possess anticonvulsant, anxioytic and sedative properties.14 the aim of this study was to evaluate the anxiogenic like activity of the ethanolic extract of sarcocephalus latifolius on animal anxiety models. 2. materials and methods: plant material the fresh fruits of sarcocephalus latifolius were obtained from makurdi, benue state, nigeria. the plant was identified and authenticated by ikechukwu chijioke of federal college of forestry jos. the collected fruits were sliced, washed and air-dried at 25oc for two weeks, then crushed into coarse powder. extraction of plant materials eighty grams of the powdered fruit was measured and dissolved in a sufficient quantity of ethanol for 24 hours with mechanical shaking (4h/day), at the end of 24hrs; the mixture was filtered with ashless filter paper. the extract was concentrated using a rotary evaporator at a temperature of 4ºc.the concentrate was heated over a water bath to obtain a solvent free extract, which was later stored in the refrigerator at 4ºc. phytochemical screening phytochemical screening of the ethanolic fruit extract of sarcocephalus latifolius was carried out using a standard procedure as described by trease and evans.15 experimental animals swiss albino mice of either sex, weighing 20-28 grams were obtained from benue state university, nigeria. the mice were acclimatized for two weeks to laboratory conditions in the animal unit of the university of jos, nigeria. the mice were housed in plastic cages in a ventilated room at a temperature of 20±0.60c, fed with standard rodent chow and allowed free access to potable water. all experiments were carried out in accordance with the experimental procedures of the animal unit of the university. oral acute toxicity study modified lorke’s method was used in the ld50 study 6 of ethanolic fruit extract of sarcocephalus latifolius. this test was carried out in two phases. in the first phase, nine mice randomized into three groups of three mice each were given 10, 100, 1000 mg / kg of the prepared extract orally. the mice were observed for the first four hours and subsequently daily for 7 days for any behavioural sign of toxicity. the same procedure as used in first one was adopted in phase two, but with different dosage levels of 1600, 2900 and 5000 mg/kg. ethic the anxiolytic study was carried out by the “principles of laboratory animal care”,17 and in accordance with standard experimental procedure approved by the ethical committee of animal house, department of pharmacology university of jos after filling out the ethic form. elevated plus-maze model the elevated plus-maze model was utilized by the method described by lister.18 the elevated plus-maze consists of two 2 all res. j.biol, 2014, 5, 1-6   open arms (25×10cm each), and two closed arms (25×10×10cm each) with an open roof. all four arms were radiated from a central platform (10×10cm). the maze is elevated to a height of 60 cm in a dimly lit room. physiological saline (10 ml/kg, orally), plant extract (200, 400 and 600 mg/kg, orally) and diazepam (2.5 mg/kg, orally) were administered to groups of 5 mice each. one hour post treatment, each mouse was placed in the centre of the elevated plus-maze, facing one of the closed arms. during a 5 min test period the following parameters were taken: the number of entries and time spent in the open and enclosed arms. entry into an arm was recorded when the mice cross the demarcation of the respective arm with its four paws, and was considered to be on the central platform whenever two paws were on it. open field model each mouse was placed in an open-field apparatus (45 × 45 × 40cm), made of a wooden floor and glass sides. the floor was carved into 9 equal sized squares (15 × 15 cm). an hour before dropping the individual mice into one of the corners of the box (i.e. 60 min. prior), the different groups were administered with respective treatments (physiological saline, diazepam 2.5 mg/kg, extract doses of 200 mg/kg, 400 mg/kg and 600 mg/kg) locomotion and number of rearings were recorded for 5min. statistical analysis the data obtained from the study was expressed as a mean±s.e.m (standard error mean). statistical significance was determined by one-way anova followed dunnett posttest and values of p<0.05 were considered significant. the analysis was performed using instant graphpad prism (version 5.02) 3. results and discussions phytochemical screening phytochemical screening of ethanolic extract of sarcocephalus latifolius showed the presence of alkaloids, carbohydrates, tannins, flavonoids, cardiac glycosides, steroids, saponins and anthraquinones. acute toxicity study the ld50 was estimated to be greater than 5000 mg/kg body weight. no mortality was recorded at all of the entire experimental dosage levels used in the acute toxicity study. effect of sarcocephalus latifolius on various parameters in the elevated plus maze model extract doses of 200, 400 and 600 mg/kg used in the study showed significant (p<0.05) anxiogenic effects, increasing the time spent in closed arms and entries into the closed arms compared to the reference standard. more so, the time spent in the centre was less in the extract treated groups compared to the physiological saline and the reference standard. 3 all res. j.biol, 2014, 5, 1-6   table i: effect of ethanolic fruit extract sarcocephalus latifolius on elevated plus maze treatment time spent in seconds number of entries open arms closed arms centre open arms closed arms physiological saline (5 ml/kg) 42.00±16.93 215.20±21.59 42.80±5.44 3.80±1.16 7.80±1.28 200 mg/kg 38.20±20.37 256.00±21.06 8.80±3.31** 0.80±0.20 1.40±0.24*** 400 mg/kg 15.20±3.92 271.80±3.11* 12.80±1.83** 1.40±0.40 1.40±0.51*** 600 mg/kg 7.40±3.50 281.40±9.40* 9.20±5.95** 1.00±0.32 2.00±0.55** diazepam 2.5mg/kg 186.00±17.26*** 80.00±11.40*** 33.40±9.56 5.60±1.36 8.40±1.21 values expressed as mean±sem, n=5, *(p<0.05), ** (p<0.01), *** (p<0.0001) effect of sarcocephalus latifolius on various parameters in the open field model decreased horizontal locomotor activity was observed in the extract groups. the degree of horizontal locomotion was less in the extract groups compared to the reference standard having the highest horizontal locomotor activity. also, there was a reduction in the number of rearings at extract doses of 400 and 600 mg/kg compared to the physiological saline and reference standard. table ii: effect of aqueous root extract of sarcocephalus latifolius and diazepam on open field model treatment no. of horizontal locomotion no. of rearings physiological saline (5 ml/kg) 10.00±6.12 5.60±0.93 200 mg/kg 13.80±4.31 6.60±0.81 400 mg/kg 3.40±1.78 1.40±0.50** 600 mg/kg 7.20±2.75 2.80±0.96 diazepam (2.5 mg/kg) 23.40±2.15 5.40±0.64 values expressed as mean±sem, n=5, **(p<0.01) phytochemical screening of the ethanolic fruit extract of sarcocephalus latifolius was carried out using standard procedure described by trease and evans.15 discussion anxiety disorders present a pattern response that display two emotional states: fear and anxiety.19 the distinction between the two emotional states lies in the concept that the former is a response to an actual threat while the latter is an anticipatory response to a potential threat.20-21animal anxiety models are widely employed in the screening of anxiolytic and anxiogenic like agents, with the view of analyzing the pathological state of anxiety and assumption that some anxiety states are essential mechanisms for survival and are a feature of all mammals.22 the present study was aimed at evaluating the anxiogenic activity of the ethanolic extract of sarcocephalus latifolius in mice with the use of elevated plus maze and open field models. the anxiogenic makers commonly associated with anxiogenic agents in the elevated plus maze model decrease the time spent in the open arms as well as decrease the frequency of crossing the intersection.23 these makers are important parameters that validate test agents with anxiogenic properties. the extract showed anxiogenic effects in all the experimental dosage levels by decreasing the time spent and number of in the open arms. animals in the extract 4 all res. j.biol, 2014, 5, 1-6   and physiological saline groups spent more time in the closed arms avoiding the open arms, probably to avoid falling off.11 avoidance of the open arm is also an index that measures anxiety in rodents.24 also, avoidance of the open arms clearly demonstrates a fear response,25 with the display of anxiety related behaviours.23 however, the extract caused a significant (p<0.05) increase in the time spent in the closed arms and entries into the closed arms at extract doses of 400 and 600 mg/kg. this increase clearly reflects the anxiogenic effects of the extract. the reference standard showed a significant (p<0.001) anxiolytic effect evidenced in the increased number of entries into the open arms and prolonged time spent in the open arms and centre axis. anxiolytic agents can increase this effect and increase the number of entries in the open arm.23 reduction in open arm activity in the extract treated group elucidates the highest level of anxiety in rodents. the decrease in the time spent in the open arm predictably illustrates the anxiogenic activity of the extract. decoction preparation of the root of sarcocephalus latifolius has been reported to possess anticonvulsant, anxiolytic and sedative properties,14 but in this present study the ethanolic fruit extract showed anxiogenic properties the horizontal locomotor activity as well as the number of rearings in the open field model decreased in the extract treated groups compared to the reference standard, with 200 mg/kg having the highest number of rearings (table ii). the reference standard produced the highest horizontal locomotor activity. locomotor activity is considered as an indicator of alertness and decreases could lead to sedation as a result of reduction in the central nervous system excitability.26 a decrease in the locomotor activity of the extract treated group elucidates the manifestation of anxiety and lack of anxiolytic activity of the extract. conclusion data from the present study suggests that the ethanolic fruit extract of sarcocephalus latifolius is not only devoid of anxiolytic activity, but it appears to exert anxiogenic like effects. references 1. brunton, l.l., parker, k.l., blumenthal, d.k., buxton, l. (2008). goodman & gilman’s manuel of pharmacology and therapeutics. united state of america: the mcgraw-hill companies, 567-600 2. costello, e.j., mustillo, s., erkanli, a., keeler, g., angold, a. (2003). prevalence and development of psychiatric disorders in childhood and adolescence. arch gen psychiatry, 60, 837–844. 3. cha, h.y., seo, j.j., park, j.h., oh, k.w. (2004). anxiolytic effects of total saponins fraction from ginserg radix rubra on the elevated plus maze model in mice. j ginseng res, 28, 132. 4. craske, m.g., waters, a.m. (2005). panic disorder, phobias and generated anxiety disorders. annu. rev. clin.psychol, 1, 197, 225. 5. kessier, r.c. (2007). the global burden of anxiety and mood disorders putting the european study of the epidemiology of mental disorders (esemd) findings into perspective. j. clin. psychiatry, 6, 819. 6. america psychological association. anxiety disorders: the role of psychotherapy in effective treatment. 1998. 750 first street ne washington, dc 20002-4242. 7. thierry, s. (2001). the biology of fear and anxiety related behaviour. dialogues in clinical neuroscience, 4, 231-249. 8. western australian psychotropic drugs committee. anxiety disorder drug treatment guideline, 2008. guidelines prepared by the psychotropic drugs committee of the western australian therapeutics advisory group. (source: http://www.watag.org.au/wapdc/guidelines.cfm) 9. baldwin, d.s., anderson, i.m., nutt, d.j., bandelow, b., bond, a. (2005). davidson jrt. evidence based guidelines for the pharmacological treatment of anxiety disordersrecommendations from the british association for psychopharmacology, 19(6), 567-596. 5 all res. j.biol, 2014, 5, 1-6   10. simpson, h.b., liebowitz, m.r., foa, e.b., kozak, m.j., schmidt, a.b., rowan, v., petkova e, kjernisted k, huppert j d, franklin m e., davies, s.o. (2004). campeas r. post-treatment effects of exposure therapy and clomipramine in obsessivecompulsive disorder. depress anxiety, 19, 225–233. 11. rang, h.p., dale, m.m. (2006). rang and dale’s pharmacology. new york: churchill livingstone, pp. 535-53. 12. orwa, c., mutua, a., kindt, r., jamnadass, r., simons, a. (2000). agroforestree database:a tree reference and selection guide version 4.0, (http://www.worldagroforestry.org/af/treedb/) 13. bum, e.n., taiwe, g.s., moto, f.c.o., ngoupaye, g.t, nkantchoua g.c.n., et al. (2009). anticonvulsant, anxiolytic and sedative properties of the roots of nauclea latifolia smith in mice. epilepsy beha, 15, 434-440 14. abbah, j., amos, s., chindo, b., ngazal, i., vongtau, h.o., et al (2010). pharmacological evidence favouring the use of nauclea latifolia in malaria, ethnopharmacy: effect against nociception, inflammation and pyrexia in rats and mice. j ethnopharmacol, 127, 85-90. 15. trease, g.e., evans, w.c. (2005). trease and evans pharmacognosy, (14th ed), wb saunders company limited, london, 357-358. 16. lorke, d. (1983). a new approach for acute toxicity testing. arch toxicol, 1983; 54, 275-289. 17. national institute of health publication. guide for the care and use of laboratory animals, revised, 1985; n0; 85-23. 18. lister, r.g. (1987). the use of plus-maze to measure anxiety in mouse. psychopharmacology, 92, 180-85. 19. davis, m. (2006). “neural systems involved in fear and anxiety measured with fear potentiatedstartle”. american psychologist, 61, 741−756. 20. belzung, c., griebel, g. (2001). “measuring normal and pathological anxiety-like behaviour in mice: a review”. behaviour brain research 125, 141-149. 21. gray, j.a, mcnaughton, n. (2000). “the neuropsychology of anxiety: an enquiry into the functions of the septo-hippocampal system” 2nd ed., oxford university press, new york 22. hendrie ca, weiss sm, eilam d (1996). “exploration and predation models of anxiety: evidence from laboratory and wild species”. pharmacology biochemistry and behavior, 54, 1320. 23. garg, v.d., dhar, v.j., sharma, a., dutt, r. (2011). experimental model for antianxiety activity: a review. pharmacology online, 394-404. 24. trullas, r., & skolnick, p. 1993. differences in fear motivated behaviors among inbred mouse strains. psychopharmacology, 111, 323-331. 25. rodgers, r.j., dalvi, a. (1997). “anxiety defense and elevated plus maze”. neuroscience and behaviour, 21, 810-810. 26. thakur, v.d. (2005). mangi sv.neuropharmacological profile of elipta alba (linn). hassk ethnopharmacol, 102, 23. 6 microsoft word layoutedit.docx signal peptide prediction suggests mycobacterium tuberculosis curli pilin subunit secretion via the sec pathway may hinder mtp overexpression in escherichia coli natasha naidoo, balakrishna pillay, martin bubb, ajit kumar, thamsanqa chiliza, manormoney pillay *, all res. j. biol., 2017, 8, 1-15 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article issue 1, vol 8, 2017, 1-15 signal peptide prediction suggests mycobacterium tuberculosis curli pilin subunit secretion via the sec pathway may hinder mtp overexpression in escherichia coli natasha naidoo a balakrishna pillay b, martin bubb c, ajit kumar b, thamsanqa chiliza b, manormoney pillay a*. a medical microbiology and infection control, school of laboratory medicine and medical sciences, college of health science, university of kwazulu-natal; b microbiology, school of life sciences, college of agriculture, engineering and science, university of kwazulu-natal; c national bioproducts institute, pinetown, 3610, south africa. * pillayc@ukzn.ac.za abstract: introduction mycobacterium tuberculosis curli pili (mtp) are novel, potential tb diagnostic biomarkers, possessing important virulence attributes, unique to the m. tuberculosis complex (mtbc). the production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. for example, the signal peptide of mtp governed by the classical secretion pathway may hinder the purification of the protein in e. coli systems. in this study, the secretion characteristics of mtp were determined and the cloning, expression and purification of mtp was attempted in e.coli. materials and methods a fragment of mtp unique to mtbc was cloned into pet101 and pgex-6p-1 vectors. the clones were confirmed by nucleotide sequencing and expression of the protein was attempted at iptg concentrations ranging from 0.1mm to 1mm and at temperatures between 25 °c to 37 °c. the pgex-6p-1/mtp clone expressed protein was purified, yielding a mtp-gst fusion protein and a free gst band that were analysed by lc/ms mass spectrometry. inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kda and below 10 kda, both of which were analysed by lc/ms mass spectrometry. transcription activity of both the clones was interrogated by real time pcr on the cdna derived from the induced clones at various time points after induction with iptg. the signal peptide and protein secretion characteristics of the mtp protein were determined by bioinformatics analysis of the amino acid sequence using publically available software. results the truncated mtp fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. expression of the pgex-6p-1/mtp clone using 0.5 mm iptg at 27 °c demonstrated the presence of the expected fragment at approximately 35 kda. this was confirmed by western blotting using anti-gst antibodies. however, the purification of mtp in adequate quantities as a pure protein fraction was unsuccessful. mass spectrometry did not detect any fragments belonging to mtp, but only those of e.coli membrane proteins for the pet101/mtp clone and fragments of the gst tag in the case of the pgex-6p-1/mtp clone. the bioinformatics secretion analyses of mtp predicted a strong sec regulated secretion pathway and the absence of non-classical “mycobacterial specific” secretion. discussion m. tuberculosis membrane and secretory proteins often contain signal peptides. in this study, excluding the signal peptide region and using a gst tag greatly enhanced the expression of the protein in the soluble fraction. however, purification of the mtp peptide remained problematic due to a lower available peptide concentration resulting from the lower molecular weight, in the purified fraction compared to the gst tag. alternately, the predicted sec regulated secretion pathway may play a role in the inhibition of mtp overexpression in e.coli. thus, alternatives to e. coli expression systems or more efficient purification strategies are required for the acquisition of high quality m. tuberculosis antigens. 1 all res. j.biol, 2017, 8, 1-15 introduction novel biomarkers for diagnostic, therapeutic and preventative strategies are pertinent to tuberculosis (tb) control 1. mycobacterium tuberculosis curli pilus (mtp), a cell surface appendage and adhesin, has recently been highlighted as an important virulence factor and biomarker for the design of such interventions 2. the potential accuracy of mtp for use in a diagnostic test was also recently demonstrated, as this adhesin is unique to m. tuberculosis complex (mtbc) pathogens 2,3. consequently, the availability of the mtp antigen is essential for studies on tb diagnostics, pathogenesis and immunological response to m. tuberculosis infection and disease. the mtp gene in its entirety has previously been cloned and expressed in escherichia coli 4. while the recombinant protein was not visible in sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page), it was detectable by western blot, reacting with rabbit anti-mtp antibodies generated using a synthetic peptide. these antibodies, confirmed with recombinant mtp were utilized in immunogold electron microscopy to detect purified pili fibres and pili involved in adhesion and invasion assays. however, the recombinant mtp protein was not purified from e. coli total protein in that study. previously reported difficulties in visualising purified mtp by coomassie blue and silver staining were ascribed to its resistance to chemical treatments such as acid hydrolysis and denaturation with formic acid, urea, triton x-100, and enzymatic digestion with proteinase k, cellulose and lysozyme 5. pili were observed either in the stacking gel or as bands that were too large to be considered as pilin proteins that are usually observed as 6-25kda fragments 5. thus, it is envisaged that recombinant mtp protein production will alleviate the difficulties encountered in the isolation and characterisation of purified mtp into subunits. e. coli cloning and expression systems are commonly used for proteins from diverse bacteria. this is largely due to the rapid and confluent growth of e.coli on inexpensive media, its well known genetics and the availability of a range of cloning vectors, host strains and soluble fusion tags 5. several m. tuberculosis proteins have been expressed in e. coli including the 38-kda, mtb81 antigens 6, esat6, cfp10, mtc28, 14-kda antigen 7, shikimate kinase and 5enolpyruvylshikimate 3 phosphate synthase enzymes 8, among others. up to 50% of m. tuberculosis transmembrane proteins cloned into e.coli compatible vectors were shown to be successfully expressed 9, but interestingly, only 25% of these clones were overexpressed, resulting in higher protein yields. whilst this study proved that e. coli compatible vectors may be a good first attempt for expression of some m. tuberculosis transmembrane proteins 9, other researchers have shown that >50% of m. tuberculosis proteins cannot be expressed in this host system 5,10. the inadequate expression of m. tuberculosis protein in e. coli may be due to the significant differences in the g-c composition of e.coli (50%) and m. tuberculosis (65-70%), resulting in differences in codon usage between the mrna of these 2 microbes 11,12. usually a small number of rare codons do not severely depress protein expression 11. however, the presence of numerous rare codons may lead to low protein yield. these problems can be overcome by enriching the cells with the essential rare trna by inserting the trna genes into multiple copy plasmids, thereby increasing the copy number available in the cell 11. commercially available e. coli strains are also available for this purpose. another major reason for inefficient expression of m. tuberculosis proteins is that mycobacteria are known to posttranslationally adp-ribosylate proteins 13, whereas posttranslational modifications are not common in e. coli proteins 5,13. in addition, proteins expressed in e. coli are sometimes located in inclusion bodies, a consequence of high-level protein production in the e. coli cytoplasm 5. however, the use of highly soluble protein partners such as glutathione-s-transferase (gst) and induction at lower temperatures usually negates this problem. monitoring of transcription levels of genes during induction in clones and confirming mrna production can elucidate possible posttranslational modifications or shunting of the protein into inclusion bodies 5. the “classical secretion pathway” is governed by the sec and tat signal peptides made up of a structured three component motif found at the n-terminal of the proteins 14. the tat motif is similar to the sec motif except that the former contains a tat motif contains a twin-arginine consensus sequence. unfolded and folded proteins are secreted through the membrane using the secand tat-dependent secretion pathways, respectively. these systems transport proteins across the inner membrane in gram-negative bacteria, whilst additional secretion systems operate in the outer membrane. signal peptides play a role in these secretion systems. little is known about pilin secretion and assembly mechanisms through the outer membrane of the “diderm mycolate” bacteria 15. thus, it is not surprising that very little information can be found on the secretion of the curli pilin subunit across the cell wall of mycobacteria. the purpose of this study was to study the pilin secretion pathway using bioinformatics and to attempt the cloning and expression of a unique fragment of mtp. in the present study, the e. coli cloning and expression system was used in an attempt to generate and purify the truncated mtp protein 2 all res. j.biol, 2017, 8, 1-15 as these are inexpensive and robust systems for the isolation of high concentrations of m. tuberculosis proteins. the truncated mtp gene was successfully cloned into the pet101 and pgex-6p-1 vectors. however, the apparent lack of overexpression of truncated mtp was insufficient for proper protein purification and subsequent detection using mass spectrometry. protein secretion characteristics of the pilin subunit determined the presence of a signal peptide governed by the classical secretion pathway and which may play a role in shunting the pilin subunit out of the cell in the presence of the signal peptide. our findings indicate that secreted and membrane protein signal peptides and secretion pathways need to be ascertained before recombinant protein expression is attempted. moreover, a more efficient system for the expression of mycobacterial transmembrane and extracellular proteins is required. materials and methods transmembrane, rare codon and secretory protein analysis of the pilin subunit the secondary structure and secretory properties of the entire mtp protein were determined using bioinformatics tools (table 1). rare codons were identified using the nucleotide and amino acid sequences of the mtp gene 12. table 1. the bioinformatics software used to analyse the mtp amino acid sequence with the details of the specific analyses. tool details website citation kytedoolittle hydrophobicity protscale on the expasy server web.expasy.org/protscale/ (38) tmmtop transmembrane domain http://www.cbs.dtu.dk/services/tmhmm/ phobius combined transmembrane topology and signal peptide predictor showing n, h and c regions http://phobius.sbc.su.se/ (39) signalp signal peptide cleavage prediction http://www.cbs.dtu.dk/services/signalp/ (40) predtat determine classical or non-classical secretion pathway http://www.compgen.org/tools/pred-tat/ (41) tatp tat motifs and associated signal peptide http://www.cbs.dtu.dk/services/tatp/ (42) tbpred m. tuberculosis specific prediction tool for subcellular localisation prediction www.imtech.res.in/raghava/tbpred/ (43) plasmids and bacterial strains cloning and expression of the mtp gene were performed in the pet101 (invitrogen) and the pgex-6p-1 (ge healthcare) cloning vectors (supplementary figure 1). the pet101 vector contained a t7 promoter, lac promoter, t7 termination site, ampicillin resistance gene and c-terminal 6× his tag (~4 kda). the pgex-6p-1 vector contained a tac promoter, lac promoter, ampicillin resistance gene, multiple cloning site containing 9 restriction enzyme sequences and an n-terminal gst tag (26kda) containing a prescission protease cleavage site. cloning and plasmid propagation were carried out in one shot top10 chemically competent e.coli cells (invitrogen). expression was performed in either the bl21star chemically competent cells (invitrogen) or the e. coli rosetta 2(de3) plysstm cells (novagen) that contained prare plasmids containing a chloramphenicol resistance gene. construct design and cloning the cloning strategies including the mtp gene-specific primers used to generate the pcr product and the dna string used for ligation into the pet101 and pgex-6p-1 3 all res. j.biol, 2017, 8, 1-15 vectors respectively, are depicted in figure 1. inserts of 64 (7.04 kda) and 33 (3.63 kda) amino acids, were cloned into the pet101 vector and pgex-6p-1 vectors respectively. the h37rv strain was used as a reference genome for the design of cloning constructs. the rv3312a gene sequence was obtained from tuberculist: http://genolist.pasteur.fr/tuberculist/genome.cgi. generation of pet101/mtp clones the insert fragment for the pet101 vector was generated by pcr in a 25 µl reaction, containing 1× taq buffer, 1.5mm mgcl2, 0.2 µm forward and reverse primers (table 2), 0.2 mm dntps, 1 u platinum® taq polymerase, 20 ng/µl m. tuberculosis h37rv genomic dna, and nuclease free water. the cycling conditions were: 94 ºc for 1 min; 35 cycles of 94 ºc for 30 s, 50 ºc for 1 min and 72 ºc for 1 min and a final extension of 72 ºc for 5 min. following size confirmation in an agarose gel and purification using the qiaquick gel extraction kit (qiagen), the pcr product was confirmed by sanger sequencing (inqaba biotech) to confirm amplification of the correct gene. ligation was performed in a 1:2 ratio of vector:insert, by combining 1 µl of salt solutiontm, 2 µl of sterile water, 1 µl of the pet101 vector (20 ng/µl) and 2 µl of pcr product (20 ng/µl) and incubated for 20 min at room temperature, according to the champion™ pet directional topo kit (thermo fisher scientific). thereafter, 6 µl of the ligation reaction were transferred into two separate vials of one shot® top10 chemically competent e. coli cells, and incubated on ice for 30 mins. this was followed by incubation in a 42 ºc water bath for 30 s, and immediately placed back on ice. two hundred and fifty microliters of soc medium were added to the vial and incubated at 37 ºc with shaking at 200 rpm for 1 hr. volumes of 10 µl, 50 µl and 100 µl of the ligation reaction mix were plated onto luria-bertani (lb) agar plates containing 100 µg/ml ampicillin, and incubated at 37 ºc, overnight. single colonies from the plates were grown in 5 ml lb broth containing 100 µg/ml ampicillin at 37 ºc with shaking (200 rpm). in addition, colony pcr was performed to screen for the insert and vector-specific primers flanking the t7 region (table 2). the region was sequenced to confirm the gene was in frame with the 6× his tag. the plasmids from the positive colonies were isolated using the probond plasmid purification kit (invitrogen) and transformed into the appropriate e. coli expression strains before expression. generation of pgex-6p-1/mtp clones a double stranded dna string (90 bp fragment of the mtp gene) was synthesized (life technologies) and cloned into the pgex-6p-1 vector as follows. the pgex vector and mtp gene insert (1 ug each) were double digested with fastdigest ecor1 (1u) and xho1 (2u) (thermoscientific) in a 20 µl reaction. the restricted dna samples were gel extracted using the qiaquick gel extraction kit (qiagen). ligation was accomplished with a 1:3 vector:insert ratio in a 20 µl reaction containing 2.42 µl pgex-6p-1 vector dna, 2.8 µl insert dna, 4 µl ligation buffer, 1 µl t4 dna ligase (invitrogen) and 9.78 µl nuclease-free water. after incubation at room temperature for 5 min, 2 µl of ligation reaction was transformed into one shot® top10 chemically competent e. coli cells as described above. positive clones were confirmed by colony pcr using primers shown in table 2, and sanger sequencing as described above. the positive colonies were treated the same as the pet101/mtp clones. figure 1 the pet101 and pgex-6p-1 cloning strategies that were designed based on the rv3312a (mtp gene) amino acid sequence, nucleotide sequence and the protein secondary structure prediction (proteinpredict). 4 all res. j.biol, 2017, 8, 1-15 table 2 primers used in this study primer sequence description mtpf mtpr 5’ caccatgtaccggttcgc 3’ 5’ gaagtcgtcatggcaggtgt 3’ generated the insert for the pet clone t7 promoter t7 reverse 5 taatacgactcactataggg 3′ 5’ tagttattgctcagcggtgg 3’ used for screening colonies and sequencing after cloning and transformation of the pet clone pgex 5’ pgex 3' sequencing primers 5' gggctggcaagccacgtttggtg 3' 5' ccgggagctgcatgtgtcagagg 3' used for screening colonies and sequencing after cloning and transformation of the pgex clone mtp_f mtp_r 5’ cagtccgcagcccaaacc 3’, 5’ cgagtcaggtgtagggatcc 3’ target mtp gene primer used for real time pcr transcript analysis of the mtp gene 16s_f 16s_r 5’ cctacgggaggcagcagt 3’, 5’ cgtttacggcgtggactac 3’ housekeeping gene primers used for real time pcr transcript analysis of the mtp gene protein expression expression of the pet101/mtp and pgex-6p-1/mtp clones was optimised using concentrations of isopropyl β-d-1thiogalactopyranoside (iptg) at 0.1mm, 0.3mm, 0.5mm, 0.7mm and 1mm, and at temperatures of 25°c, 28°c, 30°c and 37°c (table 3). protein expression was attempted in both e. coli bl21 (de3) and e. coli rosetta 2(de3) plysstm cells (novagen), and separated on either 12 or 16% denaturing sds-page while the tags were detected by western blotting using anti-his antibody (donated by dr nonhlanhla nene, national bioproducts institute) for the pet101/mtp clone and anti-gst antibody (donated by dr nonhlanhla nene, national bioproducts institute) for the pgex-6p-1/mtp clone. protein purification of the pgex-6p-1/mtp clone using glutathione agarose the sample lysate of the expressed clone was prepared by mixing the protein extract with equilibration/wash buffer to a total volume of 20 ml and transferred to a 10 ml glutathione agarose column (pierce) that had been equilibrated with 100 ml of the same buffer. the column was washed several times until the absorbance of the flowthrough fraction at 280 nm approached baseline (0 mg/ml). thereafter, the gst-tagged protein was eluted in 1ml fractions with 20 ml of elution buffer and monitored by measuring the a280. the amicon pro purification system (merck) with a 10 kda molecular weight cut off filter was used to remove glutathione and concentrate the purified protein for downstream applications. prescission protease (ge healthcare) was used to cleave the gst tag from the peptide after purification. inclusion body preparations the insoluble pellet fractions from induced samples processed after sonication were screened for expressed peptide by boiling in 5×sds-page laemmli buffer and separated by electrophoresis in sds-page in order to determine whether the cloned protein may be expressed in inclusion bodies. further analysis of the inclusion bodies was performed by inducing the pet101/mtp clone at 1 mm iptg at 30 °c and the pgex-6p-1/mtp clone at 0.5 mm iptg at 27 °c. induced cells were centrifuged at 6000 rpm for 20 min, and the pellet re-suspended in 3ml buffer a (50mm tris-hcl, ph8, 5 mm edta and 10mm nacl) per gram of cells. thereafter, 1 mm phenylmethylsulfonyl fluoride (pmsf) and 16 µl lysozyme (50 mg/ml) per gram of cells were added and incubated at 37 °c for 30 min. the solution was sonicated to reduce viscosity and centrifuged at 18 000 rpm for 30 min. the remaining 5 all res. j.biol, 2017, 8, 1-15 pellet was re-suspended in 3 ml buffer b (20 mm na2hpo4, ph 7.2, 20 mm nacl, 5 mm edta and 25 % sucrose) per gram of cells. pmsf was added to a final concentration of 1mm followed by 10 µl triton x-100 per ml of solution. following centrifugation at 20 000 rpm for 20 min, the remaining pellet of inclusion bodies was dissolved in 8 mm urea with heating at 37 °c. the urea was dialyzed in 4 l of 50 mm tris-hcl buffer at ph 8.5 using a 3.5 kda molecular weight cut off dialysis tubing (sigma) for 2 days, with a buffer replacement every 8 hrs. proteins were concentrated by dialysis with peg 20 000. table 3 the pilot protein expression conditions of the pet101/mtp and pgex-6p-1/mtp clones at various temperatures and iptg concentrations as assessed on sds-page gels bl21(de3) e.coli cells rosetta2(de3)plyss e.coli cells temp (°c)/ iptg (mm) 0.1 0.3 0.5 0.7 1 0.1 0.3 0.5 0.7 1 pet101/mtp clone 25 28 30 37 pgex-6p-1/mtp clone 25 + + ++ + + 28 + + ++ + + 30 + + + + + 37 + + + + + mass spectrometric analysis of the putative recombinant peptides bands obtained from sds-page of inclusion body preparations of the pet101/mtp clone overnight expression (10 kda and <10 kda), as well as from the pgex-6p-1/mtp glutathione agarose (pierce) purified clone were excised and analysed by mass spectrometry (central analytical facilities, proteomics laboratory, stellenbosch university). analysis of mrna levels during expression in the pet101/mtp and pgex-6p-1/mtp clones due to the difficulty in visualising mtp fibres by coomassie and silver staining on acrylamide gels demonstrated by alteri et al, 2005, transcript analysis of the pet101/mtp and pgex6p-1/mtp clones was performed to ascertain whether the pilin gene was transcribed upon induction of expression. overnight cultures of cells freshly transformed with either the pet101/mtp clone or pgex-6p-1/mtp clone were grown in 20 ml lb medium containing ampicillin (100 µg/ml) at 37 °c in a shaking incubator. cells at an od600 of 0.5 were induced with 0.5 mm iptg. prior to iptg addition, and at intervals of 5 min, 10 min, 15 min, 20 min, 30 min, 1 hr, 2 hr, 3hr, 4 hr and 5 hr after induction, samples were pelleted, frozen with liquid nitrogen and immediately stored at -80 °c. total rna was isolated using the genejet rna purification kit (thermoscientific) and treated with the rnase-free dnase (thermoscientific) according to the manufacturer’s instructions. rna quantity and quality was assessed using the nanodrop 2000 spectrophotometer (nanodrop technologies). reverse transcription of 2 µg total rna was performed with the high capacity cdna reverse transcription kit (applied biosystems) in accordance with the manufacturer’s protocol. real time pcr was performed in triplicate in 20 µl reaction volumes containing cdna corresponding to 50 ng rna and 6 all res. j.biol, 2017, 8, 1-15 jumpstart readymix sybr green (sigma), in a biorad cfx96-real-time system. the primers used are listed in table 2. amplification conditions comprised an initial activation step of 15 min at 95 °c followed by 40 cycles of 94 °c for 15 s, 58.3 °c for 30 s and 72 °c for 30 s. to ensure comparability between data for the same target obtained from different pcr runs, the pcr efficiency (e) of the mtp genespecific and 16s rrna primers was determined using a standard curve as detailed in the pfaffl method 16. an e of 1.6 and 1.8 for each gene respectively was obtained at an annealing temperature of 58.3 °c (supplementary figure s2). for the calculation of the relative changes in gene expression, the pfaffl method was applied taking the amplification efficiencies into account 16. the fold difference in gene expression after induction was calculated using the following equation 16: results bioinformatics of the mtp amino acid sequence reveals rare codons, hydrophobic and hydrophilic regions and sec-dependent pilin secretion analysis of rare codons showed 12 sites at which the trna of the e. coli differed from that of m. tuberculosis. of these 12, four occurred in the pgex-6p-1/mtp clone sequence and 5 in the pet101/mtp clone sequence. the kyte-dolittle software revealed a strong hydrophobic region at the beginning of the protein sequence and a strongly hydrophilic region thereafter, becoming slightly hydrophobic from around 80 amino acids (figure 2). this is suggestive of the putative folding of the pilin subunit into the pilus shaft. tmmtop database identified an alpha helix region in the predicted hydrophobic domain (figure 2). phobius database predicted the typical n-terminal signal sequence from substrates of either the sec or the tat systems bearing the polar n-region with a positive net charge, the uncharged hydrophobic h-region and short polar c-region 14 (figure 2). curiously, the signalp server predicted a cleavage site between amino acids 22 and 23, which is within the c-region rather than after it and also within the hydrophobic region. a possibly more accurate cleavage site between amino acids 33 and 34 was predicted by tatp. however, the prediction output demonstrated the absence of a twin-arginine (arg = r), otherwise known as a tat motif. in addition, tatp cleavage site prediction also corresponded to the pred-tat prediction with the cleavage site closer to amino acid 33, which consequently also predicted a more likely sec associated protein secretion. the bioinformatics analysis of the mtp amino acid sequence suggests that the sec-dependent pathway is used to carry unfolded pilin subunits across the inner membrane into the periplasmic space in m. tuberculosis. this is an interesting finding, as it proposes that the mtp pilin subunit secretion is affected through the classical pathway similar to the pili of other bacteria 17 rather than the non-classical pathway that is unique to m. tuberculosis. thus, even though mtp differs from other bacterial pili in terms of nucleotide sequence and gene organisation properties (not in an operon and distant from other pilin assembly components 2 pilin secretion into the periplasmic space has most likely remained the same. 7 all res. j.biol, 2017, 8, 1-15 the position specific analyses of the mtp amino acid sequence on tbpred software has predicted a protein with a lipid anchor which is highly favoured for m. tuberculosis as the bacterium has a lipid rich cell wall (figure 2). this prediction needs to be confirmed experimentally. confirmation of the pet101/mtp and pgex-6p-1/mtp clones the pet101 and pgex-6p-1 transformants were screened by colony pcr using the insert and sanger sequencing of the vector-specific pcr products. in both cases, the cloning was successful and the inserts were both in frame with the respective fusion tags (figure 3). expression of the proteins from the pet101/mtp clone, purification of expressed mtp from pgex-6p-1/mtp clones and the acquisition of products sent for protein sequencing (mass spectrometry) the pet101/mtp clone showed no expressed product in the soluble fraction of the e.coli lysate as no apparent bands could be visualized by sds-page at the expected size of 10kda in the induced or uninduced samples (figure 4a). however, western blotting using anti-his tag antibodies showed very faint bands, barely visible on images taken (data not shown). neither e.coli bl21 (de3) nor e. coli rosetta 2(de3) plysstm cells expressed detectable proteins in the pet101/mtp clone. sds-page (figure 4b) and western blotting using anti-gst antibodies demonstrated successful expression of pgex6p1/mtp clones transformed into e. coli rosetta 2(de3) plysstm cells. the expressed protein, present in the soluble fraction of the e. coli lysate, was purified using glutathione agarose and showed a strong reaction with anti-gst antibodies upon western blot analyses (figure 5). figure 2 the amino acid sequence of the mtp protein showing the rare codons, the kyte-dolittle hydrophobicity and transmembrane analysis curves, with the scores calculated according to the properties of the amino acid sequence of mtp on the protscale server (web.expasy.org/protscale/), the protein predict data with i = inside membrane, t = transmembrane and o = outside membrane, the tmmtop data with i = inside membrane, h = alpha helix and o = outside membrane, phobius data showing the n-region, h-region and c-region. the signalp data with the predicted cleavage site between amino acids 22 and 23. the c score is the prediction for the cleavage site. the s score is the prediction for the amino acids that form a part of the signal peptide. the y score is the average of the c-score and the s score, depicting a more accurate cleavage site prediction. pred-tat predicted a sec signal peptide rather than a tat signal peptide. tatp predicted a cleavage site between amino acids 33 and 34 8 all res. j.biol, 2017, 8, 1-15 e. coli bl21 (de3) cells transformed with either the pet101/mtp clone or the pgex-6p-1/mtp clone failed to show any clearly visible expressed proteins on sds-page. the flow diagram (figure 6) shows the steps that were taken in order to acquire putative mtp protein for protein sequencing by mass spectrometry. inclusion body preparation of the pet101/mtp clone revealed no overexpression of mtp protein (figure 7a). mass spectrometric analysis demonstrated that the 2 bands that were observed, at 10 kda and below this, represented membrane proteins present in e.coli, and that no m. tuberculosis proteins were detected (supplementary figure s3). protein sequencing of the pgex-6p-1/mtp clone purified bands (figure 7b) revealed fragments of the gst tag only, but not the mtp peptide upon blast analysis of the electrospray fragments to the m. tuberculosis h37rv genome (supplementary data s4). prescission protease cleaved off the peptide from the gst as indicated by the loss of the larger band on sds-page and the presence of a remaining gst band. however, there was no indication of the peptide on the sds-page gel as the peptide was too small to be seen. transcript analyses of the pet101/mtp clone and the pgex-6p-1/mtp clone the pet101/mtp clone showed a 28 fold less mtp gene expression than the pgex-6p-1/mtp clone after 5 hours of induction. overall, the transcription in the pgex-6p-1/mtp clone occurred more rapidly than for the pet101/mtp clone. thus, the data shows that the truncated mtp gene without the signal peptide is much more amenable to rapid, high level transcription as shown in the pgex-6p-1 vector compared to the loner sequence that included the signal peptide in the pet101 vector. discussion as the tb epidemic intensifies in developing countries, new diagnostic methods that are accurate, cost-effective and simple to implement are urgently required 18. the typical diagnostic algorithm takes around 2 weeks, during which an individual can infect around 10-15 people before treatment initiation 19. more sophisticated approaches in finding tb figure 3 confirmation of pet101/mtp and pgex-6p-1/mtp clones using vector-specific primers to generate pcr products and subsequent sanger sequencing. the shaded boxed sequence represents a part of the vector sequence that would have been cleaved by the restriction enzyme xho1 during cloning of the insert into the vector. 9 all res. j.biol, 2017, 8, 1-15 biomarkers have been used in recent years in order to help pinpoint epitopes that are more sensitive and specific to m. tuberculosis 19. following identification of these markers, the antigens need to be accessible in large amounts for the generation of antibodies and for evaluation using patients’ clinical samples. figure 4. the sds-page gels of clones expressed at 28 °c: (a) 16% acrylamide gel: pet101/mtp clone lane 1: uninduced sample, lane 2: 0.3mm iptg, lane 3: 0.5mm iptg, lane 4: 1mm iptg, lane 5: protein ladder (new england biolabs prestained protein ladder, cat number 7706), (b) 12 % acrylamide gel : pgex-6p1/mtp clone lane 1: uninduced sample, lane 2: 0.1mm iptg, lane 3: 0.3mm iptg, lane 4: 0.5mm iptg, lane 5: 0.7mm iptg, lane 6: protein ladder (pierce prestained protein marker: cat number 26612). although several promising antigens have been identified in previous studies, reproducible expression and purification of high-quality recombinant mycobacterial proteins is difficult and laborious 18. in this study, recombinant dna technology was used to attempt to generate high concentrations of pure mtp, an epitope that is highly specific to the m. tuberculosis complex 20. expression and purification of mtp was attempted using two vector systems containing different fusion tags (his tag and gst tag). however, the use of recombinant dna technology proved inadequate for high level purification of the truncated mtp protein. in this study, rare codons were compensated for by the use of rosettatm cells (novagen) containing “trare” plasmids. our findings demonstrated that while rosetta cells were capable of expressing mtp in the pgex-6p-1/mtp clone, they were unable to enhance expression in the pet101/mtp clone. since mtp was predicted to be a part of the transmembrane proteins and is unable to be overexpressed in e. coli, this study may have revealed a similar challenge of inadequate expression of other transmembrane proteins in e. coli. transmembrane proteins secreted via the classical secretion pathway may be shunted out of the cells in the presence of the signal peptide. this hypothesis needs to be further investigated using other transmembrane proteins with similar inadequate expression in e. coli, by exploring the signal peptides present in these proteins and monitoring transcription. in a previous study, a 5 kda product was detected by western blotting with anti-mtp antibodies on m. tuberculosis whole cell lysate, whereas a 14.5 kda product, inclusive of 6x his tag (4 kda), was detected by western blotting when expressed in e. coli 4. thus, it was postulated that e. coli lacked the post-translational modifications required to cleave the protein to 5 kda. alternatively, it could be hypothesized that a fraction of the protein may be exported from the cell without further assembly into pili on the cell surface, possibly due to the absence of the appropriate assembly factors or chaperones. thus, most of the protein was likely secreted from the cell and therefore, not visible by sdspage in the expressed total protein lysate from e.coli but detectable in the more sensitive western blot. the pet101/mtp clone that harboured the signal peptide was less successful at expressing detectable product in the sdspage and western blot than the pgex-6p-1/mtp clone. even though transcription of the pet101/mtp clone occurred in a time dependent manner at a much lower rate than the pgex6p-1/mtp clone, no apparent protein expression occurred even in the induced overnight culture, in either the soluble or insoluble fractions. thus, it can be postulated that despite the slow progression in transcription, translation of the protein and sec-associated cleavage of the protein that is observed in all bacterial species as part of the bacterial classical secretory pathway 14 may be responsible for the undetectable protein. the absence of the n-terminal signal peptide recognition region and gst tag in the pgex-6p-1/mtp clone may have resulted in protein being present in the soluble fraction of the e. coli lysate. however, the protein was not detected by sequencing using mass spectrometry, despite the discovery of several fragments of the gst tag. this inability to detect the peptide may have been due to its lower concentration following trypsin digestion from the gst tag. the peptide could not be visualised on sds-page after cleavage from the gst using prescission protease. figure 5 the expressed and purified mtp from the pgex-6p-1/mtp clone. (a) sds-page and (b) western blot, lane1: protein ladder (new england biolabs, catalogue number: 7706), lane 2: induced pgex-6p-1/mtp clone at 0.5mm iptg and 28 °c. lane 3: uninduced pgex-6p-1/mtp clone, lane 4: induced pgex-6p-1 vector control, lane 5: uninduced pgex-6p-1 vector control, lane 6: purified mtp, lane 7: purified pgex-6p-1 vector control. 10 all res. j.biol, 2017, 8, 1-15 figure 6 process by which putative mtp products were obtained for protein sequencing and the results that were obtained at each step. unfolded pilin subunits are usually secreted out of the cell using the sec dependent secretion system 17. in grampositive bacteria, sec secretion is often followed by pilin assembly mediated by assembly factors and chaperones depending on the type of pili being assembled. in gramnegative bacteria, pili need to be secreted via a second membrane before assembly on the surface. even though mycobacteria have been classified as a gram-positive actinomycete, the cell wall structure mimics that of gramnegative bacteria. thus, the pilin assembly of the gramnegative curli pili may be more closely related to that of mtp. while mtp have been reported to harbour morphological and physicochemical similarities to the curli pili of gram-negative bacteria 4, they differ in the nucleotide or amino acid sequence and genetic organisation 2. curli pili assembly are governed by two operons, one containing the pilin subunit csga and nucleator csgb 21-23. in the absence of csgb, curlin assembly is attenuated as csgb nucleates csga into a pilus fibre 23. the second operon contains four other components required for pilin assembly. all the components of both operons are secreted through the sec-dependent secretion pathway into the periplasm with the exception of csgd, a transcriptional regulator of the csgba operon. the pore through which csga and csgb proteins travel through the outer membrane is made up of the csgg protein. salmonella curli pili have similar operons with assembly also governed by the nucleation pathway 24. the components involved in pilin assembly of mtp are yet to be determined as the protein secretion through the outer membrane of mycobacteria has not been very well studied 15. the secretion mechanisms of adhesins and other proteins on the outer membrane of mycobacteria would provide insight into the mechanisms through which these bacilli survive in the host cell. nevertheless, the data in this study provides evidence that suggests that the pilin subunit is secreted through the inner membrane into the periplasm in much the same way as the pilin subunits of other bacteria. a limitation of this study was that alternative approaches in order to express mtp were not attempted. an alternate approach to generate recombinant proteins specific to m. tuberculosis included the use of the yeast pichia pastoris as a host instead of e. coli 25. this approach has been shown to produce an end product at a yield of 0.5 g per litre of expressed culture using antigen bound to his tag. this is a significant yield when compared to membrane proteins expressed in e. coli with a maltose binding protein (mbp) tag that yields 10-90 mg antigen per litre of bacterial culture over a range of 22 m. tuberculosis proteins expressed 26. in addition, the yeast derived antigen was better at detecting polyclonal antisera by western blot and antibodies in tb patient serum in elisa 27. figure 7 sds-page gels of (a) expression products of inclusion body preparation of pet101/mtp clone induced with 1mm iptg at 30 °c. lane 1: uninduced inclusion body preparation, lane 2: induced inclusion body preparation, lane 3: pageruler prestained protein ladder (cat number 26616). (b) purification products of pgex-6p-1/mtp clone induced with 0.5mm iptg at 28 °c. lane 1: purified protein, lane 2: pageruler prestained protein ladder (cat number 26616). the bands that were sequenced using mass spectrometry have been highlighted in red blocks. 11 all res. j.biol, 2017, 8, 1-15 mukherjee et al, 2003 reported the cloning of two or three copies of the same gene into a cloning vector in order to express the proteins as dimers or trimers 28. this strategy was especially effective on proteins that were too small to be resolved on acrylamide gels and therefore, not easily detected upon expression. however, the achievement of a high success rate of these experiments involved several rounds of pcr using three or four different primer sets, and restriction and ligation of the inserts in order to produce an insert that could be fused with the vector. this requires a lot of technical skill and rigorous design of the insert generating strategy. the use of water-soluble fusion tags are popular approaches and are usually effective for the expression of membrane proteins in particular 26. in this study, the his-tag and gst fusion tags have been cloned alongside the mtp protein. the maltose binding protein (mbp) has not been cloned alongside mtp protein and this fusion tag has been used to successfully express m. tuberculosis transmembrane proteins 26. future studies include using mbp tags, with yeast as a host strain instead of e. coli or using an in-tube cell free protein expression system 29 rather than a host strain. synthetic peptides have also been used for the design of elisas for diagnostic purposes to overcome the problems associated with the low yield of recombinant m. tuberculosis proteins in e. coli 30-35. contamination associated with protein purification and from cloning vectors has been reported to result in low yields of m. tuberculosis protein 36. in addition, peptides were reported to be much more cost effective than expressed recombinant proteins 37 despite the advantage of multiple propagation and protein expression of clones. the results of this study suggest that the curli pilin subunit is exported through the mycobacterial inner membrane through the sec secretion pathway. thus, we postulate that the pilin subunit is secreted through the inner membrane soon after translation by the sec machinery and assembled through the outer membrane thereafter. if the pilin is assembled through the outer membrane, there are other yet undiscovered proteins involved in this process on the mycobacterial cell wall. the alpha helix transmembrane region may indicate that the n-terminal signal peptide region may double as a lipid anchor. the removal of the signal peptide containing region of the mtp greatly enhanced expression of the peptide in e.coli. this may indicate that the signal peptides of many other proteins may hinder the detection of expressed product when the protein is exported through the classical secretion pathway. references 1. world health organisation. 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(2014). development of a poc test for tb based on multiple immunodominant epitopes of m. tuberculosis specific cellwall proteins. plos one 9, e106279. 34. shen, g., behera, d., bhalla, m., nadas, a., and laal, s. (2009). peptide-based antibody detection for tuberculosis diagnosis. clin. vaccine. immunol. 16. 1, 4954. 35. goyal, b., kumar, k., gupta, d., agarwal, r., latawa, r., sheikh, j. a., and verma, i. (2014). utility of bcell epitopes based peptides of rd1 and rd2 antigens for immunodiagnosis of pulmonary tuberculosis. diag. micro. infect. dis 78, 391-397. 36. yang, h., liu, z. h., zhang, l. t., wang, j., yang, h. s., qin, l. h., jin, r. l., feng, y. h., cui, z. l., zheng, r. j., and hu, z. y. (2011). selection and application of peptide mimotopes of mpt64 protein in mycobacterium tuberculosis. j med microbiol 60, 69-74. 37. mishra, a. r., hutke, v. r., satav, a. r., ali, s. a., daginawala, h. f., singh, l. r., and kashyap, r. s. (2016). synthetic peptides are better than native antigens for development of elisa assay for diagnosis of tuberculosis. int. j. peptide res. therap., 1-11. 38. kyte, j., and doolittle, r. f. (1982). a simple method for displaying the hydropathic character of a protein. j. mol. biol. 157, 105-132. 39. kall, l., krogh, a., and sonnhammer, e. l. (2004). a combined transmembrane topology and signal peptide prediction method. j. mol. biol. 338, 1027-1036. 40. bendtsen, j. d., nielsen, h., von heijne, g., and brunak, s. (2004) improved prediction of signal peptides: signalp 3.0. j. mol. biol. 340, 783-795. 41. bagos, p. g., nikolaou, e. p., liakopoulos, t. d., and tsirigos, k. d. (2010). combined prediction of tat and sec signal peptides with hidden markov models. bioinform. 26, 2811-2817. 42. bendtsen, j. d., nielsen, h., widdick, d., palmer, t., and brunak, s. (2005) prediction of twin-arginine signal peptides. bmc bioinform. 6, 1-9. 43. rashid, m., saha, s., and raghava, g. p. (2007) support vector machine-based method for predicting subcellular localization of mycobacterial proteins using evolutionary information and motifs. bmc bioinform. 8, 1-9. acknowledgements 14 all res. j.biol, 2017, 8, 1-15 this work was supported by the south african national research foundation (sa-nrf) for financial support (m. pillay: grant number 90508 and incentive funds for rated researchers). n. naidoo gratefully acknowledges the nrf and kwazulu-natal research institute for tuberculosis and hiv-coinfection (k-rith) for scholarships. we are grateful to ms nonhlanhla nene for providing anti-his tag and antigst antibody for western blots. 15 microsoft word 88-541-1-layoutediting.doc is coffee consumption associated with agerelated macular degeneration and diabetic retinopathy? kumari neelam, xiang li, wan-ling wong, e shyong tai, jeannette lee, rob m van dam, tien-yin wong. all res. j. biol., 2014, 5, 7-13 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com article                                                                                                                    issue 2, 5, 2014, 7-13 is coffee consumption associated with age-related macular degeneration and diabetic retinopathy? kumari neelam,1,2 xiang li,2,3 wan-ling wong,2 e shyong tai,4 jeannette lee,4 rob m van dam,4 tien-yin wong2,5,6* 1department of ophthalmology and visual sciences, khoo teck puat hospital, singapore. 2singapore eye research institute, singapore. 3department of statistics and applied probability, national university of singapore, singapore. 4saw swee hock school of public health, national university of singapore, singapore. 5singapore national eye center, singapore. 6department of ophthalmology, yong loo lin school of medicine, national university of singapore, singapore. *corresponding author email: tien_yin_wong@nuhs.edu.sg abstract coffee is among the most widely consumed beverages in the world. several epidemiological studies have evaluated the association between coffee consumption and risk of systemic diseases; however, there is paucity of data in relation to coffee consumption and risk of eye diseases. this study aims to examine the relationship between coffee consumption and risk of age-related macular degeneration (amd) and diabetic retinopathy (dr) in the multiethnic population of singapore. we analyzed the data from 4121 study participants from the singapore prospective study program (sp2) to examine the relationship of coffee to prevalence of amd and dr. sp2 was a population-based cross-sectional study inclusive of community-dwelling individuals between the ages of 21 to 95 years, selected by disproportionate stratified sampling to ensure representation of the ethnic composition of singapore’s population: chinese, malay, and indian. a standardized questionnaire that included information about the habitual amount of coffee consumed was completed by all study participants. presence and severity of amd and dr was assessed on fundus photographs by trained and certified graders in a masked fashion using the multiethnic study of atherosclerosis grading protocol. the prevalence of amd and dr in our population was 5.4% and 32.0%, respectively. a positive history of coffee consumption was present in 77.5% of amd population and 76.1% of dr population with majority of participants consuming 1-2 cups of coffee daily. no statistically significant association was observed between coffee consumption and odds of amd or dr after adjusting for confounding factors [amd: odds ratio (or) = 1.27, confidence interval (ci) = 0.88-1.83, p = 0.20; dr: or = 1.36, ci = 0.69-2.69, p = 0.37). this epidemiological study of a large multiethnic population data set does not support the hypothesis that habitual intake of coffee is associated with amd and dr among asians. keywords coffee; caffeine; age-related macular degeneration, diabetic retinopathy introduction coffee is among the most widely consumed beverages in the world and its potential health effects have been extensively studied.1,2 several epidemiological studies have demonstrated that coffee consumption is associated with decreased risk of systemic diseases, such as type 2 diabetes,3,4,5 coronary heart disease,6,7 and various cancers.8,9 however, there is paucity of data in relation to coffee consumption and risk of eye diseases. caffeine has been shown to have vasoconstrictor effect on the retinal and choroidal circulation in the eye,10 and therefore, it has been hypothesized that cumulative effect of caffeine may contribute to a higher risk of developing vascular diseases. age-related macular degeneration (amd) and diabetic retinopathy (dr), two common retinal vascular diseases, are the leading causes of blindness. the exact aetiopathogenesis of both these retinal diseases remains unknown but altered hemodynamics of retinal and choroidal circulation in the 7 all res. j.biol, 2014, 5, 7-13     form of decreased blood flow and elevated hydrostatic pressure has been proposed as one of the mechanisms in amd (hemodynamic model).11 likewise, in early stages of dr, a variety of cellular and molecular mechanisms lead to a state of hyperconstriction of blood vessels with consequential alteration in blood flow to the retina.12 to date, only one study has examined the association between coffee consumption, along with total caffeine intake, and 5-year incidence of early amd.13 furthermore, there is no published study on coffee consumption and risk of dr. this study aims to examine the relationship between coffee consumption and risk of amd and dr in multiethnic population of singapore. knowledge of the ocular effects of coffee may contribute to eye disease prevention and allow individuals to make informed choices regarding coffee consumption. materials and methods in this study, we used cross-sectional data from 4121 study participants of the singapore prospective study program (sp2), a follow-up study of participants from four previous population-based studies in singapore that were conducted between the years 2003 to 2007 [thyroid and heart study,14 the national health survey-1992,15 the national university of singapore heart study,16 and national health survey199817]. the study was approved by the singapore general hospital and the national university hospital institutional review boards, and informed consent was obtained in accordance with the declaration of helsinki. a standardized questionnaire that included information about the habitual amount of coffee consumed was completed by all study participants.18 participants were asked to choose the intake frequency of coffee from 7 pre-defined categories ranging between ‘never/rarely’ to ‘10 or more cups per day.’ the standard serving size was assigned as 1 cup that refers to ‘standard coffee-shop cup’ of 215 ml. we have assessed only caffeinated coffee because decaffeinated coffee is rarely consumed in our study population. two retinal images of each eye were obtained, one centered at the optic disc and another centered at the fovea, using a 45 degree canon digital fundus camera (cr-dgi with a 10d slr back) after pupil dilatation identical to the early treatment for diabetic retinopathy study (etdrs) standard fields 1 and 2. presence and severity of amd and dr was graded on fundus photographs (fp) using the multiethnic study of atherosclerosis grading protocol.19 the images were graded by trained and certified graders in a masked fashion at the singapore advanced imaging laboratory for ocular research. early amd was defined by 1) any soft drusen (distinct or indistinct) and pigment abnormalities (either increased retinal pigment or retinal pigment epithelium de-pigmentation); 2) large soft drusen ≥ 125 µm in diameter with a large drusen area of > 500 µm; 3) large soft indistinct drusen ≥ 125 µm in the absence of signs of late amd. late amd was defined by presence of any of the following: geographic atrophy, retinal pigment epithelial detachment, subretinal hemorrhage, visible subretinal new vessels, subretinal fibrous scar, laser treatment, and/or photodynamic therapy for amd. dr was considered to be present if any characteristic lesion, as defined by the etdrs severity scale, was present: microaneurysms, hemorrhages, hard exudates, cotton wool spots, intraretinal microvascular abnormalities, venous beading, and new vessels.20 for each eye, a retinopathy severity score was assigned according to a scale modified from the airlie house classification system.21 when two eyes of a participant were discrepant for the severity of a lesion, the grade assigned for the participant was that of the more severely involved eye. for statistical analyses, the data from 3719 study participants were used for examining the relationship between coffee and caffeine intake to prevalence of amd following exclusion of participants with dr (n = 113) and missing data (n = 289). for examining the relationship between coffee and caffeine intake to prevalence of dr, the analyses based on 353 subjects were restricted to participants with known history of diabetes mellitus (n = 412) but without amd (n = 37) after excluding 22 subjects with missing dr data. we did not exclude subjects with pre-existing history of hypertension, ischemic heart disease, stroke, dyslipidemia, and cancer; however, these diseases were adjusted in multivariable analysis as confounders. coffee intake was regrouped into four categories (never/rarely, <1cup/day, 1-2 cups/day and >2 cups per day) to avoid categories with small numbers. odds ratios (ors) and 95% confidence intervals (ci) were estimated using multivariable logistic regression model to examine the relationship of coffee and caffeine intake to prevalence of amd and dr, after controlling for confounding variables. we have reported our results in terms of coffee consumption because total daily caffeine intake correlated well with coffee consumption levels in our study population (r = 0.744, p <0.001). results a. coffee and amd the mean age of the study population was 49.48 ± 11.45 years with slight preponderance of male participants (males = 52.4%; females = 47.6%). presence of any amd (early plus late amd) was observed in 201 study participants (5.4%). a 8 all res. j.biol, 2014, 5, 7-13     positive history of coffee consumption was recorded in 155 (77.5%) amd participants with 112 (55.7%) reported consuming 1-2 cups of coffee per day (equivalent to total daily caffeine intake of 171.4 mg/day). higher coffee intake was marginally associated with smoking in amd participants (p = 0.052) controlling for age and gender. no significant association between coffee consumption and odds of amd was observed in a multivariable logistic regression model, after adjusting for confounding factors [or = 1.27, ci = 0.88-1.83, p = 0.20, table 1]. this association between coffee consumption and odds of amd remained statistically insignificant when the study population was stratified according to 3 ethnic origins [chinese: or = 1.19, ci = 0.70-2.02, p = 0.513; malay: or = 1.62, ci = 0.78-3.37, p = 0.198; indian: or = 1.03, ci = 0.49-2.17, p = 0.930, table 1]. the overall association between coffee consumption and odds of amd did not differ when analysis was restricted to study participants without pre-existing history of hypertension, ischemic heart diseases, stroke, dyslipidemia, and cancer (or = 1.17, ci = 0.72-1.89, p = 0.519). table 1: association of coffee consumption with amd and its lesion components, soft drusen and pigmentary abnormalities, stratified by ethnicity or (95% ci) p value or (95% ci) p value or (95% ci) p value chinese (n = 2223) coffee yes 1.19 (0.70, 2.02) 0.513 1.37 (0.73, 2.55) 0.325 1.29 (0.55, 3.01) 0.561 no 1.00 1.00 1.00 coffee four levels never or rarely 1.00 1.00 1.00 less than 1 cup per day 1.41 (0.68, 2.92) 0.355 1.60 (0.69, 3.72) 0.272 1.90 (0.65, 5.61) 0.243 1-2 cups per day 1.17 (0.68, 2.02) 0.577 1.35 (0.71, 2.58) 0.356 1.22 (0.50, 2.96) 0.653 more than 2 cups per day 1.03 (0.41, 2.58) 0.945 1.12 (0.38, 3.27) 0.843 0.78 (0.16, 3.92) 0.765 malay (n = 791) coffee yes 1.62 (0.78, 3.37) 0.198 2.44 (0.93, 6.37) 0.068 0.99 (0.39, 2.54) 0.997 no 1.00 1.00 1.00 coffee four levels never or rarely 1.00 1.00 1.00 less than 1 cup per day 3.21 (1.24, 8.33) 0.016 4.93 (1.52, 15.95) 0.008 1.75 (0.48, 6.34) 0.395 1-2 cups per day 1.35 (0.62, 2.96) 0.449 1.90 (0.69, 5.26) 0.214 0.84 (0.30, 2.32) 0.735 more than 2 cups per day 1.21 (0.35, 4.22) 0.765 2.37 (0.52, 10.84) 0.265 0.98 (0.23, 4.21) 0.978 indian (n = 705) coffee yes 1.03 (0.49, 2.17) 0.930 1.12 (0.48, 2.62) 0.786 0.62 (0.20, 1.92) 0.406 no 1.00 1.00 1.00 coffee four levels never or rarely 1.00 1.00 1.00 less than 1 cup per day 0.29 (0.04, 2.32) 0.244 * * 0.55 (0.06, 4.80) 0.586 1-2 cups per day 1.11 (0.51, 2.39) 0.793 1.35 (0.57, 3.19) 0.497 0.53 (0.15, 1.86) 0.321 more than 2 cups per day 1.78 (0.50, 6.36) 0.369 1.14 (0.21, 6.02) 0.880 1.57 (0.26, 9.50) 0.626 all ( n = 3719) coffee yes 1.27 (0.88, 1.83) 0.200 1.49 (0.96, 2.30) 0.075 1.09 (0.64, 1.86) 0.737 no 1.00 1.00 1.00 coffee four levels never or rarely 1.00 1.00 1.00 less than 1 cup per day 1.52 (0.90, 2.56) 0.119 1.72 (0.93, 3.17) 0.083 1.55 (0.74, 3.25) 0.243 1-2 cups per day 1.23 (0.84, 1.81) 0.287 1.46 (0.93, 2.30) 0.099 1.01 (0.41, 2.52) 0.976 more than 2 cups per day 1.18 (0.63, 2.22) 0.612 1.29 (0.60, 2.76) 0.518 1.02 (0.41, 2.52) 0.970 soft drusen pigment abnormalities coffee consumption any amd (n = 201)   adjusted for age, gender, ever smoked, body mass index, history of diabetes, hypertension, ischemic heart disease, stroke, dyslipidemia, and cancer (additionally adjusted for combined data) b. coffee and dr a positive history of dm was present in 353 study participants corresponding to a dm prevalence of 10.1%. the mean age of the study population was 59.38 ± 10.52 years with 178 females (50.4%) and 175 males (49.6%). presence of any dr was observed in 113 (32%) study participants, with non-proliferative diabetic retinopathy and proliferative diabetic retinopathy in 107 (94.7%) and 6 (5.3%) participants, respectively. of the dr subjects, a positive history of coffee consumption was recorded in 86 (76.1%) participants with 66 (58.4%) reported consuming 1 to 2 cups of coffee per day (equivalent to total daily caffeine intake of 175.9 mg/day). higher coffee intake was associated with longer duration of diabetes (p=0.039) after controlling for age and gender. no significant association between coffee consumption and prevalence or severity of dr was observed in the multivariable regression model, after adjusting for 9 all res. j.biol, 2014, 5, 7-13     confounding factors (or = 1.36, ci = 0.69-2.69, p = 0.376, table 2). the association between coffee consumption and dr remained statistically insignificant when the study population was stratified according to 3 ethnic origins (chinese: or = 0.72, ci = 0.10-5.14, p = 0.746; malay: or = 1.32, ci = 0.16-10.78, p = 0.798; indian: or = 0.83, ci = 0.27-2.58, p = 0.749, table 2). the overall association between coffee consumption and prevalence of dr did not differ when analysis was restricted to study participants without pre-existing history of hypertension, ischemic heart diseases, stroke, dyslipidemia, and cancer (or = 1.80, ci = 0.81-3.96, p=0.147). table 2: association of coffee consumption with diabetic retinopathy stratified by ethnicity coffee consumption diabetic retinopathy or (95% cl) p value chinese (n = 128) coffee yes 0.72 (0.10, 5.14) 0.746 no 1.00 coffee four levels never o rarely 1.00 less than 1 cup per day 3.46 (0.22, 55.64) 0.381 1-2 cups per day 0.44 (0.05, 3.94) 0.463 more than 2 cups per day 0.32 (0.01, 19.83) 0.592 malay (n = 89) coffee yes 1.32 (0.16, 10.78) 0.798 no 1.00 coffee four levels never o rarely 1.00 less than 1 cup per day 0.08 (0.001, 6.48) 0.266 1-2 cups per day 1.64 0.19, 11.95) 0.656 more than 2 cups per day 1.55 (0.001, 177.86) 0.927 indian (n = 136) coffee yes 0.83 (0.27, 2.58) 0.749 no 1.00 coffee four levels never o rarely 1.00 less than 1 cup per day 0.12 (0.01, 1.54) 0.105 1-2 cups per day 1.147 (0.36, 3.86) 0.792 more than 2 cups per day 0.81 (0.04, 18.20) 0.893 all (n = 353) coffee yes 1.36 (0.69, 2.69) 0.376 no 1.00 coffee four levels never o rarely 1.00 less than 1 cup per day 0.92 (0.31, 2.71) 0.883 1-2 cups per day 1.43 (0.71, 2.90) 0.316 more than 2 cups per day 1.94 (0.45, 8.43) 0.375 adjusted for age, gender, ever smoked, body mass index, hba1c, creatinine, education level, duration of diabetes, family history of diabetes, history of hypertension, ischemic heart disease, stroke, dyslipidemia, and cancer (additionally adjusted for combined data) discussion caffeine is a xanthene derivative that interacts with endogenous adenosine via receptor antagonism and prevents adenosine-mediated vasodilatation of blood vessels.22 habitual intake of coffee may be associated with an increased risk of amd and dr through a possible vasoconstrictor effect on the retinal and choroidal circulation. of note, a caffeine-induced vasoconstriction response may be additive to the pre-existing altered hemodynamics of the retinal and choroidal circulation in participants with amd and dr. however, the data from this study failed to demonstrate a significant relationship between coffee intake and risk of amd or dr. our observations are consistent with a past study by tomany et al. where neither a history of coffee nor caffeine consumption was associated with incident early amd.13 the lack of significant association between coffee intake and risk of amd and dr may be due to several mechanisms. vasoconstrictor effect of caffeine is believed to be an acute physiological response, and such responses are usually transient in nature due to adaptation phenomena (plasma half-life of caffeine = 4 hours). according to one study, adaptation to the hemodynamic effect of caffeine may appear as early as 3-5 days after the onset of caffeine consumption.23 therefore, the vasoconstrictor response may wane off with time in habitual coffee users and this may have resulted in the non-significant relationship between coffee consumption and risk of amd and dr. furthermore, the physiological effects of coffee may be different from those of caffeine. indeed, it has been shown that oral intake of caffeine in the form of capsules results in a larger increase in plasma epinephrine concentrations than intake of coffee containing the same amount of caffeine, despite similar effects on blood caffeine concentrations.24 this observation suggests that other compounds in coffee may potentially counteract the vasoconstrictor effect of caffeine. for instance, quinides may counteract caffeineinduced adenosine receptor antagonism by inhibiting the receptor transporter.25 in addition, coffee has been shown to be the major contributor to the total dietary intake of antioxidants.26,27 this is of particular relevance in the pathogenesis of amd and dr because oxidative stress may be one of the underlying mechanisms. several past studies support the hypothesis that habitual coffee consumption is associated with a substantially lower risk of type 2 diabetes.3,28 going by this hypothesis, coffee consumption should be associated with a lower risk of amd and dr; however, our study did not demonstrate an inverse relationship between coffee consumption and risk of amd and dr. it is possible that interactions among 10 all res. j.biol, 2014, 5, 7-13     bioactive compounds in coffee may have resulted in the null association that we have observed in our study. lastly, the authors believe that the associations between coffee consumption and amd or dr may have failed to reach statistical significance due to lack of adequate power of the sampled cases (i.e. participants with amd and dr). for amd, 201 cases and 3518 controls (total sample size of 3719) will be sufficient to determine coffee drinking associations with or of 1.6 and higher with 80% power and 95% significance level. similarly, 113 dr cases and 240 controls (total sample size of 353) will be sufficient to determine coffee drinking associations with or of 1.9 and higher with 80% power and 95% significance level. furthermore, digital fp was used for diagnosing the presence and severity of amd and dr in our study. a systematic protocol based grading of fp by trained and certified graders is the preferred technique in epidemiological research relating to amd and dr.29-32 indeed, drusen and retinal pigment alterations as signs for amd are best appreciated on fp; however, choroidal neovascularization lesions and activity may be missed and intermediate drusen may be diagnosed as small drusen or pigmentary changes due to lack of clarity on fp.33 similarly, fp provides a reasonably reliable diagnosis of the presence and severity of dr; however, clinical examination using slit-lamp biomicroscopy is superior to digital fp for detecting clinically significant macular edema.34 this is the first study to examine the relationship of coffee intake to risk of amd and dr. the large multi-ethnic study population and detailed assessments of potential confounders are strengths of this study. measurement error in the assessment of lifestyle exposures is unavoidable and makes the possibility of residual confounding a concern. in conclusion, this epidemiological study based on a large multiethnic study population does not support the hypothesis that habitual intake of coffee is associated with amd and dr among chinese, malay, and indian populations in singapore. references 1. ranheim, t., halvorsen, b. 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(2013). a meta-analysis of prospective studies of coffee consumption and mortality for all causes, cancers and cardiovascular diseases. eur j epidemiol, 28(7), 527-39. 7. ding, m., bhupathiraju, s. n., satija, a. et al. (2013). long-term coffee consumption and risk of cardiovascular disease: a systematic review and a dose-response meta-analysis of prospective cohort studies. circulation, nov 7 (epub ahead of print). 8. van dam, r. m. (2008). coffee consumption and risk of type 2 diabetes, cardiovascular diseases, and cancer. appl physiol nutr metab, 33, 1269-1283. 9. sang, l. x., chang, b., li, x. h. et al. (2013). consumption of coffee associated with reduced risk of liver cancer: a meta-analysis. bmc gastroenterol, 25, 13-34. 10. lotfi, k., grunwald, j. e. (1991). the effect of caffeine on the human macular circulation. invest ophthalmol vis sci, 32 (12), 3028-3032. 11. friedman, e. (1997). a hemodynamic model of the pathogenesis of age-related macular degeneration. am j ophthalmol, 124 (5), 677-82. 12. dokken, b. b. (2008). the pathophysiology of cardiovascular disease and diabetes: beyond blood pressure and lipids. diabetes spectrum, 21(3), 160165. 13. tomany, s. c., klein, r., klein, b. e. k. (2001). the relation of coffee and caffeine to the 5-year incidence of early age-related maculopathy: the beaver dam eye study. am j ophthalmol, 132 (2), 271-273. 11 all res. j.biol, 2014, 5, 7-13     14. hughes, k., yeo, p. p., lun, k. c. et al. (1990). cardiovascular diseases in chinese, malays, and indians in singapore. ii. differences in risk factor levels. j epidemiol community health, 44(1), 29– 35. 15. tan, c. e., emmanuel, s. c., tan, b. y. et al. (1999). prevalence of diabetes and ethnic differences in cardiovascular risk factors. the 1992 singapore national health survey. diabetes care, 22(2), 241–247. 16. hughes, k., aw, t. c., kuperan, p. et al. (1997). central obesity, insulin resistance, syndrome x, lipoprotein(a), and cardiovascular risk in indians, malays, and chinese in singapore. j epidemiol community health, 51(4), 394–399. 17. cutter, j., tan, b. y., chew, s. k. (2001). levels of cardiovascular disease risk factors in singapore following a national intervention programme. bull world health organ, 79(10), 908–915. 18. deurenberg-yap, m. t. l., tan, w. l., van staveren, w.a., deurenberg, p. (2000). validation of a semiquantitative food frequency questionnaire for estimation of intakes of energy, fats and cholestrol among singaporeans. asia pacific j clin nutr, 9, 7. 19. wong, t. y., islam, f. m., klein, r. et al. (2006). retinal vascular caliber, cardiovascular risk factors, and inflammation: the multi-ethnic study of atherosclerosis (mesa). invest ophthalmol vis sci, 47(6), 2341–2350. 20. early treatment diabetic retinopathy study research group (1991). grading diabetic retinopathy from stereoscopic color fundus photographs: an extension of the modified airlie house classification. etdrs report 10. ophthalmology, 98, 786–806. 21. diabetic retinopathy study group (1981). diabetic retinopathy study. report number 7. a modification of the airlie house classification of diabetic retinopathy. invest ophthalmol vis sci, 21, 210– 226. 22. fredholm, b. b. (1980). are methylxanthine effects due to antagonism of endogenous adenosine: trends pharmacol sci, 1, 129-132. 23. robertson, d., wade, d., workman, r., woosley, r.l., oates, j.a. (1981). tolerance to the humoral and hemodynamic effects of caffeine in man. j clin invest, 67, 1111-1117. 24. graham, t. e., hibbert, e., sathasivam, p. (1998). metabolic and exercise endurance effects of coffee and caffeine ingestion. j appl physiol, 85, 883-889. 25. de paulis, t., schmidt, d. e., bruchey, a. k., kirby, m. t., mcdonald, m. p. et al. (2002). dicinnamoylquinides in roasted coffee inhibit the human adenosine transporter. eur j pharmacol, 442, 215223. 26. pulido, r., hernandez-garcia, m., saura-calixto, f. (2003). contribution of beverages to the intake lipophilic and hydrophilic antioxidants in the spanish diet. eur j clin nutr, 57, 1275-1282. 27. svilaas, a., sakhi, a. k., andersen, l. f. et al. (2004). intakes of antioxidants in coffee, wine, and vegetables are correlated with plasma carotenoids in humans. j nutr, 134, 562-567. 28. odegaard, a. o., pereira, m. a., koh, w. p. et al. (2008). coffee, tea, and incident type 2 diabetes: the singapore chinese health study. am j clin nutr, 88(4), 979-985. 29. tikellis, g., robman, l. d., harper, a. et al. (2000). methods for detecting age-related maculopathy: a comparison between photographic and clinical assessment. clin experiment ophthalmol, 28, 367372. 30. van leeuwen, r., chakravarthy, u., vingerling, j. r. et al. (2003). grading of ageage-related maculopathy for epidemiologic studies: is digital imaging as good as 35-mm film? ophthalmology, 110, 1540-1544. 31. klein, r., klein, b. e., neider, m. w. et al. (1985). diabetic retinopathy as detected using ophthalmolscopy, a non-mydriatic camera and a standard fundus camera. ophthalmology, 92(4), 485-491. 32. williams, g. a., scott, i. u., haller, j. a. et al. (2004). single field fundus photography for diabetic retinopathy screening: a report by the american academy of ophthalmology. ophthalmology, 111(5), 1055-1062. 33. mokwa, n. f., ristau, t., keane, p. a. et al. (2013). grading of age-related macular degeneration: comparison between colour fundus photography, 12 all res. j.biol, 2014, 5, 7-13     fluorescein angiography, and spectral domain optical coherence tomography. j ophthalmol, 2013, 385915. 34. liesenfeld, b., kohner, e., piehlmeier, w. et al. (2000). a telemedical approach to the screening of diabetic retinopathy: digital fundus photography. diabetes care, 23 (3), 345-348. 13 microsoft word 68-393-1-proofreaded.docx pelletization by extrusion-spheronization: a detailed review nagasamy venkatesh dhandapani*, ayush shrestha, niroj shrestha, anup thapa, goti sandip, rajan sharma bhattarai. all res. j. biol., 2012, 3, 10-23 the publication cost of this article might be covered by external sponsors. more info for sponsors at: sponsors@arjournals.com review issue 2, vol. 3, 2012, 10-23 pelletization by extrusion-spheronizationa detailed review nagasamy venkatesh dhandapani*a, ayush shrestha b, niroj shrestha b, anup thapa c, goti sandip c, rajan sharma bhattarai d a) department of pharmaceutics;b) department of pharmacology;c) department of pharmaceutical analysis; jss college of pharmacy, udhagamandalam -643001, india. d) formulation, research and development, dr. reddy’s laboratory, hyderabad, india. *corresponding author email: nagasamyvenkatesh@rediffmail.com abstract oral multiparticulate drug systems (e.g. pellets, granules) in comparison to single unit dosage forms offer biopharmaceutical advantages in terms of more even and predictable drug distribution in the enteric system. extrusion spheronization is one of the most commonly used techniques in the formulation of such multiparticulate beads and pellets providing sustained and controlled release or modified release drug delivery. this review outlines the various steps involved in the extrusion spheronization process, the excipients used in such formulations along with some modifications and various processing variables affecting the quality of pellets formed. in addition, an overview of the methods available for the quality check of the pellets is reviewed. keywords: extrusion, spheronization, multiparticulate, pellet 1. introduction drugs designed in the form of uniform spherical sized pellets offer various advantages in the pharmaceutical industry. applications are found not only in the pharmaceutical industry but also in the agricultural sectors and in the polymer and biotechnology industry. by formulating drugs into pellet form, it becomes possible for developing a controlled release drug delivery, actualizing a predictable and reproducible drug over an extended period of time, thus allowing reduced dosing frequency providing constant drug levels in the blood yielding increased patient compliance and decreased adverse drug events.1, 2 pellets in addition to providing therapeutic advantages such as reduced gastric irritation and a lowered risk of side effects due to dose dumping, also offer technological advantages such as better flow properties, less friable dosage form, narrow particle size distribution, ease of coating and uniform packing.3 the manufacturing of pellets using layering processes such as solution layering, suspension layering or powder layering have been utilized over the years. these processes have limitations such as non-uniformity in the size of pellets and reduced drug loading. pellets of uniform particle size between 500-1500 µm with smooth surface morphology, narrow size distribution, good flowability, high strength and low friability are desirable for providing sustained and controlled release effects in the formulations. this can be achieved by the extrusion spheronization technique. to produce fine spherical granules of 500 µm or smaller in the spheronization process, a pore size screen of 0.4 mm or smaller has to be used. due to increased resistance caused by the narrow pore sized screen, the extrusion pressure is increased. hence, the use of lubricants such as macrogol, polyethylene glycol, poloxamer, and silicone oil was attempted.4, 5 the main objective of extrusion spheronization is to provide pellets of uniform size with high drug loading capacity. it is a composite process of wet mass extrusion followed by spheronization to produce uniform sized spherical particles called spheroids/pellets/beads/matrix depending upon the process of spheronization.6 the pellets produced by extrusion spheronization provide the following edge over conventional solid dosage forms: • small spheroids with a high loading capacity of active ingredients can be produced. • production of pellets of uniform size, smooth surface and narrow size distribution with good flow property. • due to their spherical shape and low surface area to volume ratio sucessive coatings can be applied to the spheroids. • the process can be used in preparing pellets for taste masking purposes of bitter apis(active pharmaceutical ingredients) by using taste masking polymers that create solid dispersions to prevent bitter drugs from coming in contact with the patient’s taste buds.7, 8, 9, 10 10 all res. j. biol, 2012, 3, 10-23     • enhancement of drug dissolution. • dosage forms with different doses can be produced from the same batch by adjusting the fill weight of the pellets.11 • it increases the hardness and reduces the friability of the pellet produced, hence minimizing the damage and loss during transportation. • various chemically compatible or incompatible drugs can be blended and formulated into a single unit dosage form for delivery at different sites in the gastrointestinal tract (g.i tract). • bioavailability of the drug is increased by providing sustained release. • dose dumping due to drug overload can be minimized and hence the safety and efficacy of the drug can be improved. • packaging these spheres into small containers such as hard gelatin capsules or compressing it into tablets is much more convenient than other dry forms such as powders or granules. 2. extrusion spheronization process the extrusion spheronization process was first reported by reynold (1970) and conine and hadley (1970).12, 13 1. dry mixing and granulation. 2. extrusion 3. spheronization 4. drying 5. screening 2.1. dry mixing and granulation the process involves mixing all the required ingredients in different types of mixers such as a twin shell blender, high shear mixer, tumbler mixer, and a planetary mixer to get homogenous powder dispersion.13,14, 15, 16 the dry powder is then made into fine granules by mixing with a suitable granulation liquid. different types of granulators are used for mixing the powder blend with the granulation liquid.the most commonly used granulator is a planetary mixer. 14, 17, 18, 19 during this step, the evaporation of the fluid phase should be restricted to a minimum. this could especially be a problem with the high shear mixture which produces large amount of heat. this rise in temperature will cause evaporation of the granulating fluid20 and influence the extrusion behavior of the wet mass. cooling of the granulation bowl might avoid this problem. during the granulation step, there exists a homogenous distribution of the liquid phase throughout the granulated mass. 2.2. extrusion it is the second step in the process of spheronization following granulation. this process involves shaping the wet mass into long cylindrical rods of uniform diameter by forcing it through the dies of the extruder. this process is not only used in the pharmaceutical industry but also in food, ceramics and polymer industries. 4 main classes of extruders are used: a. screw feed extruder (axial or end plate, dome and radial extruder). b. sieve and basket. c. gravity feed extruder (cylinder roll or gear roll extruder). d. piston feed extruder (ram extruder). the screw extruder consists of one or two (twin -screw) feeding the plastic mass to an axial (fig. 1) or radial extrusion screen (fig.2).21, 22 in the axial type, the screen is placed at the end of the screw, while in the radial type the screen is placed around the screw, discharging the extrudate perpendicularly to the axis of the screw.23 fig. 1: axial screw feed extruder fig. 2: radial screw feed extruder in sieve (fig. 3) and basket (fig.4) extruders, the granulate is fed by a screw or by gravity into the extrusion chamber, where a rotating or oscillating device pushes the plastic mass through the screen. the difference between the sieve and basket extruder is similar to that between the radial and axial screw extruders.23 11 all res. j. biol, 2012, 3, 10-23     fig. 3: sieve extruder. fig. 4: basket extruder. gravity feed extruders include rotary cylinder (fig. 5) and rotary gear (fig. 6) extruders, which differ mainly in the design of the two counter rotating cylinders. in the rotary cylinder extruder, one of the two counter rotating cylinders is hollow and perforated, whereas the other cylinder is solid and acts as a pressure roller. in the rotary gear extruders there are two hollow counter rotating gear cylinders with counter board holes. 23 fig. 5: cylinder roll type fig. 6: gear roll type in ram extruders (fig. 7) which are probably the oldest type of extruders, a piston displaces and forces the material through a die at the end. ram extruders are preferentially used in the development phase, because they can also be used to measure the rheological properties of the formulations.14, 15 fig. 7: ram extruder the extruded masses must have enough plasticity to deform, at the same time it must not adhere to other particles when rolled during spheronization process. this process mainly determines the final particle size of the pellets. the diameter of the extruder screen opening directly controls the diameter of the extrudate.24, 25 2.3 spheronization spheronization, also known as marumerization, is the third step in the extrusion spheronization process. it was first introduced by nakahara in 1964. it involves dumping the extruded cylinders onto the spinning plate of the spheronizer, called the friction plate, in which the extrudate is broken up into smaller cylinders with an equal length to their diameter.13 these plastic cylinders are rounded due to frictional forces14. different stages can be distinguished depending upon the particle shape, i.e. it starts from a cylinder over a cylinder with rounded edges, dumb bells and elliptical particles to eventually perfect spheres (fig. 8a). this process may be divided into 3 steps such as breaking of the cylindrical segments or extrudate, agglomeration of the broken segments and smoothing of the particles.24, 25, 26, 27 breaking of the cylindrical extrudate occurs due to the interaction of the extrudate with the rotating grooved or smooth plate, stationary wall and other extrudate particles. agglomeration occurs when the small fragments produced during the breaking stage are picked up by the larger granules during smoothing. spherical particles are created during the smoothing stage by generating rotational motion of each granule about its axis in constantly changing planes. baert and remon (1993) suggested another mechanism in which a twisting of the cylinder occurs after the formation of cylinders with rounded edges, finally resulting in the breaking of the cylinder into two distinct parts. both parts have a round and a flat side. the edges of the flat side fold together like a flower, forming the cavity due to the rotational and the frictional forces involved in spheronization process (fig. 8b).28 12 all res. j. biol, 2012, 3, 10-23     fig. 8:pellet-forming mechanism according to: (a) rowe – i) cylinder; ii) cylinder with rounded edges; iii) dumb-bell; iv) ellipse; v) sphere. (b) baert – i) cylinder; ii) rope; iii) dumb-bell; iv) sphere with a cavity outside; v) sphere.28 the most important component of the spheronizer is the friction plate, a rotating disk having a characteristically grooved surface in order to increase the frictional force. this grooved surface has two types of geometry, a cross-hatch geometry in which the grooves form right angles and a radial geometry in which a radial pattern is used. the duration of spheronization is usually 2-10 min29 and a rotational speed between 200-400 rpm of the friction plate is optimum to obtain a highly spherical pellet.30 2.4. drying to achieve the desired moisture content in the final pellet, drying is done. the pellets can be dried at room temperature31, 32 or at elevated temperature in a tray drier or fluidized bed drier.33, 34, 35, 36 bataille et al., (1993) reported the use of a microwave oven in the final phase of the production process of pellets to evaporate the slurry of the extruded mass during drying process.37 huyghebaert et al., (2005) reported the use of a freeze dryer in order to maintain the viability of living bacterial spores for the development of an oral vaccine.38 2.5 screening screening of the pellet may be done to achieve the desired size distribution, and for this purpose sieves are used. in the case of pellets being prepared by extrusionspheronization, screening is essentially required after manufacture, in order to avoid pellets with a high size polydispersity index.36,39 the overall process of extrusion spheronization is depicted in fig. 9. fig. 9: extrusion-spheronization process. hot-melt extrusion the extrusion spheronization technique usually requires a film coating to control drug release resulting in complex processing steps. to avoid this difficulty, a novel hot-melt extrusion (hme) technique was developed to prepare granules, sustained release tablets and transdermal as well as transmucosal drug delivery systems.40, 41, 42 this process does not require the use of water or solvents and few processing steps are needed, making the process simple, efficient and continuous. furthermore, by hme it is possible to improve the bioavailability of ‘‘difficult’’ actives by the formation of solid dispersions and solid solutions.43, 44, 45 3. pelletization aids to extrusion spheronization. 3.1 microcrystalline cellulose (mcc) mcc is considered the golden standard as extrusion spheronization aid because of its unique characteristics of plasticity and cohesiveness of their wet masses. furthermore, it is capable of absorbing and retaining a large quantity of water due to its large surface area and high internal porosity46, which facilitates extrusion and spheronization. mcc based pellets produced via extrusion spheronization have a good sphericity, low friability, high density and smooth surface properties. mcc has been described as a “molecular sponge”.47, 48 the mcc particles retain water in a manner similar to sponge. during the process of extrusion, these sponges are compressed and water that is squeezed from the internal structures acts as a lubricant. after extrusion, the sponge volume expands and they appear dry and brittle, which facilitates the breaking of the extrudates during the initial phase of spheronization. during the spheronization phase, the sponges are densified due to collision between particles, the spheronizer plate as well as the wall, and water facilitates the spheronization of pellets. although mcc is an ideal spheronization aid, it is associated with several limitations such as a prolonged drug release profile in the case of low solubility drugs due to the lack of disintegration of mcc-based pellets49, drug decomposition in presence of mcc50 and drug adsorption 13 all res. j. biol, 2012, 3, 10-23     onto the surface of mcc fibres.51 due to these limitations, various technological alternatives have been proposed and evaluated. to remark, wetting the mass with water–alcohol mixtures instead of water alone reduces the mechanical strength of the pellets52 or the addition to the pellets of different excipients as disintegrants53, surface active agents54 or water-soluble fillers55, 56 are the notable ones. another alternative that has been used is complete or partial replacement of microcrystalline cellulose by other diluents or excipients. incorporation of superdisintegrants (croscarmellose sodium or sodium starch glycolate) into mcc pellets prepared by extrusion spheronization does not lead to their disintegration in drug dissolution medium, however, they afford a slight increase in the drug dissolution rate.57 similarly in another study by michelle and patrick (1998), the use of hydrophilic polymers with mcc, using spray drying produced pellets in a higher yield and better sphericity. the less adhesive polymers hydroxypropyl cellulose and polyvinyl pyrrolidone appear best in regard to achieving a good yield with the proper rounding of pellets over an adequate range of water content.58 beads of acceptable physical characteristics can be produced using extrusion and spheronization with carbopol® 974p, nf, resin as a release rate-controlling polymer. drug dissolution from the beads can be slowed down by increasing the carbopol content. high carbopol and water content, a low calcium chloride level, low spheronization speed and a long spheronization time period produced the highest quality bead possessing thelongest drug release duration.59 3.1. alternative excipients for microcrystalline cellulose the excipients intended to be used for the production of pellets via extrusion spheronization require the following properties: • water insolubility • large water absorption and retention capacity • binding properties • sufficiently large surface area for interaction with water and other ingredients in the powder mixture • ability to enhance drug release. 3.1.1. biopolymers i. powdered cellulose lisardo alvarez et al., (2003) evaluated the use of powdered cellulose as the sole excipient in the preparation of furosemide pellets by extrusion spheronization.60 pellets formulated with pc and 25 or 50% furosemide showed smaller mean size, a broader particle size distribution, similar sphericity, greater surface roughness and higher friability than equivalent pellets formulated with mcc but showed rapid release, hence pellets of highly cohesive poorly water soluble drugs can be formulated using this excipient. also, the pellets formulated with powdered cellulose were difficult to prepare since it required more water for extrusion, but its water holding capacity was lower. due to water movement during extrusion, the material inside the extruder was compressed resulting in a dry mass which blocked the extruder.61, 62 hence, the powdered cellulose cannot be considered a suitable alternative to mcc compared to other pelletization aids. ii. ii. starch and starch derivates o’connor et al., (1984) reported the unsuccessful production of pellets via extrusion spheronization using starch (native and pregelatinized) as the main excipient in the formulation. otsuka et al. used a mixture of starch (27%, w/w) and lactose (63%, w/w) to produce pellets with 10% w/w of theophylline as a model drug.63 junnila et al. used up to 30% (w/w) native starch, combined with mcc and 2.5% (w/w) of anhydrous theophylline. however, no model was accepted because of poor pellet sphericity.63 recently, dukic et al., (2007) suggested the use of a modified starch (high amylose, crystalline and resistant starch) as an alternative excipient to mcc using anhydrous theophylline (25%, w/w) as a model drug. this resulted in high pellet yield (>90%), acceptable sphericity (ar<1.2), low friability (<0.01%), fast disintegration (<10 min) and complete drug release in less than 20 min in all formulations.64 in another study by the same authors, the immediate release of poorly water soluble drugs (up to 50% hydrochlorthiazide and 2.5% piroxicam) was achieved (more than 80% release after 30 min) due to pellet disintegration within 15 min.65 in another study by prieto et al. the use of starch (corn starch or wheat starch) + 20% white dextrin gave high quality pellets with good size and shape distribution.66 despite promising results for specific starch grades, starch derivatives do not meet all the properties required for an ideal extrusion spheronization aid: an additional binder had to be incorporated in the formulation in order to obtain the proper wet mass consistency, and these formulations will be less robust than mcc formulations due to a narrow range of optimal water content. iii. iii. kcarrageenan bornhoft et al., (2005) used carrageenan as a pelletization aid for extrusion spheronization. 67 three commercial subtypes of different carrageenans namely ι-, κ-, and λ-carrageenan were investigated for their pelletization behavior, and κ-carragennan was found to be a very promising substitute for mcc in extrusion spheronization. κ carrageenan required higher water content for the formation of the pellets, but the formulation was robust as the optimal range of water content was much broader. further studies on κcarrageenan were carried out to examine the effect of other ingredients on the pellet properties. four different drugs (acetaminophen, theophylline, mesalamine and hydrochlorthiazide) and four different fillers (lactose, mannitol, maize starch and dicalciumphosphate dihydrate) 14 all res. j. biol, 2012, 3, 10-23     were varied systematically with fixed ratio of κ-carrageenan (20%) in 36 formulations.67, 68 pellets with good shape and size characteristics were obtained in all formulations. κ-carrageenan pellets were characterized by a low tensile strength, fast disintegration and fast drug release and the drug release is independent of the solubility of the drug in contrast to mcc pellets. hence, κ-carrageenan could be used as a suitable pelletization aid. however, the major disadvantage of pellets formulated with κ-carrageenan is their lower mechanical stability and the possibility of ionic interactions. iv. pectinic acid tho et al. evaluated different pectin derivatives for aid to extrusion spheronization. three different drugs (riboflavin with low water solubility, and paracetamol and theophylline with high water solubility) with concentration varying from 1 to 80% wt were used as model drugs. the low soluble pectin derivative, pa (degree of methoxylation<10%) was found to be well suited as an extrusion aiding excipient in pellet preparation. this has a high drug loading capacity and produces disintegrating pellets that are suitable for the fast delivery of drugs with low water solubility. in a similar study, the low methoxylated (4%) pectin derivative was successfully pelletized in combination with lactose and 1% riboflavin as a model drug using water for pelletization. all pectinic acid pellets were mechanically stable and partly disintegrated during dissolution experiments. however, with pectinic acid, the process is more sensitive to the type and amount of drug and is not a universally accepted pelletization aid, such as the conventionally used mcc.69, 70 v. chitosan chitosan alone71, 72, 73 as well as in mixtures with mcc74, 75, 76, 77, 78 has been used for the production of pellets via extrusion spheronization. jess and steckel investigated the influence of the degree of deacetylation of chitosan on the properties of pure chitosan pellets. chitosan with a high degree of deacetylation (99.9%) and wetted with 0.2 n acetic acid provided the best wetted mass plasticity in order to obtain pellets with adequate size, sphericity, friability, mechanical strength and surface properties.72 agrawal et al., (2004)71 prepared mcc free pellets using up to 15% (w/w) chitosan and up to 10% (w/w) hydroxypropylmethylcellulose (hpmc) as an additional binder. chitosan as a pelletization aid is not ideal since it requires the addition of either a granulation liquid with a specific ph (e.g. hpmc, sodium acetate) or binder (hpmc). also, ionic interactions with drugs are possible due to the ionic nature of the chitosan. 3.1.2. semi synthetic polymers i. hydroxypropyl methyl cellulose and hydroxyethyl cellulose chatlapall and rohera (1998) evaluated the physical characteristics of hpmc and hec for their use as pelletization aids. since hpmc and hec are water soluble, it was not possible to use water as granulation liquid and it further resulted in the formation of tacky masses. however, the pellets were prepared using isopropyl alcohol (ipa) as a non-dissolving granulating fluid. it was necessary to include the binder due to the low mechanical strength of the dried pellets in the formulation. with hpmc and hec, pellets absorbed water producing a viscous gel matrix and dissolved or eroded slowly, unlike mcc pellets which stayed intact without dissolution or erosion.79 hence, these excipients find application in pellet formulation of water sensitive drugs and in those formulations where organic liquid must be used as wet massing liquid in place of water. ii. ii. cross-linked polyvinylpyrrolidone (crospovidone) crospovidone has proven to offer substantial advantages as a pelletization aid because of its ability to turn low-soluble active ingredients into fast-dissolving stable pellets. verheyen et al., (2008) prepared pellets by extrusion spheronization from crospovidone with different amounts of paracetamol, hydrochlorothiazide, and spironolactone as model drugs. only crospovidone types exhibiting small particle sizes were suitable as pelletization aid and it was possible to incorporate up to 60% (w/w) active pharmaceutical ingredients (api) into pellets with crospovidone. the pellets containing binary mixtures of the low-soluble apis and crospovidone resulted in fast release in contrast to the pellets with mcc as a pelletization aid, which exhibited a slow release.80 compared to microcrystalline cellulose (mcc), that yields non-disintegrating pellets, polyplasdone crospovidone offers enhanced drug release characteristics by combining pellet disintegration with the solubility enhancing characteristics of crospovidone. this property is highly desirable with poorly soluble drugs or to form “melt-in-mouth” pellets.81 iii. iii. polyethylene oxide howard et al. (2006) presented a means to produce extruded–spheronized beads, devoid of microcrystalline cellulose (mcc) and with a high drug load (>80%, w/w). immediate release bead product with a high yield (>60% of 1mm diameter beads) and low friability (mass loss<4.0%) that were spherical to the naked eye (roundness score<1.20) were obtained. polyethylene oxide, a highly water soluble polymer, provided sufficient plasticity to the wetted mass and a low molecular weight methoxypolyethylene glycol (mpeg) acting as a plasticizer was needed to improve the self-lubricating properties of the wetted mass.82 4. process variables and parameters influencing pellet quality 4.1. water content moisture is necessary to give plasticity to the powder mass for extrusion and spheronization into the pellets of an acceptable quality. at low moisture content, excessive pressure is required to remove the air voids during extrusion. the brittle mass thus formed does not have enough plasticity 15 all res. j. biol, 2012, 3, 10-23     to form spheres and thus breaks on spheronization generating a large amount of fines. optimal water content fills the voids and increases the tensile strength of the granule to an ideal form for extruding. on the other hand, at higher moisture content, pellets agglomerate during spheronization due to the excess water at the surface of the pellets.83 the moisture content also influences the release pattern of the entrapped drugs.84 4.2. granulation liquid water is mostly used as the granulating liquid. ethanol when used along with water produces pellets with excellent compressibility and increased friability, hardness and dissolution rate.54 glycerol solution produces more porous pellets than water.85 formulations containing viscous granulating liquids with hydrophilic polymer yields long, dumbbell-shaped pellets while those containing watery granulation liquids with calcium chloride yield short, spherical pellets due to the influence on the swelling capacity of sodium alginate.86 4.3. plasticizer lower plasticizer content in the granulating polymer produces pellets with increased tensile strength and brittle fracture under compression whereas higher plasticizer content produces pellets with a ductile property due to the transition of the polymer from a glassy to a rubbery state.87 4.4. surfactant surfactant with a high hlb value reduces the surface defects of the extrudate due to reduced friction at the die wall of the extrusion screen and produces spherical pellets.88 it may facilitate the permeability of the drug through the gastrointestinal wall.89 at optimum concentration, it ensures rapid in vitro-drug release within 30 min.90 it can also facilitate the formulation of hydrophobic drugs as pellets.91 4.5. drug solubility increase in drug solubility increases the volume of the granulating liquid, leading to an over wetting of the system.21 4.6. type of extruder pellets obtained from various extruders differ in optimal moisture content, particle size distribution and sphericity. this may be attributed to the difference in the amount of granulation liquid required, in the length to radius ratio of the extrusion screen used or in the shear rate.92, 93, 94, 95 4.7. extrusion force at lower force, the extrudate agglomerates while at higher force, it becomes dry and fails to form spherical pellets.96 so the extrusion force has to be optimized based on the characteristics of the active constituents. 4.8. extrusion speed higher extrusion speed produces extrudate with a rough surface and wide particle size distribution.18 4.9. extrusion screen change in the thickness of the extrusion screen and/or diameter of its perforations influences the pellet quality. perforation diameter determines the pellet size. 18, 93 4.10. extrusion temperature a rise in temperature during the extrusion cycle decreases the moisture content of the extrudate at the end of a batch compared to that in the beginning of a batch.97 4.11. spheronization speed higher spheronizer speed produces more spherical pellets than low spheronizer speeds.98 the spheronizer speed also affects the particle size, porosity and friability of the pellets.99, 100, 101 4.12. spheronization time extended spheronization time produces pellets with higher sphericity, narrower particle size distribution and increased diameter. 65, 100, 101 4.13. spheroniser load an increase in spheroniser load decreases the pellet size and increases hardness.102, 103 4.14. drying method microwave dried pellets are more porous, softer and rougher than oven dried pellets.39 freeze dried pellets are larger, weaker and more porous and have faster drug release than pellets dried with the other processes like hot air drying and microwave drying.104 5. evaluation of pellets a. particle size analysis mostly, pellets are analyzed for particle size by a simple sieve analysis.65,84, 85, 96, 99 b. specific area pellet specific surface area is determined by a gas adsorption technique with krypton.84, 105 c. shape sphericity or roughness of pellets is determined by an image analysis system.84, 95, 106 16 all res. j. biol, 2012, 3, 10-23     d. microstructure and surface pellet microstructure and surface morphology are assessed by scanning electron microscopy.84 e. pellet strength pellets are evaluated for crushing force using a tablet strength tester and friability by using a friabilator. 65, 84, 99 f. density and porosity the bulk and tap densities of pellets evaluate the homogeneity of the particle size distribution.107 the true density evaluates the porosity of the pellets and can be determined by an air compression pycnometer or helium pycnometer.84, 85, 106, 107 g. water content residual water content in the pellets after drying is evaluated by thermogravimetric analysis or moisture balance.84, 104 h. dissolution testing drug release is assessed by a usp rotating paddle apparatus 2.84, 106 i. flow property flow property is assessed by using the inverted funnel method or compressibility index.107, 108 6. therapeutic applications pellets formulated by the extrusion-spheronization process provide controlled drug release, targeted drug delivery and reduced side effects associated with conventional oral dosage forms. some notable examples are as follows: diclofenac sodium, having a short biological half-life, may be formulated as sustained release mini-matrices to provide zero order drug release for the long term management of rheumatic disorders.109 matrix based controlled release formulation of azithromycin may reduce the adverse effects associated with the conventional immediate release formulation.110 pellet formulation of the non-steroidal antiinflammatory drugs like indomethacin, ibuprofen, piroxicam etc. may reduce the gastrointestinal disturbances associated with conventional oral dosage forms.111, 112, 113 triamcinolone acetonide or 5-amino salicylic acid coated pellets may provide colon targeting and reduce the side effects associated with the conventional oral dosage forms used in the long term treatment of ulcerative colitis and crohn’s disease.114, 115 more recently, mucoadhesive biopolymer chitosan based pellets of 5-amino salicylic acid are prepared for topical delivery to colonic mucosa to achieve effective concentration at the site of inflammation.116 zhang et al., (2012) developed self-microemulsifying drug delivery systems (smedds) in sustained release pellets of puerarin to enhance the oral bioavailability of puerarin.117 similarly, formulation of solid self-nanoemulsifying drug delivery systems (s-snedds) of carvedilol having capability of bypassing hepatic portal route and promoting the lymphatic transport of lipophilic drugs, was made possible, thus reducing metabolism by cytochrome-p450 family of enzymes present in the gut enterocytes and liver hepatocytes and/ or inhibiting p-glycoprotein (p-gp) efflux.118 table i: commercially available marketed pellet products.23 product company bontril sr carnick laboratories, inc. brexin l.a savage laboratories, bangalore. catazyme s organon pharmaceuticals, usa. compazine smith & french, mumbai dilgard xl 180 smith kline & french, mumbai elixophyline cipla ltd, ahmedabad. fastin berlex laboratories, usa. hispril berlex laboratories, usa. ibugesic s.r. 300 cipla ltd, ahmedabad. indocrin s.r. merk sharp, mumbai. nicobid t.s. u.s.vitamin, usa. ornade smith kline. 7. some of the negative results of extrusionspheronization process 7.1 physical factors: 7.1.1 temperature oosaka et al reported that the application of a temperature sensor in the extrusion process yielded spherical granules of low sphericity. however, this is a more important parameter to be considered when the drug content is especially high, and use of fine granules is not practically possible to coat them. this ultimately causes increases in particle size and leads to a time consuming and costly process of pelletization.119 7.1.2 sphericity kanbe et al observed that the low substituted hydroxypropyl cellulose offered highest sphericity as compared with croscaramellose sodium and croscaramellose calcium. the spheroids obtained through the later excipients exhibited a poor sphericity. 120 17 all res. j. biol, 2012, 3, 10-23     7.2.role of excipients and drugs in the formulation of pellets: it was observed during the formulation of diphenhydramine hcl pellets that the low substituted hydroxyl propylcellulose offered the highest sphericity while the cross caramellose sodium and cross caramellose calcium offered unsatisfactory sphericity. it was also observed in the study that the production of 500 µm or smaller fine spherical was not succeeded with microcrystalline cellulose. the formulations prepared with microcrystalline cellulose and corn starch as excipients resulted in best quality pellets with smooth surfaces. whilst the pellets prepared using excipients such as glucose, lactose and calcium hydrogen phosphate produced pellets with much rougher surfaces.121 additionally, the incorporation of lactose at a concentration of 15% w/w with higher moisture content (> 25% w/w) lead to the agglomeration of pellets. the water soluble excipients (lactose) caused channels (or) cracks to form on the pellets surface.122, 123 in another study, panchagnula et al reported the unsuitability of the hydrophilic swellable polymer (hpmc) for water soluble drugs. a synergistic effect of the swelling property of hpmc and disintegrating property of mcc (microcrystalline cellulose) resulted in a faster drug release of 70% azithromycin within 1 hour. these combinations of excipients failed to provide a controlled release of azithromycin. however, in the same investigation the effect of waxy materials gms (glyceryl monostearate and carnauba wax) on the release rate of drugs was investigated. it is known from the literature that the waxy materials serve as potential release retardants in the controlled release systems. 124, 125 but, as compared with carnauba wax, glyceryl monostearate resulted in a better yield, crushing strength and density of pellets. this indicates that the unsuitability of spheronizing property of carnauba wax resulted in poor pellet shape as a result of hydrophobicity and poor molding capacity126 o’ connor et al reported the unsuccessful production of pellets using starch (native and pregelatinised) due to poor sphericity.63the release of caffeine from pellets was not sustained when prepared in combination with chitosan and hpmc lacking mcc.127 an increase in the amount of citric acid revealed a negative effect in the roundness of the pellets 128 indomethacin pellets prepared using sodium lauryl sulphate (sls) offered the highest bioavailability as compared to commercial product due to a better drug penetration effect .111 in another study, quinn et al. reported the inability of using poly vinyl pyrrolidine in isopropyl alcohol as liquid adhesive. the pellets had an irregular spiky appearance and required coating. furosemide powdered cellulose is an alternative to microcrystalline cellulose, however the size and size distribution of pellets are less appropriate than those pellets prepared using mcc. compared to mcc pellets, powdered cellulose showed a higher porosity, surface roughness and friability. the release of furosemide was very rapid. indomethacin indocid–r, a commercial product found to possess disappointing in vivo performance in humans. however, a competitor product developed for indocid-r by extrusion spheronization without any coating offered a high yield, adequate sphericity with better in vitro dissolution.129 diltiazem a matrix based multiunit pellet system for the controlled delivery of diltiazem was observed to be laborious due to its high aqueous solubility of the drug and larger surface area of the pellet. the outcome of the work ended with a high burst effect which may lead to a toxic level of the drug. 8. conclusion this comprehensive review hereby concludes with a 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