highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20195 highlights in bioscience october 2019| volume 2 http://bioscience.highlightsin.org/ page 1 of 2 research article open access 1 department of genome mapping, molecular genetics and genome mapping laboratory, agricultural genetic engineering research institute, giza, egypt. 2 international center for agricultural research in the dry areas (icarda), cairo, egypt. contacts of authors * to whom correspondence should be addressed: alsamman m. alsamman. citation: alsamman m.a. , habib p.t. (2019). genesyno : simple tool to extract gene sequence from the human genome despite synonymous gene terms. highlights in bioscience, volume 2 . article id 20195, dio:10.36462/ h.biosci.20195 received: august 25, 2019 accepted: september 20, 2019 published: october 25, 2019 copyright: © 2019 alsamman and habib. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. genesyno : simple tool to extract gene sequence from the human genome despite synonymous gene terms alsamman m. alsamman1* , peter t. habib2 abstract extracting gene data from the human genome is a tricky task. gene name is the key information for harvesting its sequence, annotation, and other related data.unfortunately, most human genes have different and multiple names, depending on the database and the resource in which they have been published. such an issue is delaying the ability of researchers to gather the necessary knowledge and to build their opinion on the function of genes. here we introduce genesyno, a simple, versatile and reliable tool that can be used to extract gene information from human genome data even though it is synonymous gene names. genesyno was written using c and python programming languages and could easily be integrated into another pipeline. keywords: gene information, human genome, gene name , gene annotation, gene, synonymous gene name. background human genome research is one of the most intensive research fields. synonymous terms of the name of the gene remain a major issue in human genomics (1). several human genes have different names, depending on the databases, the articles or the newly discovered function. with even more research articles published online, this information has become challenging for efficient implementation and reuse (2). such a case has been a complicated issue where genomic scientists can not form a collective and prospective conclusion by using published information on most human genes. several tools have been published to solve this problem, where text mining, and searching for databases could be used to generate symbol co-occurrences to extend information extraction capabilities (3-6).the main problem that, most of these tools require high computational skills, or are available only in online versions. most of these tools require high computational skills, or are only available in online versions. this may constrain the ability of researchers to access all available information for massive lists of genes at any time. here we introduce genesyno, a simple, versatile and reliable tool that can be used to extract gene information from human genome data even though it is synonymous gene names. genesyno was written using c and python programming languages and could easily be integrated into another pipeline. material and methods genesyno was built using c and python3 programming languages. the user’s input will be a list of human gene names. the input from the user will be a list of names for human genes. genesyno collects all available information about these genes from the grch38 database (which users could change for newer versions) and reports a tab-limited file containing gene information such as gene name, official gene name, chromosome , description, gene start, gene end, and a list of gene synonym names. furthermore, it produced a fasta file that contains sequences of all genes' proteins . if gene have more than one protein (isoforms) it will reported (figure 1). https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20195 http://bioscience.highlightsin.org/ page 2 of 2 alsamman et al., 2019 genesyno : simple tool to extract gene information despite synonymous gene terms highlights in bioscience october 2019| volume 2 http://bioscience.highlightsin.org/ genesyno core was written using c programming language to extract massive lists of information about genes in less processing time. genesyno can be installed and used on various operating systems, and has a simple gui for users. figure 1 : example of genesyn input and outputs. if a list of names of human genes is given in text format (a), genesyno can produce a table containing all gene information in tab delimited format (b) and protein sequences in fasta formats (c) . availability genesyno is available as a standalone tool at : https://github.com/alsammanalsamman/genesyno references 1. cohen kb, acquaah-mensah gk, dolbey ae, hunter l. contrast and variability in gene names. in: proceedings of the acl-02 workshop on natural language processing in the biomedical domain-volume 3. 2002. p. 14–20. 2. chen l, liu h, friedman c. gene name ambiguity of eukaryotic nomenclatures. bioinformatics. 2005;21(2):248–56. 3. cohen am, hersh wr, dubay c, spackman k. using co-occurrence network structure to extract synonymous gene and protein names from medline abstracts. bmc bioinformatics. 2005;6(1):103. 4. plotkin jb, kudla g. synonymous but not the same: the causes and consequences of codon bias. nat rev genet. 2011;12(1):32–42. 5. girish k, dubey s. eukaryotic molecular biology databases: an overview. highlights biosci. 2018;1:1–7. 6. alsamman am. the art of bioinformatics learning in our arabic world. highlights biosci. 2019;2. https://github.com/alsammanalsamman/genesyno http://bioscience.highlightsin.org/ highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20201 highlights in bioscience march 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 3 research article open access 1 department of biodiversity and crop improvement, international center for agriculture research in the dry areas (icarda), giza, egypt. 2 department of genome mapping, molecular genetics and genome mapping laboratory, agricultural genetic engineering research institute (ageri), giza, egypt. 3 department of bioinformatics & computer networks, ageri, agricultural research center (arc), giza, egypt. contacts of authors * to whom correspondence should be addressed: peter t. habib citation: habib p.t., alsamman a.m., hassanein s.e., and hamwieh a. (2020). developing convolutional neural networks-based system for predicting pneumonia using x-radiography image. highlights in bioscience, volume 3. article id 20201, dio:10.36462/h.biosci.20201 received: january 25, 2020 accepted: march 10, 2020 published: march 21, 2020 copyright: © 2020 habib et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests developing convolutional neural networks-based system for predicting pneumonia using x-radiography image peter t. habib *1, alsamman m. alsamman2, sameh e. hassanein3, and aladdin hamwieh1 abstract pneumonia is a respiratory disease caused by streptococcus pneumoniae infection. it is a life-threatening disease that causes a high mortality rate for children under 5 years of age every year. under such circumstances, we have a vital need to develop an appropriate and consistent protocol for the identification and diagnosis of pneumonia. the incorporation of computational approaches into the diagnosis of disease is extremely efficient, promising and reliable. our goal is to integrate these methods into pneumonia routine diagnosis to save countless lives around the world. we used the machine learning algorithm of convolutional neural networks (cnns) to identify visual symptoms of pneumonia in x-ray radiographic images and make a diagnostic decision. the dataset used to construct the computational model consists of 5844 x-ray images belonging to the pneumonia affected and normal individuals. our computational model has been successful in identifying pneumonia patients with a diagnosis accuracy of 84%. our model may increase the efficiency of the pneumonia diagnosis process and accelerate pathogenicity studies of the disease. keywords: cnns, artificial intelligence, pneumonia, disease diagnosis. introduction pneumonia is a bacterial disorder that causes severe symptoms such as grunting, chest retraction, central cyanosis, obtundation, lethargy, convulsions and inability to feed or drink [1,2]. each year about 1,400 cases of pneumonia occur in 100,000 children with around 1 in 71 babies. according to a recent study, pneumonia claimed the lives of over 800,000 children under the age of five last year, or one child every 39 seconds [3] . according to the american lung association, pneumonia can be diagnosed in various ways, including a blood examination, pulse oximetry, sputum analysis on a sample of mucus, arterial blood gas examination, pleural fluid culture, or bronchoscopy [4]. despite the many methods available to diagnose pneumonia, chest radiography remains the main method used for diagnosis. although x-rays are commonly used, it is difficult to diagnose them based solely on these images. perusing these images is a bottleneck problem because the area or areas of increased opacity are usually determined by pneumonia [5,6]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20201 https://doi.org/10.36462/h.biosci.20201 http://bioscience.highlightsin.org/ habib et al., 2020 convolutional nn-based system for pneumonia using radiography highlights in bioscience march 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 3 in fact, accuracy of the diagnosis of pneumonia is very limited due to certain causes of opacity which are difficult to account for. in this regard, many advances have been achieved by the application of machine learning (ml) techniques in medical diagnosis. the development of an artificial intelligence pneumonia diagnostic framework has recently become a hot topic in medical bioinformatics. such frameworks may help radiologists interpret medical images using additional perspectives developed by computer systems [7] ⁠ . continuous improvement of such frameworks using new ml algorithms and techniques could provide more accuracy and ease of use of computer platforms. in this regard, python programming language provides a hundreds of library. sci-kit learn [8] ⁠ , tensorflow [9] ⁠ , and keras [10] ⁠ are the most popular libraries ml programming. these ml libraries have been shown to have an impact on the integration of ml programming in biological data analysis [11,12]. in addition, alexnet is the name of a convolutional neural network (cnn) algorithm designed for large-scale visual recognition. alexnet has been successfully used for pathological brain detection [13–15]. in this research, we are trying to use highly specialized ml subtype for image classification to resolve many complications of routine pneumonia diagnosis. using x-images this tool may be used for diagnosis of pneumonia. this tool will also be available as user-friendly applications which can be used with the minimum programming skills. in addition, we aim to develop a diagnostic software that could be easily updated, modified and integrated into different medical diagnostic systems. materials and methods data collection the dataset is composed of 5844 x-rays images belonging to normal and pneumonia patients. the dataset consists of images with high resolutions and satisfied statistical variance (figure 1). the dataset was retrieved from kaggle database [16,17]. this data was collected from retrospective samples of one to five year-old pediatric patients. figure 1 : sample x-ray image of normal (a) and lung pneumonia (b). model construction and validation the dataset of x-rays images have been used for ml model training (5216 image) and validation (624 images). the alexnet architecture yielded 37% accuracy at the beginning of the design. our ml model design is inspired by alexnet structure, where the architecture consists of eight constitutional layers (figure 2 and table 1). figure 2 : the architecture of the ml neural network used to diagnose pneumonia from x-rays images . table 1: description of the structure of the neural network architecture. results and discussion diagnosis of pneumonia is very difficult due to hidden factors causing opacity such as pulmonary edema, bleeding, atelectasis or lung cancer. when examining an area of increased opacity in the chest radiography, it is critical to determine where increased opacity occurs [18,19]. computational analysis integration could accelerate pneumonia and enhance the routine medical diagnosis system. a highly specialized sub-type of machine learning for image classification called deep learning networks has been used in current research to detect lung pneumonia from x-ray images (figure 2). we analyzed a dataset of 5844 images of chest x-ray film to diagnose pneumonia using ml pipeline. alexnet's neural network architecture has produced an ml model accuracy of 84%. conclusion deep learning is expected to lead to a revolutionary progression in the efficacy of medical diagnosis using radiological methods around the world. we ml models and novel diagnostic approaches for various x-ray abnormalities to detect pneumonia. our pipeline may trigger the impact of artificial intelligence, which will layers feature map size strides dilation rate activation input image 1 (150, 150, 3) 1 1 relu 1 convolutional 3 (150, 150, 3) 1 1 relu max pooling 32 (148, 148, 32) 1 1 relu 2 convolutional 32 (74, 74, 32) 1 1 relu 3 convolutional 180 (72, 72, 180) 1 1 relu 4 convolutional 150 (70, 70, 150) 1 1 relu 5 convolutional transpose 100 (68, 68, 100) 1 1 relu 6 convolutional 150 (70, 70, 150) 1 1 relu max pooling 120 (68, 68, 120) 1 1 relu 7 fc 138720 softmax output fc 1000 softmax http://bioscience.highlightsin.org/ habib et al., 2020 convolutional nn-based system for pneumonia using radiography highlights in bioscience march 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 3 defiantly lead to an enormous improvement in the visual diagnosis of many diseases. availability the python source code we have used is freely available at : https://github.com/peterhabib/pneumoniaai . this code can be executed interactively in a python command line. references 1. hamborsky j, kroger a, editors. epidemiology and prevention of vaccine-preventable diseases, e-book: the pink book. public health foundation; 2015 oct 19. 2. brooks wa. bacterial pneumonia. in: hunter’s tropical medicine and emerging infectious diseases. elsevier; 2020. p. 446–53. 3. unicef. annual reports. 2020. available from: https://www.unicef.org 4. american lung association. american lung association; 2020. available from: www.lung.org 5. mattila jt, fine mj, limper ah, murray pr, chen bb, lin pl. pneumonia. treatment and diagnosis. ann am thorac soc. 2014;11(supplement 4):s189--s192. 6. makhnevich a, sinvani l, cohen sl, feldhamer kh, zhang m, lesser ml, et al. the clinical utility of chest radiography for identifying pneumonia: accounting for diagnostic uncertainty in radiology reports. am j roentgenol. 2019;213(6):1207–12. 7. al mubarok af, dominique jam, thias ah. pneumonia detection with deep convolutional architecture. in: 2019 international conference of artificial intelligence and information technology (icaiit). 2019. p. 486–9. 8. pedregosa f, varoquaux g, gramfort a, michel v, thirion b, grisel o, et al. scikit-learn: machine learning in python. j mach learn res. 2011;12(oct):2825–30. 9. abadi m, barham p, chen j, chen z, davis a, dean j, et al. tensorflow: a system for large-scale machine learning (pp. 265--283). in: 12th ${$usenix$}$ symposium on operating systems design and implementation (${$osdi$}$ 16) retrieved from https://www usenix org/conference/osdi16/technical-sessions/presentation/ abadirpy2(nd) retrieved march. 2016. p. 2019. 10. gulli a, pal s. deep learning with keras. packt publishing ltd; 2017. 11. habib pt, alsamman am, hassanein se, hamwieh a. tardict: randomforestclassifier-based software predict drug-tartget interaction. biorxiv. 2020; 12. matstubara t, ochiai t, hayashida m, akutsu t, nacher jc. convolutional neural network approach to lung cancer classification integrating protein interaction network and gene expression profiles. j bioinform comput biol [internet]. available from: https://www.worldscientific.com/doi/abs/10.1142/s02 19720019400079?af=r 13. lu s, lu z, zhang y-d. pathological brain detection based on alexnet and transfer learning. j comput sci. 2019;30:41–7. 14. lebedev v, lempitsky v. fast convnets using group-wise brain damage. in: proceedings of the ieee conference on computer vision and pattern recognition. 2016. p. 2554–64. 15. ezhilarasi r, varalakshmi p. tumor detection in the brain using faster r-cnn. in: 2018 2nd international conference on i-smac (iot in social, mobile, analytics and cloud)(i-smac) i-smac (iot in social, mobile, analytics and cloud)(i-smac), 2018 2nd international conference on. 2018. p. 388–92. 16. kaggle [internet]. 2020. available from: www.kaggle.com%0a 17. kermany ds, goldbaum m, cai w, valentim ccs, liang h, baxter sl, et al. identifying medical diagnoses and treatable diseases by image-based deep learning. cell. 2018;172(5):1122–31. 18. franquet t. imaging of pneumonia: trends and algorithms. eur respir j. 2001;18(1):196–208. 19. goodman p, prosch h, herold cj. imaging of pulmonary infections. in: diseases of the chest and heart 2015--2018. springer; 2015. p. 63–70. https://github.com/peterhabib/pneumoniaai http://bioscience.highlightsin.org/ highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20204 highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 3 perspective open access university of pécs, medical school, institute of migration health, pécs, szigeti st. 12., h-7624, hungary contacts of authors * to whom correspondence should be addressed: gábor ternák citation: gábor ternák (2020). holistic solution to an old "mystery": the global role of antibiotic consumption patterns in the spread of certain non-contagious diseases in eu countries, volume 3. article id 20204, dio:10.36462/h.biosci.20204 received: may 25, 2020 accepted: june 13, 2020 published: june 20, 2020 copyright: © 2020 ternák. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper. funding: the study was not supported by any grants. competing interests: no conflict of interest can be declared. holistic solution to an old "mystery": the global role of antibiotic consumption patterns in the spread of certain non-contagious diseases in eu countries gábor ternák abstract the discovery and extensive utilization of antibiotics are highly contributed to the considerable lengthening life expectancy of human beings. antibiotics, mixed with animal fodder, produced considerable growth promoting effect and hence, extended the indications of antibiotics at a much higher level. the indiscriminate use of antibiotics quickly resulted in the emergence of poly-resistant pathogens and the extensive antibiotic pollution of the environment, particularly of the surface water and rivers trough human and animal excreta. along with extensive and ever increasing antibiotic consumption/pollution, the pandemic-like spreading of certain non-contagious diseases like obesity, diabetes (type 1-2 t1dm, t2dm), alzheimer disease (ad), parkinson disease, multiple sclerosis (ms) etc. started unfolding, which was called as a slow moving disaster, without having any appropriate explanation of the phenomenon. the parallel appearance of those “pandemics”, which appeared simultaneously with the extensive antibiotic consumption, might indicate some kind of association. as far as several publications have reported the crucial role of altered gut flora in the development of metabolic disorders (diabetes, obesity) and neurodegenerative diseases alike (pd, ad, ms), it might be suspected that antibiotics, acting through the modification of microbiome, could influence the morbidity (prevalence) of those, non-infectious diseases. this concept, described below, might serve as a unified explanation, not excluding other, contributing causative factors, for the phenomenon, outlined above. the discovery of penicillin by fleming in 1937 and later of neomycin and streptomycin by waksman opened a new chapter of science, discovering the miraculous effect of antibiotics on infectious diseases. after the introduction of penicillin, several broad spectrum antibiotics were discovered also, like aminoglycosides, quinolones, macrolides etc., but their indiscriminate use has led to serious consequences and the rapid development of poly-resistant or panresistant bacterial species, which produced practically incurable infections (1). out of the approximately 5000 molecules considered as having an antimicrobial effect, one thousand was further investigated, and finally we have about one hundred commercially available antibiotics to treat infections. most of those drugs are produced by actinomycetes molds and different bacteria. after the „golden age” of antibiotic discovery of ’50-ies and ’60-ies and the extensive utilization of broad spectrum antibiotics in the late ’70-ies and ’80-ies, we now face the fact that the production of new classes of antibiotics practically stopped after 1987. ternák 2020 the role of antibiotic consumption patterns in the spread of non-contagious diseases highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 3 at present, only few antibiotics are in the development, but it is not of certain that they might finally be used as commercially available antibiotics (2). the accidental discovery of the growth promoting potential of antibiotics in the late ’40-ies (3) resulted in the large scale utilization of antibiotics mixed to animal fodder which contributed to the extensive environmental pollution and the emergence of multi-resistant bacteria (genes) to the environment along with the development of human infections, practically untreatable with any antibiotics (4). recently (2013) the yearly consumption of antibiotics in animal husbandry was estimated to be 131,109 tons (5). the “growth promoting effect” of certain broad spectrum antibiotics (than aureomycin) was observed and documented in premature newborns and school children also (6, 7). according to ecdc database of antibiotic consumption for 2018 expressed as the number of defined daily dose (ddd)/1000 inhabitants/day, the average total consumption (community and hospital sector) of antibacterial for systematic use (atc group j01) in the eu/eea countries was 20.1 ddd/1000 inhabitants/day (range: 9.7 – 34.0). during 2013-17, no statistically significant shift in antibiotic consumption was observed in the eu/eea countries and no significantly increasing trends were reported (8). usually, we consider as „epidemic” or “pandemic” the spreading of communicable diseases, but we have to face with the fact that the appearance and spreading of certain non-communicable diseases are similar. in the past few decades the pandemic like spread of several noninfectious diseases are alarming. the reason for the rapidly increasing amount of obesity, particularly childhood obesity, diabetes type 1-2 (t1dm, t2dm), autism, which was hardly heard of 50 years ago, the alzheimer disease (ad) of which produces more death in the usa, than cardiovascular and certain cancer deaths combined, and the parkinson disease (pd) doubled its prevalence in the past 25 years. the prevalence of multiple sclerosis (ms) has increased by approximately 10% since 1990. due to their outstandingly rapid spread, diabetes, obesity, autism, ad, pd and ms can be considered as of major importance. this phenomenon is called as a slow moving disaster (9, 10). scientists working on the explanation of this phenomenon, keep on digging deep to identify the molecular background, discovered several possible pathways and mechanisms leading to the development of those ailments and all of them emphasised the crucial role of the intestinal microbiome alteration (dysbiosis), but some external factors are suspected triggering the development of dysbiosis (11.). several sequencing efforts were made to elucidate the complete composition of gut flora. till now, 1952 uncultured bacterial species were verified by reconstructing 92,143 metagenome assembled-genomes from 11,850 human gut microbiota, which is considered 281% increase in phylogenetic diversity (12). antibiotics are powerful agents influencing the composition of the intestinal flora and producing dysbiosis, by reducing the diversity of the bacterial taxa in the gut. an extensive meta-analyses of articles dealing with the effect of antibiotics on the microbiome since the very first publication summarizes the observations. penicillin has not much effect on the gut flora, but amoxicillin, amoxicillin/clavulanic acid, cephalosporins, macrolide, quinolone, lypopolyglycopeptide, ketolide, fosfomycin; tigecycline has increased the dominance of non e. coli (mostly citrobacter, enterobacter and klebsiella spp.) enterobacteriaceae. in contrary, most of the above antibiotics (amoxicillin, macrolide, clindamycin, quinolone, and sulfonamide) reduce the presence of e. coli in the gut flora, but the combination of amoxicillin/clavulanate has increased its abundance. macrolide, doxycycline reduced the rate of enterococcus spp. in the gut flora, while amoxicillin, piperacillin, ticarcillin, cephalosporin (1-4th generations), lypoglycopep tides, carbapenem has enhanced their presence (13). antibiotics reach our body as either therapeutic agents or from the environment, particularly from the heavily polluted rivers, because of the antibiotic enriched animal fodder, industrial waste and human excreta entering the surface water and ending up in the rivers and are present in the potable water also (14-16). the decisive role of dysbiosis in the process of developing certain chronic diseases might raise the theory that the quality and quantity of antibiotic insult on human microbiome might trigger the development of different diseases and antibiotic consumption preferences in different countries might be related to the different, emerging, noncontagious diseases, showing pandemic-like spread. the issue that growth-promoting effect of antibiotics added to animal fodder of food animals might similarly affect humans as well, was first raised in 2005 (17). further research by the author has led to the discovery of the significant selective correlation between childhood obesity and certain antibiotic consumption in eu countries (18). the significant association was found between the consumption of narrow spectrum penicillin and the prevalence increase of pd. simultaneously, we observed the inhibitory effect of broad spectrum penicillin and the prevalence in pd as well (19). further comparisons of large databases derived from ecdc (20) yearly reports of antibiotic consumption by eu countries (1997-2018) and the prevalence of diabetes (21) ad (22), and ms (23) derived from different resources (publications are in progress) demonstrated the promoting and inhibitory effect of different classes of antimicrobial agents on the development of those diseases. as a conclusion, i have detected two different, very distinctive patterns of antibiotic consumption, what i have called “scandinavian type” with dominant consumption of narrow spectrum penicillin and tetracycline and higher ternák 2020 the role of antibiotic consumption patterns in the spread of non-contagious diseases highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 3 prevalence of pd (24), ms (23) and t1dm (21), and the “mediterranean type” with dominant consumption of broad spectrum antibiotics, including broad spectrum penicillins, and higher prevalence of childhood obesity (25, 26), t2dm (21), and ad (22). cross-association was observed with childhood obesity and ad. we found similar antibiotic consumption patterns in ms, and t1dm. this observation is being supported by the reported three fold higher prevalence of ms among patients with t1dm (27) and indicating the possible inhibitory effect of clavulanic acid in t1dm and ms. the holistic approach permitted us to consider the similarities of the parallel appearance and spreading of certain non-contagious pandemics, which appeared roughly at the same time as the widespread consumption of antibiotics. those findings clearly pointing to the possible common origin of the developing and spreading of certain non-contagious diseases included in the study and serve as an explanation for the “mystery” of the large scale spreading of obesity, diabetes, pd, ad and ms. references 1. hoel d, williams dn, berkelman rl. antibiotics: past, present, and future: unearthing nature's magic bullets. postgraduate medicine. 1997 jan 1; 101 (1): 114-22. https: //doi.org/10.3810/pgm.1997.01.139 2. https://www.khanacademy.org/science/health-and-medicine /current-issues-in-health-and-medicine/antibiotics-andantibiotic-resistance/a/antibiotics-an-overview 3. stokstad el, jukes th, pierce j, page jr ac, franklin al. the multiple nature of the animal protein factor. journal of biological chemistry. 1949; 180: 647-54. 4. argudín ma, deplano a, meghraoui a, dodémont m, heinrichs a, denis o, nonhoff c, roisin s. bacteria from animals as a pool of antimicrobial resistance genes. antibiotics. 2017 jun; 6 (2): 12. 5. https://www.swissre.com/dam/jcr:b8c6410f-755b-4d13-836df611c3510a3d/amr_thomas_boeckel.pdf 6. guzman ma, scrimshaw ns, monroe rj. growth and development of central american children. 1. growth responses of rural guatemalan school children to daily administration of penicillin and aureomycin. american journal of clinical nutrition. 1958; 6: 430-8. 7. robinson p. controlled trial of aureomycin in premature twins and triplets. the lancet. 1952 jan 5; 259 (6697): 52. 8. https://www.ecdc.europa.eu/en/publications-data/ surveillanceantimicrobial-consumption-europe-2018 9. https://www.who.int/publications/10-year-review/ncd/en/index 2.html 10. non-communicable diseases progress monitor, 2017. geneva: world health organization; 2017. 11. liang d, leung rk, guan w, au ww. involvement of gut microbiome in human health and disease: brief overview, knowledge gaps and research opportunities. gut pathogens. 2018 dec; 10(1):3. 12. almeida a, mitchell al, boland m, forster sc, gloor gb, tarkowska a, lawley td, finn rd. a new genomic blueprint of the human gut microbiota. nature. 2019 apr; 568 (7753): 499. https://doi.org/10.1038/s41586-019-096 5-1 13. zimmermann p, curtis n. the effect of antibiotics on the composition of the intestinal microbiota. journal of infection. 2019 oct 18. https://doi.org/10.1016/j.jinf. 2019.10.008 14. joel, l. 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https://doi.org/10.1016/j.jns.2014.11.%20019 highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20206 highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 4 research article open access 1 chemistry department, college of science, sudan university of science and technology, sudan. contacts of authors * to whom correspondence should be addressed: mohammed sulieman ali eltoum citation: eltoum m s a, elfaki a a m (2020). extraction, characterization, and usage of turmeric curcumin for color coating of metronidazole tablets. highlights in bioscience volume 3. article id 20206. dio:10.36462/ h.biosci.20206 received: may 20, 2020 accepted: june 27, 2020 published: june 30, 2020 copyright: © 2020 eltoum and elfaki .this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. extraction, characterization, and usage of turmeric curcumin for color coating of metronidazole tablets mohammed sulieman ali eltoum* 1 , almustafa ahmed mohamed elfaki 1 abstract curcumin is a chemical compound produced by curcuma longa plants that is widely used as a coloring agent and a dietary supplement and has some therapeutic activity. our aim is to evaluate the use of curcumin as color coating material for metronidazole tablets. curcumin was extracted at higher yields from three different samples of turmeric plants. the extract obtained was characterized by infrared red spectroscopy (ir), thin layer chromatography (tlc) and ash content and melting point (mp). the curcumin produced has an mp content ranging from 182 ° c to 184 ° c and an ash content ranging from 1.5 to 3.17 %. curcumin material was used as a colorful agent for the coating of metronidazole tablet pills using spray coating technology. experimental results have shown that curcumin-coated metronidazole tablets exhibit strong color stability even at higher temperatures, and that the taste of bitterness in metronidazole pills has been reduced or has disappeared entirely. keywords: curcumin, turmeric, color-coating, metronidazole tablets, pharmaceutical. introduction scientific research on color chemistry is needed to improve and maintain color stability. natural coloring additives are generally considered to be coloring additives derived from plant or animal sources by extraction or other physical processing[1,2]. examples of natural colorants include carmine, annatto extract, grape skin extract, turmeric, saffron and beta-carotene, which are the major natural color additives used in food products. synthetic coloring additives include chemically synthesized substances such as tartrazine, erythrosine and indigo carmine. natural or vegetable colors are less in use because they are expensive or difficult to extract [1, 2]. curcumin (synonyms: turmeric yellow, kurkum, ins no. 100(i)) is an orange-yellow crystalline powder. the standards of the joint expert committee on food additives (jecfa) define only curcumin extracted from natural source materials. it may also be produced by chemical synthesis, which is not used as a food additive [3]. the chemical structure of curcumin is shown in figure 1. the yellow coloring components (curcuminoids) and the primary coloring pigment are curcumin (curi), along with two additional minor curcuminoids, demethoxycurcumin (curii) and bisdemethoxycurcumin (curiii), which are the key active constituents of the turmeric (figure 2). curcumin is a major secondary metabolite of the perennial asian plant turmeric (curcuma longa l). curcumin was identified as the active principle of turmeric in 1815 and its structure was determined after crystallization in 1870 [4]. turmeric is only one representative of more than 80 species of turmeric in the ginger family, zingiberaceae [5]. eltoum m s a, elfaki a a m. 2020 usage of turmeric curcumin for color coating of metronidazole tablets highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 4 figure 1: the chemical structure of curcumin. figure 2: structure of the minor curcuminoids. turmeric is widely cultivated in many asian countries, particularly india, where it is mainly grown for dietary use and is a major component of the spice curry. in addition, turmeric is recognized for its medicinal properties and has been used for centuries in the treatment of a variety of diseases including eczema, arthritis, ulcers, asthma, anemia and many others [6]. as a result of extensive studies over the last few decades, curcumin has emerged as a promising anti-cancer agent and has been shown to target multiple and diverse pathways of disease causation and progression [4]. the attractiveness of curcumin as a therapeutic agent is enhanced by its safety, affordability, and history of longterm use [7]. molecular formula of c21h20o6, corresponding to a molecular weight of 368.37. curcumin is a yellow-orange crystalline powder with maximum absorbance at430 nm and melting point of 183 °c [6]. curcumin exhibits hydrophobic and (slight) hydrophilic properties owing to its aliphatic heptadienone linker and polar β-dicarbonyl and phenolic groups, respectively [8]. curcumin is sparingly soluble in water, but shows greater solubility in some organic solvents such as acetone, ethyl acetate, acetonitrile and ethanol. its reported partition coefficient (log p) ranges from 2.5 to 3.3 [9]. curcumin is a bis-α,β-unsaturated βdiketone and exists in equilibrium with its enol tautomer [10]. studies involving 1h, c nmr, and infrared spectroscopy have shown that the enolate form predominates in alkaline solution [11]. no previous literature has introduced the use of curcumin as a color coating for metronidazole tablets. the aim of this study is to extract and characterize curcumin obtained from the turmeric plant using different analytical techniques and to evaluate the use of turmeric powder as a coating material for metronidazole tablets. in addition, we studied the effect of this coat material on some of the properties of the metronidazole tables. materials and methods turmeric samples were collated from sudanese local market. curcumin extraction: twenty grams of ground turmeric in 50 ml of dichloromethane was magnetically stirred and heated at the reflux condenser for 1 h. the mixture was then suction-filtered and the filtrate was concentrated in a hot water bath maintained at 50°c. the reddish yellow oily residue was treated with 20 ml hexane and the resulting solid was collected by suction filtration [3]. the obtained yield was calculated as follow: yield = wpr/wtheox100 where: wpr = practical weight, wtheo = theoretical weight fourier transform infrared spectroscopy (ft –i r): two milligrams of curcumin were mixed with 300 mg of dried potassium bromide. carefully, the mixture was grinded, spread evenly in an appropriate die and subjected to a pressure of approximately 800 mpa (8 t·cm -2 ). in the case of substances that were unstable under normal atmospheric conditions, the disc was pressed into a vacuum. a disc was rejected if visual examination had been show lack of uniform transparency or when transmittance at 2000 cm -1 (5 µm) in the absence of a specific absorption band was less than 60 per cent without compensation, unless otherwise prescribed. samples were prepared by the same procedure and the spectrum was recorded between 4000400 cm -1 (2.5-15.4 µm) under the same operational conditions. the transmission minima (absorption maxima) in the spectrum was obtained with the substance to be examined correspond in position and relative size to those in the spectrum obtained with the reference substance [12]. thin layer chromatography (tlc): the solvent system which was used to characterize of curcumin powder was prepared by mixed of (3% methanol and 97% dichloromethane). the diameter of tlc plate had 10x20 cm 2 [3]. melting point (mp): a sufficient quantity of curcumin was introduced into the capillary tube to give a compact column of 4 mm to 6 mm in height. the temperature of the bath was increased to about 10°c below the assumed melting point and the heating rate was adjusted to about 1° c/min. when the temperature was 5°c below the assumed melting point, the capillary tube was introduced into the instrument. in the case of the apparatus described above, the capillary tube was immersed in such a way that the closed end is near the center of the thermometer bulb, the immersion mark of which is at the surface level of the liquid. the temperature at which the last particle was transferred to the liquid phase was recorded [12]. eltoum m s a, elfaki a a m. 2020 usage of turmeric curcumin for color coating of metronidazole tablets highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 4 ash content: the porcelain crucible was ignited at 600 ± 50 °c for 30 min and then allowed to cool over the silica gel in the desiccators. the prescribed amount of curcumin was placed in the crucible and weighed, after it had been gently heated to low temperatures as practicable, until the sample had been thoroughly charred, until white fumes had ceased to develop and ignited at 600 ± 50 °c until the residue had been completely incinerated. the crucible was allowed to cool over the silica gel in the desiccator, was weighed again and the percentage yield was calculated [12]. percentage of whiteness: the sample powder was placed in dishes, regularly highlighted and then the color was measured against the white color standard and the percentage was finally recorded. coating of metronidazole tablets with curcumin: a total of 25 g of opadry white was dissolved in 500 ml of ethanol with stirring during 30 min. and 1 g of curcumin powder was added during 10 min. the spray coating process for metronidazole tablets began by placing the tablets in the coating machine chamber, and then the main unit button was pressed.the exhaust was operated until the powder was removed from the tablets and the hot air was operated until the temperature of the tablet reached 50°c. the temperature control key was operated with hot air to control the temperature of the coating. the spray stage was then operated, and the turn sprayers were opened until the tablets were completely coated. and after coating, the other keys were turned off until the tablets had dried. the coating machine was stopped; the tablets were removed [12]. results and discussion the turmeric plant can be identified both by its characteristic tuberous root and by the leaves which extend upward from the erect, thick stems of the root. turmeric root has a fragrant aroma and a slightly bitter, peppery, biting taste reminiscent of ginger. when eaten, it colors the saliva yellow and leaves a warm feeling in the mouth. the curcumin yields obtained for the different samples are shown in table 1 and the resulting compounds were finely soft, clear yellow powder. the yield of the samples was consistent with the method described by andrew et al, [3], and the appearance of the powder was as clear as the standard colour. table 1: the curcumin yield (cy), melting point (mp), ash content (ac) and percentage of whiteness (pw) of the studied tumeric samples. figures 3 show the absorption of samples. these figures contain the following characteristic peaks: the broad band of the three oh groups at 3250-3500 cm −1 , sharp peak stretch of ch aliphatic system less than 3000cm −1 , weak peak of c-h aromatic system around 3050cm, sharp peak of c═o at 1510 cm −1 , medium peak of c-o at 1300cm −1 , and sharp peak of c=c of aromatic system at about 1510cm −1 . figure 4 shows the tlc plate of the compound. the chromatogram obtained showed that curcumin was divided into three distinct compounds. clearly, this result is in agreement with andrew et al., [3]. figure 3: ir spectrum analysis results of curcumin for samples 1 (a), 2 (b) and 3 (c). figure 4: tlc of curcumin sample. the melting point determined by the capillary method and it is the temperature at which the last solid particle of a compact column of a substance in a tube passes into the liquid phase. the results of the three samples are shown in table 1. the melting point of curcumin is 183°c (bp2012)[12]. the ash content obtained of the samples ranges from 1.5 to 3.17 % (table 1). in summary, from the three samples above, the percentage of ash in curcumin was good. the percentage of whiteness of the three samples obtained ranges from 51.08% to 53.03% (table 1). coated tablets are tablets containing one or more layers of mixtures of different substances, such as natural or synthetic resins, gums, gelatin, inactive and insoluble fillers, sugars, plasticizers, polyols and waxes. coloring matter authorized by the competent authority and sometimes flavorings and samples number cy mp ac pw 1 86.36% 183°c 1.50% 52.04% 2 81.81% 184°c 3.09% 53.03% 3 82.72% 182°c 3.17% 51.08% eltoum m s a, elfaki a a m. 2020 usage of turmeric curcumin for color coating of metronidazole tablets highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 4 active substances. the mean weight and disintegration time of metronidazole tablets prior to coating with curcumin was 323.0 mg and 23 sec and 22 sec respectively. however, the average weight and disintegration time after coating was 324.74 mg and 120 sec, 93 sec and 114 sec respectively (figure 5). figure 5: metronidazole tablets before (a) and after blistering. conclusion the primary purpose of the study in this study is to study the isolation of curcumin from turmeric and also to characterize curcumin as a color-coating martial for metronidazole tablet in pharmaceuticals. curcumin was extracted from the turmeric in a simple and easy way using dichloromethane and triturated with hexane, and the extraction yield was finely soft yellow powder. experimental results have shown that curcumin-coated metronidazole tablets exhibit strong color stability even at higher temperatures, and that the taste of bitterness in metronidazole pills has been reduced or has disappeared entirely. acknowledgments we would like to thank the climax factory for the drugs and medicines industry-khartoum for giving us the opportunity to carry out the practical work of this research in their different laboratories, and we would also like to thank the sudan university of science for accepting this work as part of the complementary research on the requirements of the m.sc. in chemistry [13]. references 1. allen l, ansel hc. ansel's pharmaceutical dosage forms and drug delivery systems. lippincott williams & wilkins; 2013 dec 23. 2. carter sj. cooper and gunn's dispensing for pharmaceutical students (12th edn) publisher. 47-50 3. anderson am, mitchell ms, mohan rs. isolation of curcumin from turmeric. journal of chemical education. 2000 mar;77(3):359 4. aggarwal bb, harikumar kb. potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. the international journal of biochemistry & cell biology. 2009 jan 1; 41(1): 40-59. 5. leong-škorničková j, šída o, jarolímová v, sabu m, fér t, trávníček p, suda j. chromosome numbers and genome size variation in indian species of curcuma (zingiberaceae). annals of botany. 2007 sep 1; 100 (3): 505-26. 6. goel a, kunnumakkara ab, aggarwal bb. curcumin as “curecumin”: from kitchen to clinic. biochemical pharmacology. 2008 feb 15; 75 (4):787-809. 7. chandran b, goel a. a randomized, pilot study to assess the efficacy and safety of curcumin in patients with active rheumatoid arthritis. phytotherapy research. 2012 nov; 26 (11):1719-25. 8. balasubramanian k. molecular orbital basis for yellow curry spice curcumin's prevention of alzheimer's disease. journal of agricultural and food chemistry. 2006 may 17; 54 (10):3512-20. 9. grynkiewicz g, ślifirski p. curcumin and curcuminoids in quest for medicinal status. acta biochimica polonica. 2012 may 14; 59 (2). 10. chignell cf, bilskj p, reszka kj, motten ag, sik rh, dahl ta. spectral and photochemical properties of curcumin. photochemistry and photobiology. 1994 mar; 59(3):295-302. 11. sun ym, wang rx, yuan sl, lin xj, liu cb. theoretical study on the antioxidant activity of curcumin. chinese journal of chemistry. 2004 aug; 22(8):827-30. 12. bp. the british pharmacopoeia 2012. 13. http://repository.sustech.edu/handle/123456789/13398?sh ow=full highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202102 research article open access 1 department of biomedical laboratory sciences , kibogora polytechnic, rwanda. 2 biomedical laboratory sciences dept, ines ruhengeri-institute of applied sciences, rwanda. contacts of authors * to whom correspondence should be addressed: yadufashije callixte received: may 30, 2020 accepted: november 2, 2020 published: january 16, 2021 citation: fulgence m, joseph m, callixte y, william n . renal dysfunction among hiv patients under highly active antiretroviral therapy attending kibagabaga district. 2021 jan 16;4:bs202102 copyright: © 2021 fulgence et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. renal dysfunction among hiv patients under highly active antiretroviral therapy attending kibagabaga district munyandamutsa fulgence1, mucumbitsi joseph2, yadufashije callixte*2, niyonzima william2 abstract antiretroviral therapy is used for the suppression of the hiv virus and stops its progression to cause disease. despite its role, it has the pathophysiologic effect to kidney function for users. the study was conducted to evaluate the renal function for hiv patients under highly active antiretroviral therapy at kibagabaga district hospital. venous blood samples (4ml) were collected by vein puncture in phlebotomy services by means of the dry tubes from 170 patients under antiretroviral therapy. blood samples were transported to clinical biochemistry department for analysis. rotor centrifuge was used to separate the serum and other blood components; creatinine level was analysed for renal function analysis. the total of 170 hiv patients were considered in the study. of the 170, 50 hiv patients were used for primary data collection, while 120 hiv patients who previously received antiretroviral therapy were considered as secondary data collection source. the patients between 25-45 years old have experienced the highest level of abnormal concentration of renal tests 25%, patients with above 45 years old was ranked the second to have the abnormal level (14.2%). females were 59% and have experienced the high risk of renal dysfunction than males, the level of glomerular filtration rate was 67 (39.4%), and was higher than creatinine 42 (24.7%). antiretroviral therapy has a negative effect on kidneys. abnormalities of the kidney parameters were prevalently high among both male and female. the serious follow is needed for this vulnerable population. keywords: renal function, hiv, haart, estimated glomerular filtration rate introduction human immunodeficiency virus (hiv) infection mostly provoke acute or chronic renal proximal renal tubule resulting in functional disturbance with mitochondrial injury being one of the most important targets [1]. it results to the failure of excretory functions of kidney, which is disease and people are living longer. hiv infection negative side effects might lead to the heart, lungs, and renal diseases, which could cause high mortality among hiv patients. africa endures greater than 60% of the global burden of hiv, however, characterized by decrease in glomerular filtration rate [2]. the advances in development of highly active antiretroviral therapy (haart) is a high risk to the development of the serious renal disease in developed world with very few studies done in developing world [3]. kidney function has been estimated to be abnormal up to 30% of all hiv patients. few outpatient renal screening studies from africa describe varying prevalence from as low 6% to a high of 50% [4]. the dysfunction of the kidney is medical kidney abnormality where this important organ gets impairment for the filtration of wastes from blood [5]. nephropathy is one of the complications characterizing the kidney dysfunction for hiv patients [6]. kidney disease was clinically diagnosed considering the presence of albuminuria but also low estimated glomerular filtration rate [7]. some antiretroviral agents can cause serious side effects that affect multiple systems; including the kidneys tenofovir (tenofovir disoproxil fumarate) and this could be associated with development of the acute renal failure (arf) and the dysfunction of proximal and distal tubules [8]. highlights in bioscience page 1 of 4 january 2021|volume 4 https://doi.org/10.36462/h.biosci.202102 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience fulgence et al., 2021 renal dysfunction among hiv patients under antiretroviral therapy the first described patient who was treated with tenofovir and developed reversible fanconi syndrome, nephrogenic diabetes insipidus and arf, renal biopsy demonstrated cytoplasmic vacuolization, apical localization of nuclei and reduction of the brush border on proximal tubule epithelial cells [9]. materials and methods study area description the study was conducted at kibagabaga district hospital. kibagabaga district hospital is located in kigali city, nyarugenge district; the hospital receives the referred patients from 16 health centers of nyarugenge from neighbouring health canters in gasabo and kicukiro. study design both cross-sectional and retrospective study design were used. the study was carried out from october to december 2019. study population and sample size all hiv patients under highly active antiretroviral therapy attending kibagabaga district hospital during the period will be targeted by the study. however, only patients who were not hiv positive did not participate. the population size was 170; the sample size was determined by the number of eligible patients who attended the hospital and accepted to participate in the study during the period of the study. collection of stool sample blood samples were collected with phlebotomy and biochemistry sops support. the patients were placed in a comfortable seat and requested to remove sleeves from the part of the arm. phlebotomist finds the arm with visible fuller vein. a stretched band tourniquet was placed around the upper arm to help the vein bloat. the place for injection was then whipped with cotton after being soaked in the antiseptic solution. the needle was slightly inserted into the swollen vein and around 4ml of blood were collected by the help of a seal dry test tube. specimen containers or collection devices was labelled with the patient record number was recorded along with the date of birth to avoid bias and errors in laboratory results. sample processing the sample collection and processing were done according to kibagabaga district hospital laboratory sops, the tubes containing blood was taken to clinical biochemistry department. the collected blood specimen was clotted for 15 min before centrifugation at 3000 rpm for 5 minutes using a rotor centrifuge for separation of serum and other blood components. creatinine level was analysed for renal function by using automated biochemistry machines (architecture machine or cobasc311), the cobas c311. data analysis after data collection, the spss version 23.0 program and excel 2016 was used for data analysis. to compare variables and association between renal function in hiv patients under haart and its clinical manifestastion unduced by this medication,and prevalence ethical considerations this study was conducted after obtaining an ethical approval from kibogora polytechnic research committee. in addition, the authorization was requested from kibagabaga district hospital, clinical research and office of education before conducting this research. the purpose of research project, sample collection procedures, as well as confidentiality of research results was explained to participants. participation was voluntarily after understanding the aim and expect outcome of research. there was no expected risks that can threaten the lives of participants. results social demographic characteristics of study population in this study the social demographic characteristics of the study population were established to characterize the population, table 1 indicates the analysis which was done on 170 hiv paatients under highly active antiretroviral therapy from kibagabaga district hospital in a period of 3 mounths. table 1 reveals the different parameters with renal function tests such as age group, gender, period, clinical manifestation and weight. the high frequency was observed in the age group between 25-45 years old. the number of females stood at 62%, mouth ulcers predominated the clinical manifestations with percentage of 29.4% followed by memory loss at 25%. over weight patients stood at 6.5% while 38.8% was in normal ranges. table 2 shows the range of of creatinine and glomerular filtration rate level according sex. clinical analyses in this study the creatinine level and glomerular filtration rate showed the high prevalence of abnormal results in males with 14.5% and 39.4 respectively. cross sectional and retrospective studies were assessed and shown in table 3; the majority of patients were females with 28 (56%) and 22 (44%) were males. moreover, in retrospective 29 (58%) were females and 22 (18.3%) were males. table 4 shows diagnosed patients based on clinical symptoms associated to renal dysfunction in hiv patients; the majority of patients was those with mouth ulcers 50 (28.6%) followed by memory loss with 43 (24.6%). discussion this study analysed biochemical parameters of hiv patients under highly active antiretroviral therapy, this medication leads to the serious side effects including kidney failure for patients under those medications. this study considered the level of creatinine and estimation of glomerular filtration rate as tests to confirm the renal function of hiv patients under highly active antiretroviral therapy. highlights in bioscience page 2 of 4 january 2021|volume 4 http://highlightsin.org/index.php/bioscience fulgence et al., 2021 renal dysfunction among hiv patients under antiretroviral therapy table 1. number of patients participated in the study and their corresponding gender, age, clinical manifestation, weight and period. parameter variables frequency percentages % age groups [5-25] years 13 9.6 [25-45] years 93 54.7 above 45 years 64 37.6 sex males 64 38 females 106 62 period 2018 retrospective 70 41.2 2019 prospective 50 29.4 2019 retrospective 50 29.4 clinical manifestation night sweats 18 10.6 weight loss 19 11.2 depression 40 23.5 mouth ulcers 50 29.4 memory loss 43 25.3 weight 40-60 kg 38 22.4 60-80 kg 66 38.8 80-100 kg 55 32.4 above 100 kg 11 6.5 total 170 100 table 2. number of patients participate the study and their corresponding results according to gender. state creatinine level (mg/dl) egfr (ml/min) females males total females males total normal 92 (54.1) 36 (21.2) 128 (75.3) 7 2(38.4) 31 (22.2) 103 (60.6) abnormal 17 (10.2) 25 (14.5) 42 (24.7) 32 (16.1) 35 (23.3) 67 (39.4) table 3. prevalence of abnormal creatinine and egfr according to age and gender in suspected patients at kdh, where the creatinine (kg/dl) (a), egfr (ml/min)(b), and prevalence (%)(c) are shown. groups variables cross sectional study n (50) retrospective study n (120) (a) (b) (c) (a) (b) (c) age groups 5-25 1 (2) 1 (2) 8% 0 (0) 2 (1.6) 13% 25-45 6 (12) 11 (22) 68% 15 (12.5) 22 (18.3) 25% above 45 2 (4) 3 (6) 2% 17 (14.1) 28 (23.3) 14.20% gender females 20 (40) 28 (56) 48% 16 (13.3) 29 (58) 59% males 22 (44) 14 (28) 36% 17 (14.1) 22 (18.3) 27% table 4. clinical manifestation and their associated abnormal level of renal test. the frequency (%) of occurrence of symptoms the frequency (% ) of renal dysfunction clinical manifestation frequency (%) abnormal creatinine abnormal egfr nigth sweats 18 (10.3%) 6 (3.5%) 7 (4.1%) weight loss 19 (10.9%) 9 (5.2%) 9 (5.2%) depression 40 (22.9) 5 (2.9%) 13 (7.6%) mouth ulcers 50 (28.6) 14 (8.2%) 25 (14.7%) memory loss 43 (24.6%) 9 (5.2%) 13 (.6%) among 170 suspected patients, 64(38%) were males and 106 (62%) females. the mean age of the population was 56.6, and the standard deviation of age was 40.5. 54.7% of patients were in the age range of [25-45] years, and were the majority in the study participants. this age is at high risk because of the habit of doing unprotected sex than elderly age. after testing creatinine level and egfr the results showed the normal females 38.4% and 23.3% males with abnormal egfr level and abnormal creatinine respectively. the study conducted on active antiretroviral therapy among hiv patients shows no difference with the current study since it revealed the high glomerular filtration rate (egfr) and creatinine in females than males under highly active antiretroviral therapy [12] the patients who had positive test results with creatinine and egfr for cross-section study, the findings show that the majority of patients were females with 28(56%) and 22(44%) were males. moreover, in the retrospective 29(58%) were females and 22 (18.3%) were males. in addition, the prevalence of renal dysfunction was high in old age in females. these results were comparable to the findings of willems and his colleagues [13], we estimated the prevalence of egfr and mortality risks in a population-based study of persons aged 85 years and older, based on these results. the patients were diagnosed based on clinical symptoms associated with renal function of hiv patients. the majority of patients induced the mouth ulcers 50 (28.6) than other clinical manifestation. the results showed that there is significant relationship between the most of clinical manifestation and renal function of hiv patients under highly active antiretroviral therapy. the similar findings were reported where self-reported strategies used by persons living with hiv/aids and their clinical manifestation induced by art such us moth ulcers, weight loss and other opportunist infections were described [14]. conclusions the study confirmed the effect of antiretroviral therapy on kidneys where the abnormalities of creatinine and egfr were more prevalent 59% and 27% in both females and males respectively. it is a paramount action of treating patients to enhance the longevity even if renal dysfunction may occur. all hiv patients under retroviral therapy should be followed up to maintain the normal function of the kidneys. acknowledgement we are with pleasure to thank kibagabaga distict hospital’s authorities to allow the study to be conducted in the hospital facilities and their abundant generosity. in addition, we once again address our thanks to kibogora polytechnic for its vision of promoting research not only in the region but also all over the world. references 1. hilton r. human immunodeficiency virus infection and kidney disease. j r coll physicians edinb 2013; 43(3):236-40. http://dx.doi.org/10.4997/jrcpe.2013.310 2. authier fj, chariot p, gherardi rk. skeletal muscle involvement in human immunodeficiency virus (hiv)–infected patients in the era of highly active antiretroviral therapy (haart). mushighlights in bioscience page 3 of 4 january 2021|volume 4 http://highlightsin.org/index.php/bioscience fulgence et al., 2021 renal dysfunction among hiv patients under antiretroviral therapy cle and nerve: official journal of the american association of electrodiagnostic medicine. 2005 sep;32(3):247-60. 3. gray f, keohane c. the neuropathology of hiv infection in the era of highly active antiretroviral therapy (haart). brain pathology. 2003 jan;13(1):79-83. 4. campbell c, nair y, maimane s, nicholson j. dying twice’ a multi-level model of the roots of aids stigma in two south african communities. journal of health psychology. 2007 may; 12(3): 403-16. 5. matiyashchuk ig, amosova km, iaremenko ob, zakharova vi, koliadenko di. impaired endothelial vasoregulatory function in patients with systemic lupus erythematosus: association with renal involvement, inflammatory markers, and autoantibodies. kidneys. 2020;9(1):14-9. 6. núñez m, saran am, freedman bi. gene-gene and geneenvironment interactions in hiv-associated nephropathy: a focus on the myh9 nephropathy susceptibility gene. advances in chronic kidney disease. 2010 jan 1;17(1):44-51. 7. romagnani p, remuzzi g, glassock r, levin a, jager kj, tonelli m, massy z, wanner c, anders hj. chronic kidney disease. nature reviews disease primers. 2017 nov 23;3(1):1-24. 8. mocroft a, kirk o, gatell j, reiss p, gargalianos p, zilmer k, beniowski m, viard jp, staszewski s, lundgren jd, eurosida study group. chronic renal failure among hiv-1-infected patients. aids. 2007 may 31;21(9):1119-27. 9. badowski me, burton b, shaeer km, dicristofano j. oral oncolytic and antiretroviral therapy administration: dose adjustments, drug interactions, and other considerations for clinical use. drugs in context. 2019;8. 10. myers gl. standardization of serum creatinine measurement: theory and practice. scandinavian journal of clinical and laboratory investigation. 2008 jan 1;68(sup241):57-63. 11. michels wm, grootendorst dc, verduijn m, elliott eg, dekker fw, krediet rt. performance of the cockcroft-gault, mdrd, and new ckd-epi formulas in relation to gfr, age, and body size. clinical journal of the american society of nephrology. 2010 jun 1;5(6):1003-9. 12. overton, et., wyatt, cm., mitsuyasu, rt., wensing, a. m., calvez, v., günthard, h. f. and richman, dd. topics in antiviral medicine. 2014;40(10):100-109. 13. willems jm, vlasveld t, den elzen wp, westendorp rg, rabelink tj, de craen aj, blauw gj. performance of cockcroftgault, mdrd, and ckd-epi in estimating prevalence of renal function and predicting survival in the oldest old. bmc geriatrics. 2013 dec 1;13(1):113. 14. sukati na, mndebele sc, makoa et, ramukumba ts, makoae ln, seboni nm, human s, holzemer wl. hiv/aids symptom management in southern africa. journal of pain and symptom management. 2005 feb 1;29(2):185-92. highlights in bioscience page 4 of 4 january 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods study area description study design data analysis ethical considerations results social demographic characteristics of study population clinical analyses discussion conclusions acknowledgement references highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 1 of 5 citation: youssef a. a. and rslan w. m. (2018) sugar beet improvement using agrobacterium-mediated transf ormation technology. highlights in bioscience, volume 1.article id 20183, dio:10.36462/h.biosci.20183 received: june 1, 2018 accepted: july 17, 2018 published: july 30, 2018 copyright:© 2018 youssef and rslan. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. sugar beet improvement using agrobacteriummediated transformation technology abo-bakr a. youssef 1 and wessam m. rslan 2 * 1, 2 agricultural genetic engineering research institute; agricultural research center, egypt. *to whom correspondence should be addressed: wessam.rslan@ageri.sci.eg abstract since discovering agrobacterium tumefaciens distinctive capacity to incorporate a specified part of their transfer-dna (t-dna) into eukaryotic cells, the bacteria were commonly used for crop transformation originally of dicotyledonous crops and subsequently of nearly all organisms. to achieve this, the tumor-inducing (ti) plasmid was changed to extract phytohormone and opine biosynthetic proteins (cytokinin and auxin) so as not to interfere with ordinary morphological growth. overall, the conversion mediated by agrobacterium was easier, more effective and less costly relative to other technologies. it also results in insertions with small copy count. tumor development in crops has also proved the susceptibility of explants from field-grown sugar beet crops to agrobacterium tumefaciens. early efforts by agrobacterium tumefaciens to transform sugar beet were unsuccessful, primarily owing to inability to regenerate crops from stably modified callus or suspended cells. a genotype-independent method was defined under which cotyledonary explants of various sugar beet genotypes are inoculated with agrobacterium tumefaciens comprising whether kanamycin tolerance and gus activity or kanamycin resistance, gus activity and glyphosate tolerance. gus expression, npt dot blot as well as epsps assays verified the presence of transgenes; progeny showed mendelian genetically modified inheritance and glyphosate tolerance at deadly concentrations to control plants. unfortunately, there was no publication of technical information of the technique. here we reviewed the concept agrobacteriummediated transformation and how to be applicable keywords: agrobacterium tumefaciens, callus, suspended cells, sugar beet, kanamycin resistance. review article open access sugar beet improvement using agrobacterium highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 2 of 5 introduction sugar beet is extremely prone to agrobacterium tumefaciens as an instance (1–5). though, genotypedependent susceptibility (1,3) can be enhanced by pre-culture explants before inoculation (6) or by extending the length of co-culture (1,3). krens et al. (4) used agrobacterium tumefaciens strains carrying an isolated cytokinine gene to enhance the development of sugar beet leaf discs, cotyledons and hypocotyls from two-week-old seedlings of nine distinct types. cotyledon and hypocotyl explants generated lowfrequency shoots; however, inoculated leaf discs did not regenerate the shoot. it was impossible to confirm a stable transformation. lindsey and gallois (1) revealed agrobacterium tumefaciens first effective regeneration of genetically modified sugar beet crops. transgenic seeds were acquired from co-cultivated shoot-base cells with agrobacterium strain lba4404 containing a bi-nary plant with a kanamycin resistance gene (nptii) and either chloramphenicol acetyl transferase (cat) or β-lucuronidase (gusa) genes. the frequency of transformation depended on the kinds of explants, genotypes and circumstances of choice. halluin et al. (2) who established crops resistant to broad-spectrum herbicides, glufosinate and sulfonylureas, as well revealed sugar beet transformation. friendly callus, originating from cotyledons, hypocotyls, petioles as well as true leaves of 2-to 3-month-old seedlings cultivated in dark, was carefully cut and inoculated with agrobacterium tumefaciens either a mutant acetolactate synthase (als) or a bialaphos resistance (bar) gene powered by multiple promoters, i.e. 35s camv, nos, tr1' or tr2.' this genotype-dependent protocol took nearly two years to get grown, which probably explains the morphologies of the aberrant plant. latest studies utilizing agrobacterium tumefaciens generated transgenic crops at frequencies that are adequate to produce crops used in experiments to evaluate herbicide tolerance and resistance to disease (7–12). for physiological and molecular research, agrobacterium tumefaciens produced transgenic crops were also used. an endogenous sugar beet gus (sb-gus) enzyme has been contrasted to e. coli in leaf disc transformation (13). variations in patterns of gene expression in roots have been investigated using distinct constructs (14) in another application. in another, development habit and accumulation of sugar were explored after transforming into a patatin gene promoter (15) with a bacterial cytokinin biosynthetic gene. although various explants of sugar beet, i.e., shoot bases, petioles, leaves and callus, were used, cotyledon explants were often more effective in transformation with efficiencies varying from 0.1 to 1.0 (4,7,9,10,16,17). with kanamycin selection (18), leaf lamina explants from shoots multiplied from apical meristems of four genotypes of sugar beet produced a 6.2 percent transformation level. small (1–3 mm) bud tips from various shoot clumps from undeveloped floral buds of five distinct genotypes resulted in hygromycin-resistant shooting rates of 13.3 to 30.6 percent. over 50% of crops from shoot-base tissue kanamycin-resistant explants produced the hpt gene (19). finally, a mixture of agrobacterium and void infiltration has been used to promote the transfer of bacterial dna to callus and crop tissue, resulting in more than 40% transgenic callus clones (20). factors influencing transformation efficiency genotype (19), explant origin (21), bacterial strain and incubation time (18) selection technique (22),type of promoter promoting selectable gene (22), light intensity (23), wound with pre-inoculation particle bombardment (10), use of acetosyringone (3), pre-treatment with drugs (6) and , preand co-cultivation interval (6). despite important advancement, the transformation of sugar beet to agrobacterium tumefaciens is still regarded to also be recalcitrant. because of the failure to absorb and integrate dna, it is impossible to be due to the small amount of morphologically competent regeneration cells. if they are integrated in big amounts of non-competent cells (24), connection to such competent cells may also be impeded. development of transgenic herbicide tolerance sugar beet competes badly with weeds, particularly in early growth, leading in dramatically elevated yield losses that can range from 50 percent to total losses (25) unless adequate weed control is attained. conventional control measures involve herbicide spraying at distinct moments and distances to decrease these losses, making weed control programs complex and challenging. the development of transgenic sugar beet resistant to broad-spectrum herbicides is therefore a significant alternative. herbicide tolerance is one of the first features that genetic engineering has effectively brought into several plant species; some types of herbicide tolerant (ht) have been on the market for further than a decade (26). ht crops have been produced using genes from microorganisms or higher plants that confer tolerance by: i changing the active site of the target protein in such a way that converted cells are less susceptible to herbicide; (ii) using an enzyme that transforms the core components of the herbicide into inactive compounds; or (iii) overproducing herbicide target proteins (27). agrobacterium-mediated transformation and in vitro cell selection have been used to evolve ht sugar beets sugar beet improvement using agrobacterium highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 3 of 5 tolerant either to non-selective, broad-spectrum herbicides, i.e., glyphosate, roundup ® active ingredient and glufosinate, basta ® active ingredient, liberty ® and herbiace ® main ingredient, or specific herbicides such as imidazolinone, chlorsulfurone and sulfonylurea (28). in plants, encoded in the nucleus, the enzyme 5enolpyruvylshikimate-3-phosphate synthase (epsps) performs a main part in chloroplast responses arising in synthesis of phenylalanine, aromatic amino acids, tyrosine, and tryptophan. after finding in the early 1970s that perhaps the analog amino acid glyphosate [n-(phosphonomethyl)-glycine] could preferentially inhibit epsps activity, shut down aromatic amino acid synthesis and ultimately kill the plant, strategies are established to produce glyphosate-tolerant plants (or roundup ready ®). epsps occurs only in crops and certain microorganisms, not in livestock or humans, probably explaining why glyphosate toxicity is exceptionally small in human cells (29). to create glyphosate-tolerant sugar beet, two methods listed above i and ii) have been utilized. in the first scenario, a gene agrobacterium sp. was used for an altered protein, cp4-epsps, which is extremely glyphosate tolerant (30); in vitro assays and in vivo herbicide sprays (16) verified glyphosate tolerance. the second approach used a gene encoding achromobacter sp. glyphosate oxidoreductase, gox, and catalyzing glyphosate degradation in and out of non-toxic compounds, glyoxylate and aminomethylphosphonic acid. however, sugar beet plants were transformed in the second approach with both cp4-epsps nd gox genes (9) and evaluated with distinct roundup ® spraying systems in the greenhouse and field. extremely tolerant transformants have been acquired without phytotoxic or any other harmful phenotypic effects (31). inversely linked with the transgenic copy number (9), herbicide tolerance emerged. glufosinate (ammonium salt l-phosphinothricin, ppt) and bialaphos (ppt plus two alanines; l-phosphinothricinyl-l-alanyl-lalanine) are extremely toxic to plant cells; they behave as competing glutamine synthetase inhibitors that are critical to the transformation of glutamine acid and ammonia onto glutamine. inhibition contributes to toxic ammonia accumulation, leading to death of the cell. enzymes encoded in bars and pat and extracted from various streptomyces sp., detoxify ppt and have been broadly used as selectable markers for the production of transgenic ht plants. bar, motivated by the proponents of camv 35s, nos, tr1' or tr2, was used to achieve glufosinate-tolerant sugar beet crops (2) assessed in the sector of gene flow and agricultural efficiency research (32). like glyphosate, glufosinate has a very low toxicity to mammals (29). joersbo (33) assessed both glyphosate-and glufosinatetolerant sugar beets environmental efficiency, including economics and utilization flexibility. by deactivating the first enzyme in the pathway, acetolactate synthase (als), sulfonylurea compounds prevent the biosynthesis of clustered amino acids, valine, leucine and isoleucine. sugar beet has been transformed with a mutant sulfonylurea-insensitive als gene (2). in vitro cell selection (34) was also acquired from mutant sulfonylurea-and imidazolin one-tolerant sugar beet crops. however, many ht transgenic beet varieties have been approved for discharge in the u.s. (1996, 1998, 2005), canada (2001, 2005) and japan (2007) (26). roundup ready sugar beets were grown for the first season in 2008 by sugar beet growers in michigan. for ten years, michigan state university researchers have been collaborating with these varieties to determine implementation rates and timing and policies to delay glyphosate-resistant weed growth. references 1. lindsey k, gallois p. transformation of sugarbeet (beta vulgaris) by agrobacterium tumefaciens. j exp bot. oxford university press; 1990;41(5):529–36. 2. halluin k, bossut m, bonne e, mazur b, leemans j, botterman j. transformation of sugarbeet (beta vulgaris l.) and evaluation of herbicide resistance in transgenic plants. bio/technology [internet]. 1992;10(3):309–14 3. jacq b, lesobre o, sangwan rs, sangwan-norreel bs. factors influencing t-dna transfer in agrobacteriummediated transformation of sugarbeet. plant cell rep. springer; 1993;12(11):621–4. 4. krens fa, verhoeven ha, van tunen aj, hall rd. the use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation. vitr cell dev biol. springer; 1998;34(2):81–6. 5. zakharchenko ns, kalyaeva ma, bur’yanov yi, others. the method for genetic transformation of different sugar beet varieties. russ j plant physiol. 2000;47(1):70–5. 6. krens, trifonova a, keizer lcp, hall rd. the effect of exogenously-applied phytohormones on gene transfer sugar beet improvement using agrobacterium highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 4 of 5 efficiency in sugarbeet (beta vulgaris l.). plant sci. elsevier; 1996;116(1):97–106. 7. pedersen hc, steen p. the stability of transgenes inserted into sugarbeet (beta vulgaris l.). 58th congress international institute for beet research, paris (france), 19-22 jun 1995. 1995. 8. tenning p, bensefelt j, fouillard p, mannerloef m, tuvesson s. glyphosate tolerance in transgenic sugar beet. 58th congress international institute for beet research, paris (france), 19-22 jun 1995. 1995. 9. mannerlof m, tuvesson s, steen p, tenning p. transgenic sugar beet tolerant to glyphosate. euphytica. springer; 1997;94(1):83–91. 10. snyder gw, ingersoll jc, smigocki ac, owens ld. introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet (beta vulgaris l.) by agrobacterium or particle bombardment. plant cell rep. springer; 1999;18(10):829–34. 11. adawy ss, mokhtar mm, alsamman am, sakr mm. development of annotated est-ssr database in olive (olea europaea). int j sci res. 2015;4(9):1063–73. 12. mokhtar mm, adawy ss, el-assal se-ds, hussein eha. genic and intergenic ssr database generation, snps determination and pathway annotations, in date palm (phoenix dactylifera l.). plos one. public library of science; 2016;11(7):e0159268. 13. wozniak ca, owens ld. transformation of sugarbeet cell suspension cultures mediated by agrobacterium tumefaciens. j cell biochem suppl d. 1989;13:272. 14. vitha s, phillips jp, gartland js, gartland kma, beneš k, elliott mc. activity of β-glucuronidase in root tips of different types of transgenic sugar beet plants. biol plant. springer; 1998;40(4):531–41. 15. ivic sd, smigocki ac. transformation of sugar beet cell suspension cultures. vitr cell dev biol. springer; 2003;39(6):573–7. 16. fry je, barnason ar, hinchee m. genotypeindependent transformation of sugarbeet using agrobacterium tumefaciens. molecular biology of plant growth and development, third international congress of plant molecular biology, 6--12 october 1991, tucson, az, usa. 1991. p. 384. 17. zhang c-l, chen d-f, elliott mc, slater a. thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (beta vulgaris l.). vitr cell dev biol. springer; 2001;37(2):305–10. 18. norouzi p, malboobi ma, zamani k, yazdi-samadi h. using a competent tissue for efficient transformation of sugarbeet (beta vulgaris l.). vitr cell dev biol. springer; 2005;41(1):11–6. 19. hisano h, kimoto y, hayakawa h, takeichi j, domae t, hashimoto r, et al. high frequency agrobacteriummediated transformation and plant regeneration via direct shoot formation from leaf explants in beta vulgaris and beta maritima. plant cell rep. springer; 2004;22(12):910–8. 20. kishchenko em, komarnitskii ik, kuchuk n v. production of transgenetic sugarbeet (beta vulgaris l.) plants resistant to phosphinothricin. cell biol int. wiley online library; 2005;29(1):15–9. 21. bekheet sa. a synthetic seed method through encapsulation of in vitro proliferated bulblets of garlic (allium sativum l.). arab j biotech. 2006;9:415–26. 22. joersbo m, mikkelsen jd, brunstedt j. relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet. mol breed. springer; 2000;6(2):207–13. 23. joersbo m, guldager petersen s, okkels ft. parameters interacting with mannose selection employed for the production of transgenic sugar beet. physiol plant. wiley online library; 1999;105(1):109–15. 24. luo y, guo z, li l. evolutionary conservation of microrna regulatory programs in plant flower development. dev biol. 2013;380(2):133–44. 25. may mj, wilson rg. weeds and weed control. sugar beet. wiley online library; 2006;359–86. 26. james c. global status of commercialized biotech/gm crops, 2011. isaaa ithaca, ny; 2011. 27. lathouwers j, weyens g, lefebvre m. transgenic research in sugar beet. adv sugar beet res genet modif sugar beet int inst beet res brussels. 2005;5–24. sugar beet improvement using agrobacterium highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 5 of 5 28. yang x, scheffler be, weston la. recent developments in primer design for dna polymorphism and mrna profiling in higher plants. 2006;10:1–10. 29. tomlin cds. the pesticide manual, british crop protection council, alton. hampshire, united kingdom. 2003; 30. hinchee maw, padgette sr, kishore gm, delannay x, fraley rt. herbicide-tolerant crops. in “transgenic plants vol. 1” kung sd and r. wu. academic press, inc., san diego; 1993. 31. brants i, harms h. herbicide tolerant sugar beet. comptes-rendus des congres de l’institut international de recherches betteravieres (belgium). 1998. 32. meier p, wackernagel w. monitoring the spread of recombinant dna from field plots with transgenic sugar beet plants by pcr and natural transformation of pseudomonas stutzeri. transgenic res. springer; 2003;12(3):293–304. 33. joersbo m. chimeric genes for selection systems and reporter makers. bentham science publishers: oak park; 2011. 34. ming r, vanburen r, wai cm, tang h, schatz mc, bowers je, et al. the pineapple genome and the evolution of cam photosynthesis. nat genet. 2015;47(12). highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20202 highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 5 research article open access 1 department of nucleic acid and protein structure, agricultural genetic engineering research institute (ageri), arc, giza, egypt. 2 department of microbiology, faculty of science, ain shams university, 11566, abbassia , cairo, egypt. contacts of authors * to whom correspondence should be addressed: ghada abu el-heba citation: hassan a.h., a-bouzeid m., and abu el-heba g. (2020). dna variations between medicago truncatula symbiotic mutant line and native variant using fluorescence-based aflp marker, volume 3. article id 20202, dio:10.36462/h.biosci.20202 received: march 3, 2020 accepted: march 20, 2020 published: april 1, 2020 copyright: © 2020 hassan et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. dna variations between medicago truncatula symbiotic mutant line and native variant using fluorescencebased aflp marker asmaa h. hassan 1 , mohamed a-bouzeid 2 , and ghada abu el-heba * 1 abstract genetic mutagenesis is a very efficient tool in studying genes function. because of great benefits of legumes as human food and animal feed worldwide, we used a model plant medicago truncatula for identification gene function related to nitrogen fixation process. our mutant is a medicago mutant line contains a tobacco tnt1 retrotransposon mobile element with the two long terminal repeats (ltr) inserted within the genome. our mutant is predicted to contain a mutation in gene/s belonging to symbiotic interaction between legume and rhizobia. a novel technique was used based on using fluorescent oligonucleotide primers against oligonucleotide primers for tnt1-ltrs of our mutant. this novel protocol was very successful in detection the polymorphism between our mutant line and the wild variant r108 using biosystems 310 genetic analyzer. electropherograms of the mutant line and wild type gave a total of 561 wellresolved aflp peaks, 357of which were polymorphic peaks and 204 were monomorphic peaks. this novel technique enables the calculation percentage of polymorphism between the mutant line and the wild type. additionally primers combinations amplified more bands from others to detect polymorphism between the plants. keywords: medicago truncatula, aflp, polymorphism, symbiotic mutant, nitrogen fixation. introduction legumes played vital roles in improving agriculture and considered as onethird of the world’s major crop production. they represent about 27% of the world’s crop production. human consumes approximately 33% of the dietary protein needs in the form grains. many food industries based on legumes like treated vegetable, oil production, and chicken industry. animals depend on forage legume plants as major source of food like medicago stavia and trifolium spp (1). this group of plants has unique character as they have ability to make symbiotic relationship with soil bacteria called rhizobia. rhizobia are beneficial soil bacteria that belong to gram negative group (2) . in this relation legumes can fix nitrogen in specific organ called root nodule (1,3). nitrogen fixation process occurs naturally during growing this family of plants and so we can decrease our usage of industrial nitrogen fertilizers and related ecosystem pollution (1). many studies focused on genes responsible for nitrogen fixation in legumes and rhizobia (4-9). lotus japonicus and medicago truncatula are considered as more suitable two plants for studying genes responsible for symbiotic relationship between both organisms. hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 5 numerous technological platforms like; ethyl methanesulfonate (ems) mutagenesis (10), fast neutron bombardment and g-rays (11) were developed and used for model plants genome studying. dna-insertion mutantgenesis populations save a rapid method for isolating genes responsible for symbiotic nitrogen fixation through both reverse and forward genetics (12-14). retrotransposon is a type of mobile elements (class i) that transpose to new sites in the genome via an rna intermediate. their primary transcripts are retro-transcribed into dna copies by a reverse transcriptase (15). tobacco retrotransposon tnt1 is an effective mutagen in m. truncatula that is used for molecular studies worldwide (16). this transposone element (tnt1) belongs to retrotransposon elements family (17) , its transposition was induced during tissue culture of tobacco protoplast(18) and during in vitro transformation of a. thaliana. tnt1 insertions of are stable in the plant and transfer to the progeny because of the replication cycle of ltr retrotransposons (19). samuel roberts noble foundation has the largest collection of dna-insertion mutants of all legumes specially tnt1-insertion mutant populations in medicago truncatula. numerous mutant phenotypes have been screened such as mutant loss the ability to form nodules, mutants produce dysfunctional nodules, mutants produce large number of nodules and mutants with delayed and decreased nodulation but efficient in nitrogen fixation (9). among molecular technique, aflp protocol provides a very powerful dna finger printing method for nucleic acid of any organism (20). fluorescence-labeled pcr products were sensed on an automated dna sequencer instead of the standard polyacrylamide gel electrophoresis tool (21-22). capillary electrophoresis has more advanced characters from slab gels in speed, resolution and availability of numerical information in an electronic format following the end of a reaction. in this study, the genetic polymorphism between medicago truncatula symbiotic mutant line and the wild type of the same plant has been estimated using amplified fragment length polymorphism (aflp) marker analyses. this mutant contains tnt1 reterotrasposon element that have mutation in the gene/s involving in nitrogen fixation process. materials and methods plant materials the mutant line of medicago truncatula was provided from noble foundation tnt1 mutant collection that transformed from m. truncatula r108-1 ecotype (23). the wild type plant r108has been used in this study as a reference. seed sterilization and germination seeds of medicago truncatula wild type and mutant line were scratched with sand paper and surface sterilize with 6.25% (v/v) sodium hypochlorite for 10 minutes. after seven washes using sterilize water, seeds were put on murashige and skoog (ms) basal medium (murashige and skoog 1962) supplemented with 0.8% agar (grade j3, gelita) in dark over night at 29° c. seedlings were transferred to growth chamber with this condition at stable 21° c during a 16-h and 8-h night photon flux density of 100 ϻ mol m -2 s -1 (24). dna isolation and quantification total genomic dna was extracted from mutant plants and control for using in aflp analysis. healthy leaves from both line were used for dna isolation using qiagen genomic dna isolation kit, (qiagen science,valence , ca , usa). the quality and quantity of genomic dna were estimated using spectrophotometric measurement of ultra violet absorbance at wave length 260 and 280 nm in thermo scientific nanodrop 2000™) and were electrophoresed at 0.5% agarose gel electrophoresis. aflp protocol a) genomic dna digestion and ligation amount of genomic dna from mutant line and wild type were digested with 10u ecor1 and 10u mfe1 (new england biolabs, ipswich, ma, usa). the total volume was incubated for three hrs at 37°c then the enzymes were inactivated by incubation for 20 mins at 65°c. to assess the digestion, 5 μl from each sample were electrophoresed on a 1.5 % agarose gel. the digested fragments dna were ligated by eco and ase adaptor ligation using t4 dna ligase (new england biolabs) to produce modified digested fragments that were used as a template dna for amplification. c) pre-selective amplification reaction the reaction was performed on ligated dna using two aflp oligonucleotide primers using takara la taq kit. then the product was run on 1.5% agarose gel. d) selective amplification reaction selective amplification was performed using two aflp oligonucleotide primers. the first one was fluorescence-labeled primer: eco (dye-primer-axx) and unlabeled primer (ltr4&ltr6). three primer combinations were used: e-acc (ned)/ltr4, e-acc (ned)/ltr6, e-aag (6-fam)/ltr4, e-aag (6-fam)/ ltr6, e-aac (hex)/ltr4 and e-aac (hex)/ltr4. aflp procedure was applied according to (25) with a modification which is using three florescence labeled oligonucleotide primers (e-aac, e-aag, and e-acc) (20). 1ul of selective amplified product was added to 12ul hi-di formamide and 0.5 microliter of genescan500 rox internal size standard (applied biosystems, foster city, california, usa). the mixture of these components was denatured and inoculated to the single capillary of applied biosystems 310 genetic analyzer. gene mapper analysis software version 4.1 (applied biosystems) was used for analyzing the generated electropherograms for mutant and r108 from the raw date according to aflp system analysis getting started guide. hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 5 results pre-selective amplification was achieved using preselective oligonucleotide primer pair and the product was run on 1.5% agarose gel. successful pre-selective amplification was confirmed as a smear product that was interrupted by a number of bands and the polymorphism cannot be detectable at this stage. aflp electropherograms of mutant line and wild type selectively amplified using primer combination as illustrated in (table 1). oligonucleotide primer combinations examined for selective amplification of dna fragments of mutant line and wild type gave a total of 561 wellresolved aflp peaks of which 357 were polymorphic peaks and 204 were monomorphic peaks. the highest number of amplified dna fragments was 218 peaks were obtained with primer combination (eagg/ltr4); however the lowest number of amplified dna fragments was 31 peaks from combination (e-acc/ltr6). aflp electropherograms of both lines were produced using the illustrated primer combination e-acg/ltr4 (as an example) was shown in (figure 1). table 1: showing that the total number of peaks (tnp), number of polymorphic peaks (npp) and number of monomorphic peaks (nmp) were produced by each primer combination (pc). discussion legumes have a fundamental role in sustainable agriculture for decades. they are able to reduce the atmospheric n2 to ammonia through symbiosis interaction with rhizobia which is nitrogen-fixing bacteria. this criterion enables legumes to propagate well in soils poor with nitrogen without any fertilizer addition. symbiotic nitrogen fixation (snf) in legumes produces nitrogen about fifty million tons into the agricultural systems every year. both legumes and non-legumes production is sustained by such nitrogenous compound in the soil (21). legumes are essential source for food and feed (1). in our research we used medicago truncatula which is a model plant for studying snf because it has a small diploid genome, short life cycle and it performs symbioses with (sinorhizobium meliloti and sinorhizobium medicae). mutant collections production was necessary to identified genes function. large scale mutagenesis was generated in m. truncatula via tnt1 retrotransposon (23). tnt1 is a ty1copia type retrotransposon with long terminal repeat (ltr), isolated from tobacco (26). tnt1 transpose via copy & paste mechanism . each mutant line has from four to fifty insertions per genome (23). retrotransposon mediated mutagenesis was used in the other plant species like rice (27), lettuce (19) , and soybean (28). samuel roberts noble foundation has produced about 21,000 tnt1 mutant lines (23). we used aflp-type protocol in combination with abi 310 genetic analyzer to determine the genetic polymorphism between our mutant line which is putatively defects in nodule development and r108 wild type. figure 1: aflp electropherograms of r108 and mutant line amplified selectively by the primer combination e-aag/ltr6. red row indicates a polymorphic peak example that is present in mutant line and absent in r108. hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 5 aflp electropherograms of both lines produced a total of 561 aflp peaks were recorded, 357 of which were polymorphic and 204 were monomorphic .this corresponds to a level of polymorphism of 63.7%. [the percentage of polymorphism was calculated as [(the total no. of polymorphic peaks / the total no. of peaks) x 100]. aflp has the capability to discover the polymorphisms between these types of plants without knowing the sequence of genome. so, this is one of the best advantages of using aflp technique. the established website (http://bioinfo4.noble.org/mutant/) encloses information about 11,000 tnt1 mutant lines with images of diverse phenotypes. our perspective work will include aflp-type pcr to isolate all the tnt1-flanking sequence tags (fts) for more mutant characterization and identification. all fragments will be analyzed and the mutated gene/s will be recognized. acknowledgement the authors appreciate dr. pascal ratet for providing mutant line & r108 wild type. references 1. graham ph, vance cp. legumes : importance and constraints to greater use update on legume utilization legumes : importance and constraints to greater use. plant physiol. 2003;131:873–7. 2. zakhia f, de lajudie p. taxonomy of rhizobia. edp sci. 2001;21:569–76. 3. smil. nitrogen in crop production : an account of global flows adds recycled in organic up by harvested and quantification of n losses from crop to 26-60. glob biogeochem cycles,. 1999;13(2):647–62. 4. fuhrmann ‎‎m. hennecke h. rhizobium japonicum nitrogenase fe protein gene (nifh). j bacteriol. 1984;158(3):1005–11. 5. roche p. maillet f. plazanet c. debellé f.ferro m et al. the common nodabc genes of rhizobium meliloti are host-range determinants. proc natl acad sc. 1996;93(december):15305–10. 6. radutoiu s. madsen lh.madsen eb. felle hh. umehara y. grønlund m.et al. plant recognition of symbiotic bacteria requires two lysm receptor-like kinases. nature. 2003;425:585–92. 7. arrighij-f, barre a, benamorb, bersoulta, soriano lc, mirabella r, de carvalhoniebel f, journet e-p, ghérardim, huguet t et al. the medicago truncatula lysine motif-receptor-like kinase gene family includes nfp and new. plant physiol. 2006;142:265– 79. 8. mergaert p, uchiumi t, benoıt a e, cheron a, catrice o, mausset a-e, et al. eukaryotic control on bacterial cell cycle and differentiation in the rhizobium – legume symbiosis. pnas. 2006; 9. pislariu ci d. murray j, wen j, cosson v, muni rrd, wang m, et al. a medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation. plant physiol [internet]. 2012;159(4):1686–99. 10. penmetsa rv, cook dr. production and characterization of diverse developmental mutants of medicago truncatula 1. plant physiol. 2000;123(august):1387–97. 11. rogers c, wen j, chen r, giles o. deletion-based reverse genetics in. breakthr technol deletionbased. 2009;151(november):1077–86. 12. tadege m, ratet p, mysore ks. insertional mutagenesis : a swiss army knife for functional genomics of medicago truncatula. trends plant sci. 2005;10(5):229–35. 13. young nd, udvardi m. translating medicago truncatula genomics to crop legumes. 2008;12:193201. 14. urban´ ski d f, małolepszy a, j stougaard , andersen su. genome-wide lore1 retrotransposon mutagenesis and high-throughput insertion detection in lotus japonicus. plant j. 2012;69:731–41. 15. wessler sr. transposable elements and the evolution of eukaryotic genomes. nat rev genet. 2006;103(47):17600–1. 16. erfurth i, cosson v, eschstruth a, lucas h, kondorosi a, ratet p. ef ficient transposition of the tnt1 tobacco retrotransposon in the model legume medicago truncatula. plant j. 2003;34(1):95–106. 17. grandbastien, marie-angele as& mc, laboratoire. tntl, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. nature,. 1989;337:5–9. 18. pouteau s, huttner e, grandbastien m a, caboche m. specific expression of the tobacco tntl in protoplasts. embo. 1991;10(7):1911–8. 19. mazier m, botton e, flamain f, bouchet j-p , courtial ´, et al. successful gene tagging in lettuce using the tnt1 retrotransposon from tobacco. 2007;144(may):18–31. 20. thom‎as cm, vos p, zabeau m, jones da, norcott ka, chadwick bp, et al. identification of amplified restriction fragment polymorphism (aflp) markers http://bioinfo4.noble.org/mutant/ hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 5 tightly linked to the tomato cf‐ 9 gene for resistance to cladosporium fulvum. plant j. 1995;8(5):785–94. 21. weising k, nybom h, wolff k kg. dna fingerprinting in plants: principles, methods, and applications, 2nd edn. boca raton: crc press. 2005. 472-477 p. 22. karudapuram s, larson s. identification of hedysarum varieties using amplified fragment length polymorphism on a capillary electrophoresis system. j biomlecular tech. 2005;16(4):316–24. 23. tadege m, wen j, he j, tu h, kwak y, eschstruth a, et al. large-scale insertional mutagenesis using the tnt1 retrotransposon in the model legume medicago truncatula. plant j. 2008;54:335–47. 24. shao c, russinova e, iantcheva a, atanassov a, mccormac a, chen d, et al. rapid transformation and regeneration of alfalfa ( medicago falcata l .) via direct somatic embryogenesis. 2000;31:155–66. 25. ratet p, porceddu a, mysore ks. insertional mutagenesis in m . truncatula using tnt1 retrotransposon insertional mutagenesis in medicago truncatula using tnt1 retrotransposon . 2010;1-12. 26. kumar, bennetzen jl. plant retrotransposons. annu rev genet. 1999;33:479–532. 27. greco r, ouwerkerk pbf, sallaud c, kohli a, colombo l, puigdome p, et al. resources and opportunities transposon insertional mutagenesis in rice 1 , 2. plant physiol. 2001;125:5–7. 28. cui y, barampuram s, stacey mg, hancock cn, findley s, mathieu m, et al. tnt1 retrotransposon mutagenesis : a tool for soybean. 2013;161:36–47. highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 1 of 5 citation: rslan w. m. (2018) sugar beet artificial seeds an overview. highlights in bioscience, volume 1.article id 20181, dio:10.36462/ h.biosci.20181 received: march 22, 2018 accepted: june 17, 2018 published: june 26, 2018 copyright:© 2018 rslan. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. sugar beet artificial seeds an overview wessam m. rslan* agricultural genetic engineering research institute; agricultural research center, egypt. *to whom correspondence should be addressed: wessam.rslan@ageri.sci.eg abstract artificial seed propagation of crops broadens the horizon of plant biotechnology and farming. the technology offers techniques for micropropagulated seed analogs such as axillary leaves, embryogenic calli, somatic embryos, apical shoot tips, and protocorm-like organs. micropropagules are embedded in gelling medium and carboxyl methyl cellulose active coatings. a variety of plant species, such as mulberry, sandalwood, cardamom, banana, sugar beet, maize, and relative, have recorded encapsulation of micro shoots and somatic embryos and subsequent recovery of full plantlets. this knowledge has shown that artificial seed manufacturing is possibly helpful for the propagation of economically significant species ' inferior hybrids on a big scale. artificial seed development can only succeed with effective upstream manufacturing of micropropagules and downstream germination procedures for an elevated proportion of plant regeneration as one of the significant value-added plant tissue culture goods. different micropropagules were regarded for the manufacturing of artificial seeds; however, mostly favored were somatic embryos and axillary stem buds. as micropropagules, somatic embryos were used to create artificial seeds in a wide range of fruit and plant organisms, which include daucus carota, picea abies, arachis hypogaea, medicago sativa, psidium guajava, and vitis vinifera. the review illustrated the concept of synthetic seeds and encapsulation procedure of sugar beet. keywords: artificial seed, plant biotechnology, embryogenic, sugar beet, micropropagulated. review article open access highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 2 of 5 introduction ara et al. (1) described artificial plants as synthetically encapsulated somatic embryos, shoot tips, axillary flowers or another meristematic tissue employed for seed sowing and capable of converting into whole plants under in vitro and in vivo circumstances and maintaining their potential even after storage. the first one to suggest and handle encapsulated somatic embryos was murashige (2). he also noted to the transfer option and used it as natural plants. reddy et al. (3) clarified that the cultivation of plants using artificial plants from somatic embryos or other vegetative propagules opens up new agricultural and forestry technologies. a variety of plant species synthetic seeds have been effectively researched in plant propagation. encapsulating agents kitto and janick (4) discovered that the most appropriate medium for the encapsulation of somatic embryos was' polyox,' water-soluble resin. redenbaugh et al. (5) suggested that magnesium alginate should be the most appropriate to encapsulate somatic embryos in alfalfa, celery, cauliflower and rose. artificial seed performance relies on the spatial, qualitative, quantitative availability of growth regulators and nutrients together with an ideal physical setting (6). mariani (7) indicated that gibberellic acid (ga3) and saccharose had an adverse impact on eggplant germination of synthetic seeds. in the meantime refouvelet et al . (8) used ba (5 mg / l) + 1⁄2 ms + naa (0.01 mg / l) to encapsulate syringa vulgaris. pattnaik and chand (9) axillary buds, murashige and skoog (10) medium (ms) without hormones and ms + 6-benzyladenine (ba, 4.4 μm) have been used as artificial endosperm in morus species. saiprasad (11) revealed that sodium alginate would be the most widely recognized hydro-gel and was regularly used as a matrix for artificial plants due to its small toxicity, low price, rapid gellation and bio-compatibility. many researchers noted that the introduction to the encapsulation solution of aquatic cyanobacterial samples (12), bactericides and activated carbon (13), pesticides, fertilizers, microorganisms (rhizobia), mycorrhiza fungi (14), fungicides (15) can safeguard encapsulated propagules from microorganisms, decrease the discharge of toxic compounds and improve the germination ability of seeds. encapsulation procedure the hydro-gel encapsulation technique established by (5) was the most suitable technique for producing synthetic seeds. in this technique, by combining with calcium free liquid ms medium, sodium alginate of varying levels (2 to 5 percent) was formed and then the explants were blended with the solution. together with the sodium alginate solution, explants were sucked with a pipette and dropped into calcium chloride pool in which the ion exchange reaction happens and sodium ions were substituted by calcium ions comprising alginate beads. it is necessary to complete the entire method under aseptic circumstances. the capsule size relies on the pipette nozzle's inner diameter. the beads structure and size relies on sodium alginate quantity, calcium chloride solutions, and complexion duration. redenbaugh (16) proposed using a multi nozzle pipette in this embryo stream through the internal pipette and the solution of the alginate flows via the exterior pipette. artificial seeds germination and field planting several researchers (17–20) recorded successful field cultivation and transformation of natural plants. the synthetic plants in the future, particularly for the extremely requested species (21), may be an option planting material intended for the forestry sector. artificial plants would enable plant propagules to be planted directly into the greenhouse or field, circumventing many of the additional phases (21). fujii et al. (22) discovered that the maturation of aba somatic alfalfa embryos yielded an elevated soil transformation rate of 48% to 64%. adding fungicide to alginate beads avoids contamination and increases the sustainability of mulberry seeds in soil (23) when sown. fujii et al. (24) revealed effective field planting with 23 percent crop transformation of alfalfa artificial plants obtained from calcium alginate embryoids. nieves et al. (25) revealed that artificial plant sugarcane crops were larger and had a lower diameter at eight months, but at 12 months these distinctions faded. no variations in all parameters assessed among both artificial seed-derived plants and plants based from the other two techniques (traditional and isolated plant techniques) were discovered with regard to sugar assessment and yield. asmah et al. (21) and ma et al. (26) recorded the efficient germination level in acacia hybrid (73.3 to 100%) and pseudostellaria heterophylla (80%). in podophyllum peltatum, sugar beet and stevia rebaudiana, rizkalla et al. (27) and nower (28) noted that crop development improved by adding mannitol and/or sorbitol to the medium. types of synthetic seeds two forms of synthetic seeds have been established, i.e. desiccated and hydrated synthetic plants, according to the current literature. the synthetic desiccated seeds were first launched either directly or encapsulated in polyox from somatic embryos, followed by their desiccation (29). highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 3 of 5 desiccation has been accomplished either linearly through chambers of decreasing relative humidity slowly over a period of one or two weeks or swiftly by leaving the petri dishes overnight on the bench at the laminar airflow room (30). the hydrated artificial seed technology was first developed by encapsulating medicago sativa (16) hydrated somatic embryos. these artificial hydrated seeds are employed to create plant species that are recalcitrant and susceptible to desiccation in their somatic embryos. hydrated artificial seeds are usually ready in a hydrogel capsule by encapsulating somatic embryos or other propagules. several techniques for producing hydrated artificial seeds were investigated, mostly using calcium alginate encapsulation (16). the genetic stability of synthetic seeds artificial seeds were commonly used in many plant species for micro-propagation. molecular researches have began from the last decade to determine genetic stability of plantlets derived from synthetic seeds, but no changes have been revealed at the biochemical and/or molecular scales. many studies (31) endorsed the prospective benefit of synthetic seeds for genetically identical to natural seeds. gangopadhyay et al. (32) investigated the genetic structure of plantlets obtained from encapsulated ananas comosus micro shoots using rapd and issr technologies (33, 34). bekheet (35) indicated that both plantlets obtained from encapsulated bulblets and usually in vitro were genetically comparable to those obtained from in vivo in allium sativum. narula et al. (36) used rapd assessment to explore in vitro plantlet genetic structure obtained from dioscorea bulbifera encapsulated plant advice. srivastava et al. (37) reported that the analysis of cineraria maritana's rapd patterns revealed a median ratio of resemblance of 0.944, confirming the molecular consistency of crops extracted from encapsulated micro-shoots followed by six months of storage. tabassum et al. (38) investigated the genetic consistency of synthetic seeds obtained from mother crops and somatic embryos and discovered similarity in cucumis sativus using rapd markers. mishra et al. (39) also studies the genetic consistency of crops obtained from encapsulated microshoots in picrorhiza kurrooa using rapd profile cluster analysis. lata et al. (40) used issr and gas chromatography (gc) study of six significant cannabinoids to examine the genetic structure of synthetic seed based crops of cannabis sativa and demonstrated homogeneity in the regrown clones and the mother plant. shoot tips are by far the most genetically consistent, but in callus and protoplast culture there is a strong probability of genetic shift (41–43). references 1. ara h, jaiswal u, jaiswal vs. germination and plantlet regeneration from encapsulated somatic embryos of mango (mangifera indica l.). plant cell rep. springer; 1999;19(2):166–70. 2. murashige t. plant cell and organ cultures as horticultural practices. symposium on tissue culture for horticultural purposes 78. 1977. p. 17–30. 3. naga blri, mangamoori ln, subramanyam s. identification and characterization of est-ssrs in finger millet (eleusine coracana (l.) gaertn.). j crop sci biotechnol. springer; 2012;15(10):9–16. 4. kitto sl, janick j. hardening treatments increase survival of 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transformation and plant regeneration via direct shoot formation from leaf explants in beta vulgaris and beta maritima. plant cell rep. springer; 2004;22(12):910–8. 43. krishna h, alizadeh m, singh d, singh u, chauhan n, eftekhari m, et al. somaclonal variations and their applications in horticultural crops improvement. 3 biotech. springer; 2016;6(1):54. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20221 highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 4 review article open access department of biomedical sciences, college of health sciences, qatar university, doha, qatar. contacts of authors * to whom correspondence should be addressed: ibrahim mustafa citation: roan zaid and ibrahim mustafa (2020). the distribution of the abo and rh blood groups among different populations in the mena region: a review. highlights in bioscience volume 3. article id 20221. dio:10.36462/h.biosci.20221 received: june 13, 2020 accepted: august 18, 2020 published: september 10, 2020 copyright: © 2020 zaid and mustafa. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. the distribution of the abo and rh blood groups among different populations in the mena region: a review roan zaid and ibrahim mustafa* abstract abo and rh (rhesus) blood grouping is one of the most widely available laboratory tests that could prevent possibly deadly mismatches following blood transfusion and organ transplantation. the distribution of the blood group antigens varies between different populations and ethnic groups. our objective was to showcase these variations within the middle east and north africa (mena) region and to outline the relationship between blood type and disease risk association to determine the current findings and outline possible future study areas. in healthy blood donors, blood group o was found to consistently be the most prevalent blood group and ab the least prevalent blood group except in turkey where a is the most prevalent and, in the united arab emirates (uae), where b is the least prevalent blood type. keywords: abo blood groups, rh blood groups, blood donors introduction according to the international society of blood transfusion (isbt), there are 33 blood group systems that represent more than 30 antigens as of date [1, 2]. the four common blood group antigens (a, b, ab and o) of the abo system, along with the d (rhesus/rh) and kell antigens are the most important in blood transfusions and organ transplantation and are commonly tested due to their clinical significance [3]. the abo gene codes for glycosyltransferases that transfer complex oligosaccharide antigens to the h antigen on the surface of red blood cells (rbcs). n-acetylgalactosamine and galactose are transferred in the case of a and b blood groups, respectively. individuals with the o blood group do not produce the glycosyltransferase due to a single point deletion (guanine-258) and, as a result, lack these antigens on the surface of their rbcs [4, 5]. the distribution of the abo/rh blood groups among different populations within the mena region as well as some the diseases that are associated with these blood groups will be addressed in this review. the distribution of the abo blood groups in the mena region ever since the discovery of the abo blood groups by landsteiner in 1900, the studies on human population genetics were on the rise [6]. blood group typing entails finding the antigenic properties of rbcs. these antigenic properties are found to vary between different racial/ethnic groups. population and prevalence studies are therefore important to help elucidate the distribution of the abo/rh blood groups and to help prevent possibly deadly mismatches before blood transfusions or organ transplantation [7]. table 1 shows the distribution of the abo and rh blood groups among different populations in the mena region. https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20221 https://doi.org/10.36462/h.biosci.20221 http://bioscience.highlightsin.org/ zaid and mustafa, 2020 the distribution of the abo and rh blood groups in the mena region highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 4 in healthy blood donors, blood group o was consistently found to be the most prevalent and ab the least prevalent blood group. turkey and the uae were the only exception where a was found to be the most prevalent in turkey and b was the least prevalent in the uae. blood group antigens and disease association abo blood groups have been statistically associated with many diseases including diabetes, venous thromboembolism, ovarian cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer and other gastrointestinal cancers [15, 18-22]. however, reports have been variable and inconsistent between studies. some of these diseases and their associated blood group antigens will be addressed. venous thromboembolism and blood group antigens one of the most described associations between non-o blood groups (a, b, and ab) and other diseases is that of venous thromboembolism (vte). multiple studies have established a relationship between the abo blood groups and its influence on normal haemostasis [19, 23]. this relationship is attributed to the expression of the abo antigens on the surface of other human cells, including cells of the epithelium, vascular endothelium and platelets [24]. in addition, the blood group antigens largely determine the plasma levels of the von willebrand factor (vwf) where vwf levels are reported to be 25% higher in non-o blood groups [23, 25-27]. elevated vwf would prompt platelet adhesion as well as maintain the coagulation factor ⅷ (fⅷ) in the plasma increases the risk of vte [24]. it is also important to note that individuals of the a2 blood groups are reported to have low levels of the vwf and fⅷ. the risk of developing vte in these individuals is said to be lower when compared to the other blood groups (a1, b and ab) [19]. in concordance with this phenomenon, higher risk of myocardial infraction and ischemic strokes were also previously described in individuals with blood group a and ab [28]. table 1. distribution of the abo and rh blood groups among different populations within the mena region. type 2 diabetes mellitus and blood group antigens a study done in qatar revealed that type 2 diabetes mellitus (t2dm) patients are significantly more likely to be of blood type b as compared to healthy individuals (25.7% vs. 20.4%; p < 0.001) (table 1). in the same study, blood group o was significantly higher in healthy individuals than in t2dm patients (38.5% vs. 45.4%; p < 0.001) [10]. a study done in kingdom of saudi arabia (ksa) found a 52.2% decrease (2.1 fold reduction) in rh (-) blood groups in t2dm female patients as compared to healthy controls (4.21% vs. 8.8%; p = 0.043) [12]. similar findings were reported for male t2dm patients in ksa where males had a 3.4 fold reduction in rh (-) blood group in comparison to healthy controls (3.13% vs. 10.6%; p = 0.028) [13]. blood group b was higher in both male and female t2dm patients in ksa, however, these findings were not statistically significant [12, 13]. in iraq, it was reported that blood group o individuals had significantly higher levels of total cholesterol, glucose and blood pressure in comparison to other blood groups (p < 0.01). these parameters showed a decreasing trend starting from group o to a to b and then ab [22]. although all the reported results were of high significance in all populations, it is clear that these findings cannot be extrapolated to other populations due to the conflicting reports. these variations between populations could be explained by the multifactorial nature of t2dm. cancer and blood group antigens multiple studies consistently report that individuals with the o blood group have reduced risk of developing pancreatic cancer than non-o individuals [4, 20, 29, 30]. one study reported that the risk of pancreatic cancer increases with each addition of non-o allele. in this study, individuals with ao and aa genotypes had odds ratios (ors) of 1.33 and 1.66 respectively when compared with individuals of the oo genotype. whereas bo and bb individuals had ors of 1.45 and 2.42, respectively [30]. it was also recently reported that the genetic variant of the abo gene, rs505922 (t allele), which is said to confer protective properties against pancreatic cancer, is in complete linkage disequilibrium (r2= 1.0) with the guanine-258 deletion that encodes the o allele [4, 31]. this variant entails a single nucleotide polymorphism in the first intron of the abo gene. comparable findings were reported for hepatocellular carcinoma (hcc) where blood group o was found to improve the disease’s prognosis after hepatectomy [21]. country population studied sample size a (%) b (%) ab (%) o (%) rh+ (%) rh(%) ref. iraq blood donors 5,000 32.6 22.8 7.6 37.0 91.3 8.7 [8] pakistan blood donors 3679 31.87 21.71 12.22 34.2 88.86 11.14 [9] qatar blood donors t2dm patients 1,650 1,633 27.6 29 20.4 25.7 6.5 6.8 45.4 38.5 [10] [10] saudi arabia blood donors male blood donors t2dm female patients t2dm male patients 57,396 944 214 128 26.0 33.4 24.8 (a+) 22.6 (a+) 18.0 6.0 27.1 (b+) 30.5 (b+) 4.0 3.8 7.0 (ab+) 4.7 (ab+) 51.0 56.8 36.9 (o+) 39.1 (o+) 92.0 92.8 95.8 96.88 8.0 7.2 4.21 3.13 [11] [7] [12] [13] sudan blood donors 3,087 19.14 14.0 66.8 74.4 25.6 [14] turkey blood donors blood donors lung cancer patients 3,022,883 86,797 2,044 42.2 44 43.9 16.4 16.2 17.3 7.5 6.5 8.1 33.9 33.3 30.7 87.7 88.2 86.2 12.3 11.8 13.8 [15] [16] [15] united arab emirates blood donors 661 24.0 22.8 31 48.4 91 8.9 [17] http://bioscience.highlightsin.org/ zaid and mustafa, 2020 the distribution of the abo and rh blood groups in the mena region highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 4 studies conducted by wu et al., revealed the mean overall survival (os) of hcc after hepatectomy to be 55, 39, 34 and 34 months for blood groups o, a, b and ab, respectively. the overall hazard ratio for non-group o individulas in this study was 1.485 (95% ci, 1.204–1.830; p < 0.001) [21]. studies done by urun et al. in turkey found that having o and rhblood decreased the risk of lung cancer by 14% and 13% respectively [15]. however, another study, also done in turkey, showed no correlation between blood groups and lung cancer status of patients [32]. finally, zhang et al. compared findings for 89 studies with 30 different cancer types. for these studies, the pooled or for a blood groups vs. non a blood groups was 1.12 (95%ci: 1.09-1.16) showing a significantly increased risk of blood group a. in the other hand, individuals with the o blood group had a significantly reduced risk of cancer compared to other blood groups with an or of 0.84 (95%ci: 0.80-0.88) [33]. in all the reported cases, the exact mechanism by which blood group o causes a risk reduction is not fully understood. however, many possible explanations were suggested. for instance, the intracellular adhesion molecule-1 (sicam-1) levels were found to be higher in group o individuals [34]. it is possible that the reduced levels of sicam-1 promote tumor metastasis in non-group o individuals [35]. furthermore, the single nucleotide polymorphism (snp) of the abo gene (rs505922) was found to be associated with higher levels of tumor necrosis factor (tnf) levels. [31] these findings could explain the relationship between specific blood groups and carcinogenesis [36]. conclusion in conclusion, an understanding of blood group data between different populations is important for effective management of blood bank inventory and for more efficient transfusion practices. the present study collected data from different populations of the mena region to reveal that blood group o is the most common blood group type, while ab is the least prevalent. some of the diseases associated with specific blood groups were also addressed. however, more studies are required to fully understand the reported associations and to address the inconsistencies between studies. author contributions im conceived and planned the manuscript writing. rz took the lead in writing the manuscript with equal contribution. all authors provided critical feedback and helped shape the research, analysis and manuscript. references 1.logdberg l, reid me, lamont re, zelinski t. human blood 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(2018) sugar beet artificial seeds an overview. highlights in bioscience, volume 1.article id 20182, dio:10.36462/ h.biosci.20182 received: may 22, 2018 accepted: july 17, 2018 published: august 12, 2018 copyright:© 2018 rslan. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. date palm biotechnology: recent research an overview wessam m. rslan* agricultural genetic engineering research institute; agricultural research center, egypt. *to whom correspondence should be addressed: wessam.rslan@ageri.sci.eg abstract date palm (phoenix dactylifera l.) is among the earliest fruit crops cultivated in the arid arab peninsula, north africa, and middle east territories. dates are a significant source of food and revenue for middle east and north africa's local communities. it has distinctive features of biology and development that require special methods of reproduction, culture and governance. in varying dategrowing regions, there are thousands of date plant cultivars and varieties. the lengthy life cycle, long juvenile lifespan, and date palm dioecism produce cultivation difficult. every year, the percentage of crop genomes sequenced has continued to increase. the incredible rate at which dna samples become accessible is mainly due to the enhancement in cost-and speed-related sequencing techniques. modern sequencing techniques enable the sequencing at realistic price of various cultivars of tiny plant genomes. although many of the published genomes are deemed incomplete, they have nevertheless proven to be useful instruments for understanding significant plant characteristics such as fruit maturation, grain characteristics and adaptation of flowering time, here we review date palm genomic studies and determine its genomics element. keywords: date palm, genome-wide association, snp, genomics element, genome annotation . review article open access date palm biotechnology an overview highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 2 of 5 introduction several of the plant genome sequences have been fully sequenced (collected, annotated and released) and have been openly accessible in the database, such as oryza sativa (1), zea mays (2), hordeum vulgare (3), phoenix dactylifera (4), triticum aestivum (5), eucalyptus grandis (6), capsicum annuum (7), beta vulgaris (8) and solanum (9). yang et al. (1) presented a full date palm chloroplast genome sequence of approximately 158,462 bp. they revealed that there are 112 distinctive genes and 19 duplicated parts in the ir areas in the date palm chloroplast genome. they recognized 78 snps within the population of a particular chloroplast genome as significant intravarietal polymorphisms, most of which were found in genes with essential tasks. they also discovered 18 polycistronic transcription blocks and 3 extremely expression-based genes-atpf, trna-ugc, and rrn23 based on rna-sequencing records. al-dous et al. .(4) used the illumina gaii sequencing platform to present the first date of the palm assembly draft genome sequence for the khalas cultivar. they reported a date palm genome with size of 658 mb, 58% assembled sequences (382 mb) and forecast 25,059 genes. among the nine cultivars of date palms trees (deglet noor, deglet noor bc5, khalas, khalas bc2, medjool bc4, medjool, alrjfem, khltmale, khfxfem) they recorded more than 3.5 million polymorphic locations. in (http://qatar-weill.cornell.edu/ research/datepalmgenome/), this sequence was stored. fang et al. (12) provided date palm full sequence of mitochondrial genome with an estimated size of approximately 715.001 bp. al-mssallem et al. (13) introduced the second khalas nuclear genome assembly, which is 605.4 mb in size, spanning > 90% of the genome (~671 mb) and > 96% of its functional genes (~41,660 genes). this sequence was stored with bioproject id prjna83433 in genbank (http://www.ncbi.nlm.nih.gov/) and produced the first genetic map of the date palm (phoenix dactylifera) and recognized the putative gender chromosome. they submitted ~4000 maps on the map using a total of 1293 cm of nearly 1200 molecular markers. these molecular markers were divided into 18 linkage clusters (lg) and proposed that the sex chromosome could be lg12. hazzouri et al. (14) launched an extensive catalog of about seven million single nucleotide polymorphisms in date palms based on a set of 62 cultivars re-sequencing the entire genome. analysis of population structure suggested a significant genetic gap between north africa and the date palms of middle east/south asia with proof of admixture in egyptian and sudanese cultivars. candidate mutations for trait variation were recorded, including polymorphisms of nonsense and variety of presence/absence of gene material in important agronomic trait mechanisms. in the r2r3 myb-like orthologue of the oil palm virescens gene connected with fruit color variability, they also recognized a copy-like retrotransposon insertion polymorphism. the first mirna segments of the palm species elaeis guineensis, elaeis oleifera and phoenix dactylifera were recorded by silva et al. (15). in the genomes of these species, they recognized mirna precursors and defined their potential biological functions proposed by the mature mirna-based target gene regulation. three hundred and thirty-eight precursors were recognized, varying in sequence size from 76 to 220 nucleotides dispersed in 33 households. in addition, they also recognized musa acuminata 266 mirna precursors that were phylogenetically near to palm species. detection of resistance genes in date palm al-mssallem et al. (13) monitored phoenix dactylifera protein samples using the pfam nbs (nb-arc) family pf00931 databases (e-value cutoff of 1.0) hidden markov models (hmms). using an hmm model with pfam tir pf01582 (e-value cutoff 1.0) domains as well as lrr motifs in the c-terminal domains, the 144 expected amino acid sequences were used to identify tir domains. in total, 144 non-redundant nbs-encoding proteins have been recognized and validated manually, accounting for about 0.35% of the gene designs in the phoenix dactylifera genome. simple sequence repeats in date palm billotte et al. (16) built a library (ga) n enriched with microsatellite and described 16 nuclear simple sequence repetition (ssr) loci in phoenix dactylifera. they studied amplification and genotyping across taxes. this experiment reveals the usefulness of most ssr indicators in 11 other phoenix organisms and the applicability of some of them in elaeis guineensis, 11 pritchardia species, pritchardiopsis jeanneneyi species, and six astrocaryum species. akkak et al. (17) separated from two microsatellite enriched date palm libraries forty-one easy sequence repeats (phoenix dactylifera). of the 41 ssrs, 17 chosen microsatellite loci were identified and assessed on a collection of 31 algerian and californian germplasm cultivars and clones. all primer pairs generated an amplification pcr pattern of the expected size and high polymorphism was detected among the samples analyzed. tax amplification across the genus phoenix indicates the usefulness of most ssr indicators in 14 other species. hamwieh et al. (18) researched the prevalence of microsatellite patterns in the first edition of the new genome profile of the date palm assembly produced by the next generation dna sequencing of the entire genome shotgun. http://qatar-weill.cornell.edu/%20research/datepalmgenome/ http://qatar-weill.cornell.edu/%20research/datepalmgenome/ http://www.ncbi.nlm.nih.gov/ date palm biotechnology an overview highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 3 of 5 they structured a total of 1,091 primer combinations in simple/perfect microsatellite motifs flanking regions. from it 377 primers flanked dinucleotide, 352 primers flanked trinucleotide, and 362 primers pair flanked tetranucleotide repeats out of these primer combinations fifty primer pairs used for 8 iraqi date palm varieties screening. results showed that 28 combinations of primers were usable (56%) and 18 (36%) disclosed polymorphic alleles. also (19) developed date palm molecular markers database. elmeer et al. (20) constructed thirty primer pairs of microsatellite genomic dna markers for ssr. these molecular markers were used for eleven date palm genotypes to evaluate genetic diversity. results stated that the targeted pcr fragments were not amplified by only seven (23.3 percent) of the thirty primers. while thirteen pcr primers (43.3%) enhanced monomorphic banding patterns and polymorphic banding motifs were produced by the remaining ten primers (33.4%). arabnezhad et al. (21) formed two ssr-enriched date palm genomic databases, including repeat motifs (ag) n and (aag) n, using 22 ssr pcr primer pairs to evaluate the genetic connection between 16 date palm cultivars cultivated in separate geographic areas (iran, iraq and africa). based on nei's genetic distance they used, the cluster analysis put african date palms in a group other than the genotypes of iran and iraq. they stated it appears that african date palms ' domestication has taken a distinct path than those cultivated in the middle east. bodian et al. (22) used easy sequence repeat markers to analyze genetic variation among 128 date palm specimens from the figuig desert (morocco). 121 females belonging to 11 cultivars were among those palms, and 7 were males. eighteen ssr primers have been used, but all samples have been effectively amplified by only 15 primers. the dendrogram acquired showed three populations and male and female genotypes did not detect any genetic distinctions. zhao et al. (23) evaluated 28,889 samples of est from the database of the date palm genome and recognized 4,609 ests as ssrs. trinucleotide patterns (69.7%) were the most common among these ssrs, followed by tetranucleotide (10.4%) and dinucleotide patterns (9.6%). they also engineered a total of 4,967 primer pairs from sequencing data for est-ssr markers. twenty of these primer pairs from twelve date palm cultivars were tested with genomic dna. one-third of these primer pairs identified polymorphisms of dna that distinguished the cultivars of the twelve date palms. aberlenc-bertossi et al. (24) used the new sequence draft of the date palm genome al-dous et al. (11) to identify microsatellites and layout primers for ssr. they recognized 204 genes with microsatellite coding sequences, 150 of which were appropriate for primer construction, but only 103 had annealing locations that were not duplicated. with six or more repeats, such microsatellites had perfect trinucleotide motifs, and hexanucleotide motifs with at least four repeats. of the 47 first pairs lastly maintained, 33 in a preliminary experiment with eight phoenix dactylifera samples produced expected pcr amplification. the 33 loci have been further evaluated on 16 individuals comprising phoenix roebelenii o'brien(2), phoenix dactylifera (7), phoenix reclinata jacq .(2), phoenix rupicola t. anderson (2), phoenix theophrasti greuter (2), and phoenix canariensis interspecific hybrid phoenix sylvestris . zehdi-azouzi et al. (25) used both nuclear ssr and chloroplast microsatellites to evaluate the variety and genome composition of date palm accessions from mauritania to pakistan in ten regions. a total of 295 accessions were evaluated using 18 nuclear ssr loci revealing > 200 alleles for every locus. they recorded analyzes of the date palm population's genetic composition using the bayesian clustering strategy, hierarchical ranking of nj and dacp. their findings were obviously compatible with a two-cluster geographic structure. the first, called the eastern pool, includes djibouti, iraq, oman, pakistan, and the uae date palm accessions. while the western pool was appointed the second cluster, it included the residual accessions from africa, including egypt, algeria, mauritania, tunisia and morocco (26). snps studies in date palm al-dous et al. (11) analyzed 3.5 million snp across the genomes of male and female date palms trees to recognize gender-segregating polymorphisms. the findings noted best fit an xy sex-determination model with the heterogamous sex being males. using a male heterogamete model 1,605 snps were noted segregating with gender. analyzing two scaffolds with the most gender-segregating snps, they noted an estimated threefold distinction in divergence between male and female haplotypes from the reference sequence. across all genome sequences in these regions, the cultivars of 867 polymorphic sites have been observed. comparing the females of deglet noor and medjool with the female reference of khalas disclosed that 253 and 271 sites varied from the reference of khalas and that only 24 (9%) and 19 (7%) locations were heterozygous. at the same sites, their backcrossed males illustrated 736 and 770 different locations from the khalas reference, of which 584 (79%) and 578 (75%) were heterozygous, respectively. al-mssallem et al. (27) researched genetic variety of snps between many date tree species (khalas, fahal, sukry, agwa, deglet noor, deglet noor bc5, medjool, medjool bc4, khalsfx, alrijalf and khalt) and recognized snp date palm biotechnology an overview highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 4 of 5 (indel) abundances of 6.10 (0.25), 5.51 (0.15) and 6.24 (0.20) per kb. they explored how evolutionary selection left its marks in genomes of various date palm species by first assessing the allocation of snps on big scaffolds and then pinning down obviously chosen functional genes, particularly those strongly engaged in the metabolisms of sugar and energy. they found that there is no random distribution of snps, but a bimodal curve. depending on snps and the frequency distributions, they built the phylogenetic tree of 11 phoenix dactylifera cultivars. in nine saudi arabian date plant cultivars (sukkariat al-madinah perny al-riyadh, sukkariat qassim, rabia almadinah, dekhaini al-riyadh, ajwa al-madinah,, shalaby al-madinah, moshwaq al-riyadh and moshwaq hada alsham), sabir et al. (28) evaluated the entire maternal and plastid genome snps. they detected 188 snps in the mitochondrial genome at 37 distinct locations. for all nine cultivars, the amount of mutual snps was 16. while there were 30 of the nine date palm cultivars in the amount of plastid snps, they were situated in 20 distinct genome locations with 13 in genes, 2 in introns, and 5 in intergenic spacers. hazzouri et al. (14) established an extensive catalog of about seven million snps in date palms depending on a set of 62 cultivars re-sequencing of the entire genome. assessment of population structure indicates a significant genetic divide between north africa and middle east/south asian date palms, with evidence of admixture in egyptian and sudanese cultivars. at least 56 genomic regions correlated with selective sweeps which may underlie regional adaptation were proposed by genome-wide selection scans. candidate mutations for trait variety were recorded, including polymorphisms of nonsense and variety of presence/absence of gene material in important agronomic trait mechanisms. in the r2r3 myb-like orthologist of the oil palm virescens gene connected with fruit color variability, they also recognized a copy-like retro-transposon insertion polymorphism. referances 1. yu j, hu s, wang j, wong gk-s, li s, liu b, et al. a draft sequence of the rice genome (oryza sativa l. ssp. indica). science (80). 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al-mssallem is, hu s, zhang x, lin q, liu w, tan j, et al. genome sequence of the date palm phoenix. nat commun. nature publishing group; 2013;4:1–9. 28. sabir jsm, arasappan d, bahieldin a, abo-aba s, bafeel s, zari ta, et al. whole mitochondrial and plastid genome snp analysis of nine date palm cultivars reveals plastid heteroplasmy and close phylogenetic relationships among cultivars. plos one. 2014;9(4): e94158. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.20226 research article open access contacts of authors * to whom correspondence should be addressed: pramod prasad dahal 1 agriculture extension officer,governemnt of nepal,nepal. 2 agriculture and forestry university, agronomy, rampur, chitwan. 3 agriculture and forestry university, agronomy, rampur, chitwan. 4 nepal agricultural research council, singhdurbar plaza. revised: august 20, 2020 accepted: november 24, 2020 published: march 28, 2021 citation: dahal pp, basnet kb, sah sk and karki tb . effect of weather parameters on growth stages of winter maize explain the grain yield. 2021 mar 28;4:bs20226 copyright: © 2021 dahal et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. effect of weather parameters on growth stages of winter maize explain the grain yield pramod prasad dahal*1, komal bahadur basnet2, shrawan kumar sah3 and tika bahadur karki4 abstract a study was conducted on research farm of national maize research program (nmrp), rampur, chitwan, nepal during winter season of 2015/16 laid out in split-split plot design with three replications consisting of two fym levels (fym at 10 t ha-1 and no fym application), three nitrogen levels (100, 75 and 50% n of recommended dose p and k remaining constant) and two levels of seed inoculation (azotobacter chrococcum seed inoculation and no inoculation) with rampur-2 maize hybrid in nepal. one of the major factor affecting maize production is weather and this paper is prepared to assess the suitability of weather condition at different growth stages of winter season hybrid maize in condition of western chitwan, nepal at nmrp. the result shows that the requirement of the maize crop during different growth stages i.e. temperature, rainfall, relative humidity were fulfilled which helped to obtain remarkably higher grain yield (4.26 t ha-1) in the experiment compared to average national grain yield (2.84 t ha-1) of maize. keywords: integrated plant nutrient management, winter maize, weather parameters, maize growth stages introduction maize (zea mays l.) can be grown easily in any season and is important cereal crop due to fast growing nature, higher yield, palatability and nutritiousness resulting easily accessible food and feed for humans as well as animals [1]. in nepal maize is grown as a staple food crop for many years. in terms of area and production, maize ranks second to rice [2]. it shares about 6.54 % in agricultural gross domestic product (agdp). maize can grow in a wide range of environment; however, warm climate with temperature ranging from 21 to 30 °c is most favorable [3]. moreover, it was reported [4]that maize growth is best with temperature between 18 °c and 27 °c during day time and around 14°c at night. the experimental site falls under the sub-tropical humid climatic belts of nepal with three distinct seasons i.e. november to february (winter), march to may (hot spring) and june to october (rainy). the maximum temperature during the coldest month of year (december to february) reaches to 27 °c whereas the minimum temperature during this period ranges from 6 to 10 °c. likewise, the maximum temperature during hottest month of year (may to june) is measured up to 42 °c. recent studies conclusively proved that maize is a potential winter season crop having three times higher yield potential than kharif crop [5]. according to annual report of nmrp [6], the productivity of winter maize is about 6-7 t ha-1 while that of summer maize is around 4.0 t ha-1. highlights in bioscience page 1 of 5 march 2021|volume 4 https://doi.org/10.36462/h.biosci.20226 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield the mean annual temperature trend at rampur, chitwan during 1968 to 2008 showed that the increase of temperature seemed to be more in the recent decade than the former decade and the trend was 0.039 °c per year [7]. thus the changed weather condition can create impact upon the maize production as yield of maize is primarily influenced by sunlight, temperature, available plant nutrients and water supply [8]. one of the major factors affecting maize production is weather and this paper is prepared to assess the suitability of weather condition at different growth stages of winter season hybrid maize in condition of western chitwan, nepal at nmrp. materials and methods a experiment was conducted during winter season (october to march) of 2015 at the research field of national maize research program (nmrp), rampur, chitwan, nepal with rampur hybrid-2 variety. the experimental soil was sandy loam in texture, having slightly acidic ph (5.4). organic matter (4.02), total nitrogen (0.11), available phosphorus (58.0 kg ha-1), available potassium (253.8 kg ha-1). the experiment was laid out in strip-split plot design consisting of 12 treatments with three replications. fym levels as main plots (fym at 10 t ha-1 and no fym application), three nitrogen levels as sub plots (100, 75 and 50% n of recommended dose p and k remaining constant) and two levels of seed inoculation (azotobacter chrococcum seed inoculation and no inoculation) as sub-sub plots. farm yard manure (fym) was applied two weeks before sowing and was incorporated into soil. chemical fertilizers: urea, single super phosphate (ssp), diammonium phosphate (dap) and muriate of potash (mop) were also applied as main source of nitrogen, phosphorus and potassium, respectively. urea was applied in three splits; at sowing, knee high and tasseling stages. azotobacter was applied as seed inoculation preparing of slurry of 10% sugar solution mixing 100 gm of sugar on 1000 ml of water and was boiled and azotobacter were applied after cooling on shade. harvesting was completed on march of 2014, from net plot area. weather data were collected from meteorological data center near (100m) the research field. weather condition during the experimentation the experiment field receives regular precipitation of 1919.5 mm (nmrp, 2015/16).the average weather parameters regarding minimum as well as maximum temperatures, relative humidity and total rainfall are presented at an interval of two weeks in figure 1. the weather condition during the period of experimentation presented in the figure 1 and table 1 shows that the higher value of maximum temperature (33.85 °c) was recorded on 1st week of september which gradually declined up to 3rd week of december (21.1 °c). thereafter, it increased up to 24.69 °c on 1st week of january and then declined to 21.05 °c on 4th week of january. further, the values of maximum temperature were found to increase up to 32.84 °c on 2nd week of march and then remained at the same level at the 4th week of march (32.51°c). figure 1. weather condition during the course of experimentation at nmrp, rampur, chitwan, nepal, 2015/16. on the other hand, the minimum temperature was found to decrease from first week of september (26.60 °c) to 4th week of december (6.36 °c) and then remained almost at the same level (8.16 to 9.64 °c) from 1st week of january to 1st week of february and thereafter increased to 18.43 °c on 2nd week of march. further, similar minimum temperature (18.61 °c) was recorded on 4th week of march. in spite of it, the maximum rainfall of 43.6mm was recorded on 2nd week of september and thereafter it declined to zero precipitation on 1st week of october. however, a remarkable rainfall of 25.83mm was recorded on 4th week of october. further, there was no rainfall from the 1st week of november to 3rd week of february with the exception of january 3rd week where there was a rainfall of 7.7 mm. the relative humidity was found to increase from 1st week (81.86%) to 3rd week (89.14%) and then declined on 4th weeks (87.33%) of september. similarly, it increased from 1st (82.14%) to 2nd (88.29%) and then remained at the same level in 3rd (82.29%) and 4th (82.10%) weeks of october. further, the values of relative humidity were increasing from 90.86% on 1st week of november to 100% on 1st week of december and thereafter remained at this level up to 4th week of january with exception of 3rd week of january where it was a little decreased (98.43%). thereafter, the highlights in bioscience page 2 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield table 1. weekly maximum and minimum temperature, relative humidity, rainfall and solar radiation during research period at nmrp, rampur, chitwan, nepal, 2015/16. stage date min temp max. temp avg rainfall rh% germination 2nd week of oct. 23.125 32.845 27.99 8.3mm 85.21 knee high 1st week of nov. 17.75 30.31 24.03 0 90.86 grand growth 1st-4th week of nov. 16.18 28.305 22.24 0 94.92 tasseling and silking 1st-2nd dec. 13.75 24.39 19.07 0 100 milking 4th of january 8.11 21.05 14.58 0 100 dough 4th of feb 15.04 28.45 21.75 0.96 mm 95.03 grain filling 3rd jan-3rd feb 10.33 24.35 17.34 7.7mm 93.615 physiological maturity 3rd march week 16.59 31.69 24.14 26.8mm 79.43 total rainfall during crop season 14.35 27.23 20.79 72.99mm 93.86 relative humidity gradually declined from 98.14% on 1st week of february to 70.60% on 4th week of march. statistical analysis analysis of variance (anova) was used to test the row, column and interaction effect of fym, nitrogen levels and biofertilizer on yield and yield attributing characters of maize at 5% level of significance. values were computed using software program mstatc, gen stat and microsoft excel 2010. results analyzed data of phenological stages (table 2) showed that on an average, the emergence of maize seeds occurred at 6.36 das whereas the maize plants attained knee high stage (34.06 das) after around one month of seed emergence further, tasseling stage (73.10 das) was recorded after one month of the knee high stage. there was only four days difference between tasseling and silking stages. the silking, milking and dough stages were observed at 76.94, 126.18 and 144.17 das, respectively. finally, the maize crop was physiologically matured after about 20 days of dough stage (164.53 das). discussion the average minimum and maximum temperature during the cropping period were 14.35 and 27.23 °c suitable for growth and development of maize, respectively (table 1). further, the annual precipitation of experimental area was 1919.5 mm (nmrp, 2015/15), however, maize requires rainfall of about 600 1,200 mm throughout the year with uniform distribution [15]. thus, the availability of water was sufficient for growth and development of the maize crop. the minimum temperature required for maize seed germination is 10 °c. in addition, emergence gets far more rapid and uniform above 16 °c and around the temperature of about 20 °c, maize usually emerges within 5-6 days after sowing [9]. thus, the minimum and maximum temperatures recorded during the germination of maize seed in the experimental site were 23.13 °c and 32.84 °c with the average of 27.99 °c. therefore, the germination of seeds took place within 6 days. [10] stated that for uniformity of initial stand, a minimum temperature of 17 °c to 20 °c is required but during knee high stage, the average temperature was 24.03 °c with minimum and maximum temperatures of 17.75 and 30.31 °c, respectively. this indicates that average temperature was suitable for the establishment of plant stand in the experiment. further, during vegetative growth stage (grand growth stage) the minimum, maximum and average temperatures were 16.18, 28.31 and 22.24 °c, respectively. in this context, joshi [11] reported that optimum temperature for vegetative phase ranges from 28 to 34 °c. venkataraman and krishanan [4] also mentioned that range of temperature for the growth of maize is from 9°to 46 °c with the optimum around 34 °c during vegetative growth stage. thus, the minimum temperature was found higher but maximum temperature was slightly lower due to winter season. however, it was close to the temperature given by joshi [11]. moreover, in the experimental site, the average temperature during tasseling and silking stages equaled to 19.07 °c. with respect to it, venkataraman [12] reported that the optimum temperature for tasseling ranges from 21 to 30 °c and temperatures above 32 °c during reproductive stage reduced the yield. thus average temperatures during tasseling and silking were close to optimum level. further, minimum and maximum temperatures during these stages were 13.75 and 24.39 °c, respectively. in respect of it, berbecel and eftimescu [13], mentioned that the maximum temperature above 32 °c around tasseling and pollination increases the differentiation process of the reproductive parts and consehighlights in bioscience page 3 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield table 2. weekly maximum and minimum temperature, relative humidity, rainfall and solar radiation during research period at nmrp, rampur, chitwan, nepal, 2015/16. table shows treatments (tr), germination (gr), knee high (kh), tasseling (tas),silking(si), milking (mi), dough (do), physiological maturity (pm), and grain yield (t ha-1) (gy) paramter phenological stages (das) tr gr kh tas si mi do pm gy fym levels (t ha-1 ) 0 6.56 34.56 74.21 77.39 126.33 145.06 164.4 3.91 10 6.17 33.56 73.35 76.5 125.61 145.28 164.61 4.58 statistical analysis f test ns ns ns ns ns ns ns ns lsd (=0.05) 1.04 1.9 2.89 2.43 3 1.57 2.91 2.2 sem (±) 0.17 0.31 0.48 0.4 0.5 0.26 0.82 0.36 nitrogen levels tr gr kh tas si mi do pm gy 50 6.41a 35.67a 74.50a 78.92a 126.08a 146.17a 166.58a 2.95c 75 6.42a 34.92a 72.00b 75.67b 126.42a 145.16a 165.42a 4.64b 100 6.25a 31.58b 72.08b 75.24b 125.41a 144.17b 162.58b 5.16a statistical analysis f test ns * * * ns ns * ** lsd (=0.05) 0.42 2.83 1.72 2.57 1.67 1.76 2.23 0.41 sem (±) 0.13 0.87 0.53 0.79 0.51 0.54 0.68 0.12 biofertilizer tr gr kh tas si mi do pm gy no inoculation 6.33 34.17 73.11 77.28 126.54 145.28 164.72 3.95 with inoculation 6.39 33.94 72.61 76.61 125.89 145.06 164.33 4.55 statistical analysis f test ns ns ns ns * ns ns ** lsd (=0.05) 0.21 1.01 0.67 0.72 0.48 1.1 0.5 0.4 sem(±) 0.07 0.35 0.22 0.23 0.17 0.35 0.162 0.13 cv, % 4.5 2.1 1.5 1.3 1 0.5 0.6 12.9 grand mean 6.36 34.06 72.88 76.94 126.18 144.17 164.53 4.25 recommended dose of nitrogen: 120 kg n ha-1 azotobacter chroococcum seed inoculation: 40 g kg-1 seed quently the rate of kernel abortion. further, according to adjetey [3] at high temperatures the pollens gets shed before silk become receptive or cause either death of tassel or drying out of silk. therefore, to achieve higher grain yield the temperatures of the late vegetative and reproductive phases should be relatively lower than 30 °c which was also recorded in this experiment. finally, during maturity, the minimum, maximum and average temperatures were 16.59, 31.69 and 26.8 °c respectively. joshi (2015) stated that optimum temperature during ripening stage ranges from 32 to 35 °c. thus, the maximum temperature of the experimental site was close to it but the average temperature was a little lower which might be related to the winter season and subtropical climatic condition. thus, on the basis of above discussion it is obvious that in general, the weather condition related to temperature was suitable for growth and development of maize. further, a rainfall of 43.60, 10.3 and 9.6 mm were recorded at three, two and one week before germination, respectively (figure 1) which provided enough moisture for germination of maize seeds. moreover, a rainfall of 8.3mm was recorded at sowing which also helped in better germination of seeds. germination of maize seed was completed within 6 days. no rainfall was recorded during knee high and grand growth stages. others [10] stated that moisture stress in early growing season causes a large reduction in grain yield. therefore, the experimental plots were irrigated at this stage. kranzet al., [14] highlights in bioscience page 4 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield mentioned that corn requires most of the water during the early reproductive growth stages i.e. tasseling and silking. moreover, the requirement of maize in water is more prominent during the tasselling to silking stage [15] which is considered critical as grain formation initiates during this period. thus the availability of soil moisture at the time of tasselling is therefore vital for the production of high yield in maize [16]. futher, period of silking and ear growth the corn is most sensitive to moisture deficiency as far as the grain yield is concerned [4]. however, there was no rainfall during this period in the experimental site due to which the crop was irrigated at this phase. another critical period with respect to crop requirement in moisture is grain filling period. corn requires one third part of seasonal water requirement at beginning of dough stage. drought or water stress at dough stage results in acceleration of maturity preventing grains from gaining full size as well as weight [14]. however, there was 7.7 mm rainfall during grain filling period and 0.96mm at dough stage which might had helped to some extent fulfill the crop requirement in water. thus, in general, the requirement of the maize crop in water was fulfilled either through rainfall or irrigation which helped to obtain remarkably higher average grain yield (4.26 t ha-1) in the experiment as compared to average national grain yield (3.09 t ha-1) of maize (moad-abpsd, 2015). it concludes from above discussion that weather recorded during different key growth stages of winter maize in the experimental site were resultant and it is essential to give emphasis on weather conditions in improvement of grain yield of winter maize. references 1. shinde sa, patange mj, dhage sj. influence of irrigation schedules and integrated nutrient management on growth, yield and quality of rabi maize (zea mays l.). international journal of current microbiology and applied sciences. 2014;3(12):828-32. 2. moad. statistical information on nepalese agriculture, 2015. 3. adjetey ja. maize physiology and growth requirements. 1994. 4. venkatraman s, krishnan a. crops and weather publications and information division. icar, new delhi, pp.1119, 1992. 5. desai sn, deore dd. performance of maize cultivars in rabi season. journal of maharashtra agricultural universities. 1980;5(2):181-2. 6. annual report nmrp. performance of maize cultivars in rabi season. nep al agriculture research council, rampur, chitwan, nepal., 2015. 7. nayava jl. impact of climate change on production and productivity: a case study of maize research and development in nepal. journal of agriculture and environment. 2010 sep 16;11:59-69. 8. huzsvay l, nagy j. effect of weather on maize yields and the efficiency of fertilization. acta agronomica hungarica, 53(1):31-39, 2005. 9. raemaekers rh, editor. crop production in tropical africa. belgium: dgic; 2001. 10. purseglove, jw. crop production in dry regions. vol. ii: systematic treatment of the principal crops by i. arnon london: leonard hill (1972), pp. 683,10.50. experimental agriculture, 9(4):380-380, 1973. 11. joshi kd, conroy c, witcombe jr. agriculture, seed, and innovation in nepal: industry and policy issues for the future. washington, dc, united states: international food policy research institute, pages 1-60, 2012. 12. venkataraman, s. crops and weather. publications and information division, indian council of agric. res., 1992. 13. berbecel o. effect of agrometeorological conditions on maize growth and development. 1972. 14. kranz wl, irmak s, van donk sj, yonts cd, martin dl. irrigation management for corn. neb guide, university of nebraska, lincoln. 2008 may;10(5):1-8. 15. awuku ka , brese gk, ofosu gk, baiden so. senior secondary school agriculture and environmental studies. evans brothers ltd.: london, uk, pp.85-86, 1991. 16. tweneboah ck. modern agriculture in the tropics. a textbook on animal production. accra: co-wood publishers, 2000. highlights in bioscience page 5 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods weather condition during the experimentation statistical analysis results discussion references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20215 highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 6 research article open access 1 department of science laboratory technology, biological sciences unit, microbiology option, the oke ogun polytechnic, saki, nigeria. 2 department of pure and applied biology, p.m.b 4000, ogbomosho, ladoke akintola university of technology, nigeria. 3 department of zoology, parasitology unit, university of ibadan, ibadan, nigeria. 4 genetics and molecular biology unit, department of botany, university of ibadan, nigeria. 5 department of zoology, cellular parasitology unit, university of ibadan, ibadan, nigeria contacts of authors * to whom correspondence should be addressed: adeoti o. micheal citation: micheal ao, olayemi aa, adenike ak, olabiyi aa, olufemi os, olajumoke ae, ademola ad, joy oo. (2020). antibiotic effects of selected leaf extracts and molecular profile of klebsiella pneumoniae and enterobacter bugandensis strain (adm2). highlights in bioscience volume 3. article id 20205. dio:10.36462/h.biosci.20215 received: may 20, 2020 accepted: july 18, 2020 published: august 05, 2020 copyright: © 2020 micheal et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. antibiotic effects of selected leaf extracts and molecular profile of klebsiella pneumoniae and enterobacter bugandensis strain (adm2) adeoti o. micheal 1, 2,5 *, aboladale a. olayemi 1 , adeoye k. adenike 1, 3 , abiola a. olabiyi 1 , olufemi s. olufemi 1,4 , adedokun e. olajumoke 1 , adesina d .ademola 1 and olaoye o. joy 1 abstract only healthy surface tissues of healthy animals that are constantly in contact with the environment can easily be colonized by various microbial species, including pathogenic ones. however, these microorganisms are usually the cause of opportunistic infections in animals. two plants; vernonia amygdalina (taxonomic id number 112605) and ocimum gratissimum (taxonomic id number 112606) were identified at the nigerian federal research institute in ibadan and subsequently concentrated with methanol, water and n-hexane. twelve pre-slaughter healthy cattle in the same ratio of male to female were selected for this study. samples from the mouth, skin, nose, vaginal and gastrointestinal tract were collected. susceptibility patterns for antimicrobial agents were performed using disk-diffusion agar method. all isolates were subjected to bacteriological and molecular identification (pcr and sequencing techniques). preliminary identification was carried out based on standard bacteriological, microscopic identification, biochemical criteria, while pcr and dna sequencing techniques were used for molecular identification and analysis. basic local alignment sequence tools (blast) were used to compare retrieved bacterial sequences to a set of previously published strains in the database. the morphological and biochemical profiles of six out of eight normal flora from different sites were gram-negative. all bacterial cultures were oxidase-positive, including 7 catalase-positive cultures. the majority of isolated bacteria were moderately sensitive to all the extracts tested but resistant to amoxylin. noticeable amplification of 16s rrna genes from klebsiella pneumoniae strain emb and enterobacter bugandensis strain adm2 was obtained. the amplification of the resistant genes of the two isolates was approximately 1,500 base pairs; however, some of the suspected virulence and antibiotic genes were within this base pair range. keywords: vernonia amygdalina, ocimum gratissimum, anti-bacterial, 16s rrna, cattle introduction antibacterial are kinds of antimicrobial agent used mainly against bacteria, for the treatment of bacterial infections [1]. the invention of antibiotics has assisted in the control of pathogenic bacteria until when most pathogens become resistant to the treatment. increased alarming rate of antibiotic resistance worldwide is causing difficulty and posing danger to humanity as various diseases and ailments are now difficult to treat and control [2]. https://creativecommons.org/licenses/by/4.0/ micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 6 in addition, majority of available synthetic antibacterial drugs are becoming less effective due to their side effects such as tendonitis, seizure, and steven-johnson syndrome [3]. the emergence of antibiotic resistance and the unbearable side effects (toxicity) of some of the commercially available antibiotics makes it imperative to search for newer, more effective and cheaper drugs that could serve as alternative therapy for the treatment of various infections and diseases [4]. plants are reservoir of therapeutic substances which have a vital role in the sustenance of human health in the time past. world health organization (who) opines that plant parts or their potent make up are used in the productions of array of drugs in traditional therapies [5]. these plants have highly contributed to the development of human health and welfare. simultaneously, there is an increase in data and huge patronage to herbal products round the world [6]. medicinal plants such as ocimum gratissimum have been affirmed to provide different culinary and medicinal properties which effect can be bacteriostatic and bacteriocidal on some bacteria. these effects have been attributed to the peptides, alkaloids, essential oils, phenols and flavonoids which are main constituents in these plants [7]. it is a therapeutic plant which has been used traditionally for the treatment of various disorder [8]. cattle are reared for various reasons for consumption and economic purposes .they are used to pull farm implements in farmland and rural areas. cattles are herbivores which mean they do not eat only plants, grass and cereals. microbiota is the total of microorganisms that resides on or within any of a number of human tissues and bio-fluids. in new born, it is a powerful stimulus for the development of the immune system. unlike humans which are also colonized by many microorganisms, some microbes are normal flora in human they are commensal, they co-exist without being detrimental to humans; others to humans host are mutualistic [9]. this study was to provide an insight into molecular identification of two pathogenic organisms and document their antimicrobial susceptibility profiles to locally available plants. gene sequencing technology will be used for strain identification, and classification. in addition, we aim to use some nucleotide sequence analysis tools to perform a basic phylogenetic and diversity analysis in order to evaluate the genetic variability of the flora studied. materials and methods plant collection and identification plants were selected based on ethno botanical information obtained from traditional medicine practitioners in saki. all plants were collected around challenge ii area in saki. parts of the plants (leaves) were used for extraction. the identification was carried out in forest research institute of nigeria (frin), ibadan with voucher specimen of 112605 and 112606 for vernonia amygdalina and ocimum gratissimum respectively. extraction and isolation of active agents the materials of each was washed with distilled water and dried at room temperature by exposure to atmosphere for weeks. the dried materials were pulverized into fine powder using electric blender and packed into a plastic container with screw cap for further use. four types of extracts (aqueous, methanol, methanol plus water, n-hexane) were done. 100 g of each bitter leaf powder were weighed into three different sterile bottles and was soaked with 500 ml of methanol; n-hexane and 250 ml of methanol plus 250 ml of distilled water. 50 g of each scent leaf powder were also weighed into three different sterile bottles and was soaked with 250 ml of methanol, n-hexane and 175 ml of methanol plus 175 ml of distilled water. the mixture was stirred carefully with sterile stirrer and was covered with foil paper. the bottles were kept at room temperature for 3 days. the aqueous solution was prepared on the night of extraction by weighing 100g of bitter leaf powder in another bottle and was soaked with 500 ml of water and 50 g of scent leaf powder was also weighed in another sterile bottle and soaked with 250 ml of distilled water and covered with foil paper. the mixtures were extracted by sieving through muslin cloth, cotton wool and later filtered using whatman filter paper into clean sterile bottles. the aqueous prepared was boiled for 15 minutes and it was allowed to cool and was extracted like it was done to the previous ones. the extracts filtrates were concentrated using rotary evaporator at 45°c at the department of pharmaceutical chemistry laboratory in university of ibadan, oyo state, nigeria. determination of the antibacterial activity of the extract normal flora samples were collected from twelve cattle (6 males and 6 females) from different sites (nose, mouth, anus, vagina and skin) from the abattoir situated in foofo, saki, ibadan, oyo state, nigeria. swab sticks were used for the collection of specimen from different site from each cattle and were well labeled for recognition. nutrient agar was prepared according to manufacturer’s instructions. the specimens were directly streak on the prepared agar in different plates after solidifying and then incubated for 24 hours at room temperature. after 24 hours, the growth from the media incubated was transferred on macconkey agar and eosin methylene blue for sub-culturing till pure culture is formed. isolates were also tested on mrs and ssa agar but there was no growth. minimum inhibitory concentration each test organisms from serial dilution were streaked onto various plates and carried out in a septic condition and the plates were labeled accordingly. disc diffusion method were introduced by using disc(sterilized perforated disc 6 micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 6 mm in size seeded with different concentrations of the extract of 2 g, 4g and 6 g which is diluted into 10 ml of each methanol, n-hexane, methanol + aqueous and aqueous and amoxylin which is the control used was also diluted in 100 ml of distilled water. then the seeded disc was placed on the surface of the inoculated agar medium. the inoculated plates were incubated at 37 0 c the organism’s susceptibility test with plant extracts were recorded after 24 hours by measuring the mean diameter of the clear zone of inhibition using vernier caliper in millimeter (mm). biochemical tests for bacterial identification isolates were subjected to catalase and oxidase tests, which was identified using bergey's determinative bacteriology manual [10]. in order to perform a catalase test, the production of gas bubbles has been used as an indication of a positive result. isolate was placed on a glass slide with an inoculating loop 2 % of hydrogen peroxide was drop on the isolates and bubble isolates were recorded positive and vice versa. the oxidase test was used to detect the enzyme cytochrome oxidase activity. a purple coloration indicates positive results while no color change indicates oxidase negative results. inoculums of each isolate were smeared on a clean filter paper and oxidase reagent (tetramethylphenylenediaminedihydrochloride) was drop on each isolate. then colour changes were observed. dna extraction and 16s pcr bacterial cells broth was prepared and lysed through a zr bashing tm lysis tubes. 750µl lysis was pipetted in form of solution to the tubes. dna extraction from bacterial samples was carried out using the zymo bacterial dna extraction kit according to the protocol used by the manufacturer. the dna quantity and quality was measured using gel electrophoresis techniques. conventional pcr was applied to all the samples to amplify the 16s rrna gene using previously reported protocol (figure 1) [11]. figure 1: agarose gel electrophoresis showing positive amplification of 1,500 bp fragment of 16s rrna gene of klebsiella pneumoniae performed with strain-specific primer. lane m: 200-1037 bp dna ladder. strain lane g1:1500bp from cattle while g2: adm2 (1000-1700) isolated from cattle. g3 and g4 (isolates from other sources: rain and well water respectively). the pcr results were sequenced by using sanger sequencing technique and its products were subjected to analysis by basic local alignment search tools [12] (blast) and similar sequences were retrieved from the national centre for biotechnology information (ncbi) database [13]. the phylogenetic analysis was performed using mega 5.2 version [14]. ethical considerations experiments on animals have been conducted, according the research protocol reviewed by animal ethics committees of microbiology option, the oke ogun polytechnic, saki, nigeria. results and discussion the resultant nucleotides sequences of the 16s rrna was matched to klebsiella pneumonia and enterobacter bugandensis (adm2) with accession number mk719814 and mn213349 (figure 2). figure 2: the blast results of 16s bacterial sequences obtained in the current study. the antibacterial activity of both v amygdalina (bitter leaf) and ocimum gratissimum (scent leaf) were found to be dosage and extraction solvent dependent. methanolic extract of both plants at their highest tested doses possessed more antibacterial activities compared to the other extracts from different solvents when tested against the bacteria strain of klebsiella pneumoniae and e. bugandensis (adm2). this might attributed to the fact that methanol micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 6 extracted more of the bioactive constituents of the plant compared to other solvents. the methanolic extract of v amygdalina appeared more effective in the concentrations of 6g/ml when tested on k. pneumonia and showed moderate zone of inhibition on enterobacter bugandensis compared to the other tested doses, though the trend revealed dose dependent activity, i.e the higher the concentration the higher the effectiveness of the plant extract. the aqueous extract showed little to no inhibitory activity on the tested organisms while ciprofloxacin used as control inhibited the growth than the extracts (table 1). zones of inhibition produced by methanolic extract of ocimum gratissimum ranged from 7. 00 ± 0.00 at 2g/ml to 14.75±0.05 at 6g/ml against on k. pneumonia, followed by n-hexane extract ranged from 4.30±0.15 at 2g/ml to the highest dose of 6g/ml with value of 5.75±0.20, methanol + aqueous extract also showed moderate inhibitory activity while aqueous extract was found not to inhibit the growth of the test organisms at lower concentrations. however, at higher concentration, zones of inhibition were observed for the enterobacter bugandensis ranges from 1.30±0.02 at 4g/ml and 2.85±0.03 at 6g/ml (table 2). table 1: antibacterial activities of bitter leaf extract on k. pneumonia and e. bugandensis (adm2). leaf extract concentration (g/ml) mean diameter of zone of inhibition of bacteria in mm (±sem) methanol methanol + aqueous nhexane aqueous only control (ciprofloxacin) k. pneumonia 2.0g/ml 9.75 ± 0.03 4.75±0.02 3.30±0.15 1.00±0.00 22.00± 0.00 4.0g/ml 11.35±0.02 5.00±0.00 3.10±0.22 1.40±0.08 24.00±1.00 6.0g/ml 16.75±0.05 5.25±0.02 3.75±0.03 1.50±0.05 27.30±1.00 e. bugandensis 2.0mg/ml 4.50±0.05 2.50±0.08 2.00±0.00 0.00±0.00 10.00±0.00 4.0g/ml 5.00±0.00 3.60±1.06 2.90±0.14 1.00±0.00 11.00±0.00 6.0g/ml 6.30±0.10 4.30±0.14 3.30±0.00 1.00±0.02 15.00±1.00 table 2: antibacterial activities of scent leaf extract on k. pneumonia and e. bugandensis (adm2). leaf extract concentration (g/ml) mean diameter of zone of inhibition of bacteria in mm (±sem) methanol methanol + aqueous nhexane aqueous only control (ciprofloxacin) 2.0g/ml 9.75 ± 0.03 4.75±0.02 3.30±0.15 1.00±0.00 22.00± 0.00 k. pneumonia 4.0g/ml 11.35±0.02 5.00±0.00 3.10±0.22 1.40±0.08 24.00±1.00 6.0g/ml 16.75±0.05 5.25±0.02 3.75±0.03 1.50±0.05 27.30±1.00 e. bugandensis 2.0mg/ml 4.50±0.05 2.50±0.08 2.00±0.00 0.00±0.00 10.00±0.00 4.0g/ml 5.00±0.00 3.60±1.06 2.90±0.14 1.00±0.00 11.00±0.00 6.0g/ml 6.30±0.10 4.30±0.14 3.30±0.00 1.00±0.02 15.00±1.00 veronia amygdalina produces a variety of flavonoids and bitter lactones which contribute to the bioactivities of this plant [15]. v. amygdalina serves well as a low cost are readily available source of important nutrients to human [16]. the result of the current study demonstrated that the six out of eight normal flora from different sites isolated from cattle were bacterial culture while others were positive according to the standard bacteriological identification and microscopic identification. the sensitivity tests show that there is high resistance of test organism against antibacterial activity of plant extracts. this means that scent leaf and bitter leaf extracts are more effective as antibacterial agent against test organism. sequencing analysis was done to subject a deoxyribonucleic acid, polymerase chain reaction to test organism so as to understand its features, structure, function and evolution [17,18].some of the suspected antibiotic resistance genes and virulence genes were (hyla, teta, tete and blatem) respectively with base pairs of 1-1.5kb [19–21]. phylogenetic analysis of retrieved bacterial isolates indicates a high level of identification of 16s rrna sequences for bacterial genus and species (figure 3). such results could confirm the usefulness of using such genomic regions in the identification and characterization of bacterial isolates. micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 6 figure 3: phylogenetic trees of enterobacter bugandensis (a) and klebsiella pneumoniae (b) isolates from normal cattle flora compared to similar 16srrna gene sequences. conclusion most of the sensitive extracts were unable to inhibit the growth of the organisms and are presumably drug-resistant. scent leaf and bitter leaf extract appeared more effective than their previously reported actions. notwithstanding, v. amygdalina possesses various bioactivities with low or absence of side effects having great health promoting effects. it is however more advantageous to incorporate v. amygdalina into health supplement for both human and animal benefits. acknowledgement the authors wish to acknowledge the abattoirs attendants for the supports during the course of this research. also, fehintola oluwaseun deserves to be mentioned; for processing and retrieval of sequences from ncbi database. references 1. jakovac h. covid-19 and vitamin d-is there a link and an opportunity for intervention. american journal of physiology-endocrinology and metabolism. 2020 may 1; 318(5):e589. 2. nanasombat s, lohasupthawee p. antibacterial activity of crude ethanolic extracts and essential oils of spices against salmonella and other enterobacteriacea. current applied science and technology. 2005; 5(3):527-38. 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buffalo and cow mastitic milk. pathogens and global health. 2014 jun 1; 108(4):191-9. 19. li gy, li j, xiao p, guo yh, mo zl. detection of type iii secretion gene as an indicator for pathogenic edwardsiella tarda. letters in applied microbiology. 2011 mar; 52(3):213-9. 20. akinbowale ol, peng h, barton md. diversity of tetracycline resistance genes in bacteria from aquaculture sources in australia. journal of applied microbiology. 2007 nov; 103(5):2016-25. 21. chung th, yi sw, kim bs, kim wi, shin gw. identification and antibiotic resistance profiling of bacterial isolates from septicemic soft-shelled turtles (pelodiscus sinensis). veterinární medicína. 2017 mar 15; 62(3):169-77. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202103 research article open access 1 natubhai v. patel college of pure and applied sciences , sardar patel university, vallabh vidyanagar, anand, india. contacts of authors * to whom correspondence should be addressed: yachana jha received: august 23, 2020 accepted: january 15, 2021 published: january 25, 2021 citation: jha y . macrophytes as a potential tool for crop production by providing nutrient as well as protection against common phyto pathogen. 2021 jan 25;4:bs202103 copyright: © 2021 jha. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. macrophytes as a potential tool for crop production by providing nutrient as well as protection against common phyto pathogen yachana jha*1 abstract applications of synthetic chemical fertilizers and pesticides lead to several environmental hazards, causing damages to entire ecosystem. to reduce damage caused by such chemical inputs in agriculture and environment required a serious attention for replacement of chemicals input with eco-friendly options. in this study decaying macrophytes were selected as an option for organic agriculture, by analyzing its ability to provide important mineral nutrient to the maize crop grown in low nutrient soil as well as for providing resistant towards many common phyto-pathogens to enhance yield. the results of the study showed that decaying macrophytes have high concentration of stored important mineral nutrient in their body mass, which get released in the soil during its decay and to be used by the maize plant. the decaying macrophytes leaf extract have considerable amount of phenolic and flavonoids also having antimicrobial activity. the antimicrobial activity of the leaf extract has been analyzed against the common phyto-pathogen pseudomonas aeruginosa , and s. aureus by agar disc method and the formation of clear zone indicate its potential as bio-control agent. so under intensive agricultural practices, application of such biological waste is of particular importance for enhancing soil fertility without chemical input, to ensure sustainable agriculture. keywords: macrophytes, phytochemicals, phenolic, flavonoids, antimicrobial activity, mineral nutrient. introduction aquatic plants are adapted to live in aquatic environments and are referred to as hydrophytes or macrophytes. these plants require special adaptations for living and can only grow in water or soil that is perfectly saturated with water. aquatic plants generate one of the most productive ecosystems of the world and are essential life supporting systems, which provide a wide array of benefits to the aquatic life and human kind. however, these ecosystems are today’s fast declining and rapidly deteriorating ecosystems in many parts of the world. ignoring the economic and ecological significance of aquatic plants is often regarded menace [1]. due to continuous pollution of water bodies and urbanization resulted in eutrophication, which disturbed the aquatic ecosystem. so to maintain the aquatic ecosystem, cleaning of water bodies and dumping of macrophytes is a global problem. global climate change causes a series of extremities like frost, salinity or drought, which adversely affect the plant nutrient dynamics as well as world food requirement. the availability of nutrients in the soil and its acquisition, assimilation, distribution within the plants or in the crop fields are directly disturbed by climatic stress factors [2]. low soil nutrient has deleterious effect on nutrient dynamics within the plant root as well as in shoot, including transportation of nutrient to reproductive structures via xylem and phloem, or mobilization of nutrient from senescing leaves. water fluxes, assimilatory activities, and the redistribution of inorganic nutrients are also affected by environmental stress. macrophytes have the ability to accumulate large amount of water and nutrient in their leaves, can serve as a good option for the plant nutrient under stress/low soil nutrient condition. macrophyte decomposition is important for carbon and nutrient cycling in lake ecosystems or in the low nutrient soil [3]. highlights in bioscience page 1 of 5 january 2021|volume 4 https://doi.org/10.36462/h.biosci.202103 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production it act as a important source of essential nutrient like phosphorus, carbon, potassium, zinc and nitrogen in the form of ammonia etc., due to the ability of macrophyte plant to absorbs and accumulate these nutrients from soils or water in their body, which ultimately disposes during its decomposition in the ecosystem. such nutrient rich macrophytes plants can be used efficiently in organic agriculture/bio-composting that can be applied to obtain required mineral nutrient effectively and successfully for the proper growth of plant, results in increased plant growth and yield. as bio-composting is an economically and ecologically important necessity in resent agriculture, which in return increases the soil quality and crop yield. biological agents are nowadays drawing attention because of its increased organic efficiency in terms of physical and chemical characteristics of the soil [4]. in nature, plants species grow together and interact with each other by inhibiting or stimulating the growth and development through various interactions. a special form of competition among plants is known as allelopathy, also shown by aquatic plants. such interaction is a biological phenomenon in which an organism produces many bio-chemical compounds that influence the growth, and reproduction of other organisms. such interactions are primarily based on the synthesis and release of secondary metabolites, known as allelochemicals or allelotoxins [5]. such allelochemicals initiate a wide array of biochemical reactions and induce several biological changes, which inhibit the growth of target pathogenic organism, which is not possible solely by physical or chemical agents. the macrophytes also have ability to overcome stress arise due to multiple symbiotic or pathogenic interaction. the aim of the work is analyze the effect of decaying macrophytes on the plant nutrient status, growth and its ability for the production of antibiotics and related secondary metabolites, to protect host maize plant against the potential phyto-pathogens. materials and methods aquatic plants collection and preparation of samples macrophyte plants were collected from nearby lake and ponds from anand-tarapur district of gujarat. it was immediately packed in polyethylene bags to prevent the degradation of bioactive compounds of the collected plant samples and was brought to the laboratory for further study. the healthy and disease free plants were used for further study. the collected leaf samples were thoroughly washed with water to eradicate surface dust and foreign materials and used for sample preparation. the washed leaves were properly dried at room temperature for 7 days under shade and were ground into a powder with the help of grinder. estimation of mineral nutrient concentrations of soil, hydrophytes and maize leaves the physio-chemical property of the soil, hydrophytes and maize leaves were analyzed by saturated water extraction method in sicart lab. the leaves powder sample were digested for 6 hours in nitric-perchloric acid (5:3) and used to estimate the concentration of phosphorus by colorimetric. similarly the concentration of nitrogen was estimated after the kjeldahl digestion by colorimetry and 2g of plant material were digested in mixture of sulphuric acid, nitric acid and perchloric acid (tri-acid) in the ratio of 9:3:1 and were used to determine the concentration of n, p, k, zn and ca. the aliquots of digested filtered plant material were analyzed on digital flame photometry by using specific filter. preparation of leaf extract the leaf extract was prepared by soaking 10g of leaves material powder in 50 ml of distilled water for 24 hours under shaking condition at 130-140 rpm at room temperature. after that the extract was filtered from eight layers of muslin cloth and filtrate was collected in petri-dish. the obtained filtered extracts were dried at room temperature in petri-dish, and was scraped, transferred to tubes for further use. similarly, plant material was re-extracted with 50 ml of ethyl acetate, and methanol also. finally obtained extracts were dried under reduced pressure and were stored in a refrigerator at 4 °c until use. qualitative analysis of phyto-chemical screening the phyto-chemical tests to detect the presence of alkaloid, saponins, quinones, steroids, phenols, flavonoide, tannins, coumarines and reducteurs compound were performed. the visual observations like formation of a precipitate or change in color after the addition of specific reagents were used to determine the presence of specific phyto-chemical in the plant extract. quantitative analysis on phytochemicals constituents total phenolic content the total phenolic content of leaf extracts was measured using colorimetric folin–ciocalteu method [6]. the leaf extract (1ml) was mixed with 5ml of distilled water and 250 μl of 1n folin-ciocalteau reagent. the mixture was covered and allowed it to stand for 3min at 25°c. in this mixture, 1ml of saturated na2co3 and 1ml of distilled water were added. the mixture was incubated for 1 hour at 25°c for color development and measured at 725 nm using spectrophotometer. standard graph was prepared by using different concentration of phenol crystals. total flavonoid content the colorimetric assay was used to estimate total flavonoid content in presence of aluminum chloride [7]. in 10 ml volumetric flask 1ml of leaf extract, 4 ml of distilled water and 0.3 ml of 5% nano2 was added. after proper mixing for 5 minutes 0.3 ml of 10% alcl3 and 2 ml of 1 m naoh was added in it. finally 3.7 ml of distilled water was added to made up total 10 ml volume. the obtained mixed solution was used estimate total flavonoid content by taking absorbance at 510 nm against blank and calculated by the help of standard graph of gallic acid. phyto-pathogenic microorganism two most common plant pathogenic microorganisms pseudomonas aeruginosa and s. aureus has been selected to analyze highlights in bioscience page 2 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production the antibacterial potential of the leaf extract. these isolates were obtained from a biotechnology research lab of biosciences department sp university. antimicrobial activity assay the agar disc diffusion method was used for this analysis. the culture suspension was spread all over plate by placing 0.1ml of culture at center of nutrient agar plate and spreading uniformly with sterile spreader. then, 6 mm in diameter filter paper discs containing the different types (water, ethyl acetate, and methanol) of leaf extract at a concentration of 100µl were used with distill water as control. and 4 filter paper discs were placed on the nutrient agar medium surface for the diffusion of leaf extract in the nutrient agar. the petri discs were then incubated in incubator at 37 °c for 24 h, plates were observed for zone of inhibition by measuring the colony diameter. a 100 % dmso was used as control. effect of decaying macrophytes on minerals concentration under low soil nutrient condition in maize leaves for this analysis, the maize seedlings were planted in the field with low soil nutrient supplemented with decaying macrophytes without chemical fertilizer for 5 weeks. after 5 weeks plant sample were used determined the foliar phosphorus and nitrogen content by colorimeter, after digesting the plant sample with nitric-perchloric acid (5:3) for 6h kjeldahl digestion respectively. the foliar potassium, calcium and zinc content were estimated by digesting 1 g of plant sample with tri-acid mixture, which contain mixture of sulphuric acid, nitric acid and perchloric acid in the ratio of 9:3:1. the filtrates of digested material were used for the further analysis by using specific filter on digital flame photometry. effect of decaying macrophytes on plant growth parameters macrophytes were collected from the polluted water bodies and dumped at a site to initiate its decaying process. after 3 days of collection 10 cm thick bed was prepared on the crop field and was masked by 5 cm soil layer in a low soil nutrient field. after 10 days maize seed were sown in this field to analyze the effect of decaying macrophytes on plant growth parameters. then seedlings were collected carefully with root after 35 days of sowing to measure the shoot and root length and dry weight. the plants were dried in an oven at 80°c for 72 hrs for dry weight. statistical analysis all experiments described were performed three times independently, with measurements taken from three (for all other measurements) or ten (for dry weight measurements) different plants for each treatment in each of the three independent experiments. one-way analysis of variance (anova) test was used to analyze all data and mean (of three independent experiments) was compared at 5 % level of significance. results and discussion soil hardly ever supports crop production at their full potential yield due to lack of sufficient nutrient. so use of chemical fertilizers is a regular mechanism to achieve the yield potential. but low use of chemical fertilizers may be results in reduced yield and excessive application pollute the environment including water bodies, if applied without prior information about the nutrient content of the soil. at the same time pollution of water bodies resulted in overgrowth of macrophytes having harmful affect on water life, so such macrophytes are problem for water bodies. therefore use of such macrophytes as a source of nutrient in low nutrient soil status is the best option to overcome the problem, as it accumulates extra nutrient and water in their bodies. so in the present study, the macrophytes have been used as a source of supplement nutrient in the low nutrient soil in place of chemical fertilizer. so the initial major mineral nutrients (n, p, k, zn and ca) has been analyzed in the soil used for maize cultivation, control maize leaves and decaying macrophytes leaves and result of the study, showed significant variation in the initial mineral nutrient concentration in these three. mineral nutrient concentration was considerably high in decaying macrophyte as shown in table 1, in compare to control maize leaves as well as soil under consideration. the decomposition of macrophytes release higher concentration of phosphorus and nitrogen and its assimilation was also higher in plants as reported by lu et al. [8]. the synthesis of the phenolic acids, flavonoids, quinones, flavonols, phenols, coumarins and tannins like aromatic secondary metabolites in plant takes place to acquire resistant against the adverse environmental stress [9]. and in the present study, such compounds present in significantly higher amount in macrophytes leaves. phytochemical constituents of the macrophytes showed presence of different phytochemical constituents as alkaloid, saponins, cardiac glycosides, steroids, phenols and flavon -oides (table 2), but there was considerable variation in its constituents in fresh and decaying macrophytes. phenolics having broad spectrum biochemical activity like antimicrobial, antitumors, antioxidant and also has the ability to alter the expression of related gene in plant under stress. the majority of the antioxidant activities in plants have been carried out by phenolics, which is a largest group of phytochemical [10]. flavonoids are type of phenolic compounds naturally produced in the plant either in free or as glycosides have numerous biological functions like protein kinase inhibition, antioxidants, antimicrobial and mitochondrial adhesion inhibition etc.with the development of resistant towards the pesticides, there is requirement for ecologically sustainable natural alternatives or plants derivates as a choice to enhanced crop production. plant induced phyto-chemicals as well as plant extracts have antimicrobial properties against different phytopathogen including bacteria. plants interact with its changing surrounding environment efficiently due to production of secondary metabolites, which helps in acquiring tolerance towards harsh environment for sustainable adaptation. biotic stress in plants resulted in retarded highlights in bioscience page 3 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production table 1. concentration of minerals nutrient concentration in soil, hydrophytes and maize leaves (n=5). s.no sample n (mgkg-1 ) p (mgkg-1 ) k (mgkg-1 ) zn (mgkg-1 ) ca (mg kg-1 ) 1 soil 19.5±0.12 952.1±1.12 26246±0.11 285.4±1.27 11782±0.16 2 decaying hydrophytes 32.6±1.3 1372.4±2.11 58362±0.23 429.2±1.32 14467±1.37 3 maize leaves 27.9±1.21 1068.2±0.16 31583±0.21 363.3±2.01 12947±0.13 values are mean of three replications (p≤0.05; lsd test). . table 2. qualitative analysis of phytochemicals in fresh (a) and decaying (b) macrophytes. phytochemicals a b alkaloid + + saponins + + quinones steroids + phenols + + flavonoides + + tannins + coumarines + + reducteurs + +: present and : absent. plant growth with enhanced secondary metabolites production as phenolic compounds to defend biotic stress [11]. phenolic and flavonoids metabolites of plants are organic compounds having aromatic rings with one or many hydroxyl groups. in this study, the phytochemicals constitutes as total phenolic and total flavonoid contents were quantitatively analyzed in the macrophyte and result showed that, the total phenolics content was higher in decaying macrophyte, while total flavonoids content was higher in fresh macrophyte (table 3), and play major role in attaining maximum yield potential by providing nitrogenous product in normal state as well as in low soil nutrient. also presence of significant amount of phenolic and flavonoids provide protection against wide range of phytopathogen. table 3. quantitative analysis of phytochemicals in in fresh (a) and decaying (b) macrophytes. phytochemicals a b total phenolic content (mg/g of extract) 3.34±0.01 4.76±0.12 total flavonoid content (mg/g of extract) 6.83±0.02 5.37±0.02 for each parameter, values are mean of three replications (p≤ 0.05; lsd test). the use of synthetic pesticides is a quick and effective tool to manage plant diseases. but nonstop and arbitrary use of such synthetic pesticides in the crop field has several deleterious effects on human health and ecosystem, due to its incorporation in food chain and residual toxicity. use of synthetic pesticides attributes to long degradation periods, elevated acute toxicity, and gathering in the food chain. so the use of biological agents is an economically viable and safe option, to reduce the use of chemicals application. in the present study, the antibacterial activity of the decaying macrophytes showed significant effect on the both pseudomonas aeruginosa and s. aureus potential phytopathogenic bacteria observed during agar disc diffusion method, by formation of clear zone around the disc having different leave extract (figure 1). figure 1. effect of macrophytes plant extract (water, ethyl acetate, and methanol extract) with distill water as control on the bacterial pathogen on nutrient agar plate as zone of inhibition (zoi). where a for pseudomonas aeruginosa and b for s. aureus. plant growth is a complex process and the plant production depends on availability of sunlight, chemical energy, carbon source, mineral nutrients and water from the soil. many mineral nutrients in proper proportion are necessary for plant growth to attain its full strength yield. the plant having limited above ground resources like sunlight, and carbon source, then plant enhance the biomass allocation to the shoots and when there is limited below ground resources like mineral nutrients and water, then plant enhance the biomass allocation to the roots resulted in shunted growth according to functional balance theory[12]. so for proper plant growth the main nutrients required are potassium, phosphorus and nitrogen. these primary nutrients are accountable for limiting crop growth and among them nitrogen, is most intensively used element. nitrogen is often unavailable in the correct form to be utilized by the plant. in the present study, concentration of important mineral nutrients was analyzed in maize leaves grown in different condition. in this study, the nutrient content of the maize leaves has been assayed and result showed that concentration of mineral nutrient was significantly high in the maize leaves grown in low soil nutrient state supplemented with decaying macrophytes (table 4). so, use of decaying macrophytes as under soil bed has ability to enhance the nutrient content of maize for its the growth and development in low nutrient soil. such organic agriculture plays an important role in minimizing the use of inorganic fertilizers highlights in bioscience page 4 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production table 4. effect of decaying macrophytes on minerals concentration under low soil nutrient condition in maize leaves (n=5). s.no sample n (mgkg-1 ) p (mgkg-1 ) k (mgkg-1 ) zn (mgkg-1 ) ca (mg kg-1 ) 1 maize in low nutrient soil 26.3±1.02 2383.1±1.02 73246±0.21 724.4±1.04 13782±1.43 2 maize in low nutrient soil + decaying macrophytes 32.6±0.11 3272.4±0.78 78362±0.64 729.2±2.11 14467±0.87 3 maize in low nutrient soil + chemical fertilizer 28.9±0.21 2668.2±0.65 76583±0.73 733.3±1.87 13947±1.23 values are mean of three replications (p≤ 0.05; lsd test). and consequently minimizes potential pollution and improves soil quality. it is important to develop new applications which are sustainable both agronomically and economically, as well as environmentally to produce safe food for human consumption. so the use of biological based compounds of plant extracts is considered successful and environment friendly option for sustainable agriculture. references 1. reitsema re, meire p and schoelynck j. the future of freshwater macrophytes in a changing world: dissolved organic carbon quantity and quality and its interactions with macrophytes. front. plant sci.2018, 9: 629. 2. etienne p, diquelou s, prudent m, salon c, maillard a, ourry a. macro and micronutrient storage in plants and their remobilization when facing scarcity: the case of drought. agriculture. 2018, 8: 14. 3. pravaiz ahmad l, bhardwaj ak, shah kw. macrophyteas powerful natural tools for for water quality improvement. research journal of botany. 2014, 9:24-30. 4. vidya s, girish l. water hyacinth as a green manure for organic farming. international journal of research in applied natural and social sciences 2014, 2(6):65-72. 5. zandalinas si, sales c, beltrán j, gómez-cadenas a, arbona v. activation of secondary metabolism in citrus plants is associated to sensitivity to combined drought and high temperatures. front plant sci. 2017, 7:1954. 6. salawu so, ola-salawu bb, ogundare ao, akindahunsi aa. antimicrobial activities of phenolic containing extracts of some tropical vegetables. afr j pharm pharmacol 2011, 5: 486-492. 7. mathur r, vijayvergia r. determination of total flavonoid and phenol content in mimusopselengi linn. int j pharm sci res. 2017, 8(12): 5282-85. 8. lu j, bunn se, burford ma. nutrient release and uptake by littoral macrophytes during water level fluctuations. sci total environ. 2018, 622-623:29-40. 9. jha y, subramanian rb, sahoo s. antifungal potential of fenugreek coriander, mint, spinach herbs extracts against aspergillus niger and pseudomonas aeruginosa phyto-pathogenic fungi. allelopathy journal 2014, 34 (2): 325-334. 10. okpuzar j, ogbunugafor h, kareem gk, igwo-ezikpe mn. in vitro investigation of antioxidant phenolics compounds in extract of sennealata. res j phytochem. 2009, 3:68–76. 11. compean kl, ynalvez ra. antimicrobial activity of the plant secondary metabolites; a review. j med plant res 2014, 8: 204213. 12. hendrik p , karl jn, peter b, reich j, oleksyn p, poot liesje m. biomass allocation to leaves, stems and roots: meta-analyses of inter specific variation and environmental control. new phytol. 2012, 193:30–50. highlights in bioscience page 5 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods aquatic plants collection and preparation of samples estimation of mineral nutrient concentrations of soil, hydrophytes and maize leaves preparation of leaf extract qualitative analysis of phyto-chemical screening quantitative analysis on phytochemicals constituents phyto-pathogenic microorganism effect of decaying macrophytes on minerals concentration under low soil nutrient condition in maize leaves effect of decaying macrophytes on plant growth parameters statistical analysis results and discussion references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20207 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 6 research article open access 1 department of quantum electronics, faculty of physics, uzhhorod national university, ukraine contacts of authors * to whom correspondence should be addressed: alexander shuaibov citation: shuaibov a, minya a, shevera i, malinina a, gritsak r, malinin a, gomoki z, danilo v. (2020). characteristics of bipolar nanosecond discharges in air formed in the electrode system “blade-surface of nonmetallic liquid -blade”. highlights in bioscience volume 3. article id 20207. dio:10.36462/ h.biosci.20207 received: may 20, 2020 accepted: june 27, 2020 published: july 4, 2020 copyright: © 2020 shuaibov et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. characteristics of bipolar nanosecond discharges in air formed in the electrode system “blade-surface of nonmetallic liquid -blade” alexander shuaibov* 1 , alexander minya 1 , igor shevera 1 , antonina malinina 1 , roksolana gritsak 1 , alexander malinin 1 , zoltan gomoki 1 , vladislav danilo 1 abstract curcumin the design of the device for producing a high-current, bipolar nanosecond discharge over the surface of a non-metallic liquid (water, electrolytes, alcohols, etc.) in air is given. air pressure is ranged from 5 to 101 kpa. the distance between the tip of the blade and the surface of water or liquid (5% solution of copper sulfate in distilled water) was 4 mm, and the distance between parallel metal blades was 40 mm. the conditions for uniform plasma overlapping of the electrolyte surface between the metal blades are established. the spatial, electrical, and optical characteristics of the discharge are investigated. it is shown that the discharge under study allows obtaining colloidal solutions of copper nanoparticles in distilled water in a macroscopic amount (1 liter or more). the developed reactor is of interest for use in poisonous chemical solution disinfection systems, solutions based on dangerous bacteria and viruses for which the use of traditional systems with a point spark discharge or a barrier discharge becomes ineffective. the rector is also promising for the synthesis of colloidal solutions of transition metal oxide nanoparticles from solutions of the corresponding salts. these solutions can be used in micro-nanotechnology and for antibacterial treatment of plants in greenhouses, processing of medical instruments and materials. keywords: nanosecond discharge, distilled water, copper sulfate, electrolytes, alcohols, colloidal solutions, copper nanoparticles. introduction recently, intensive use of various gas-discharge sources of ultraviolet (uv) radiation in medicine, microbiology, chemistry, sanitation and ecology has been observed [1–9]. promising for these purposes is the use of “cold” plasma jets, which are also formed mainly in different gas-discharge devices [10]. when using such uv emitters, including open ones, operating in atmospheric air and not having a quartz shell, for the action of biologically or chemically active solutions on the surface, the distance between the lamp and the liquid is several centimeters. the duration of the radiation pulses of such gas-discharge emitters is tens to hundreds of microseconds, and the operating voltage of their power sources does not exceed 10-11 kv. this leads to a decrease in the pulsed power of radiation compared with nanosecond lamps. simultaneously, the fluxes of active particles (such as hydroxyl radicals oh, hydrogen atoms h, and oxygen atoms -o from the plasma are eliminated because they do not have time to shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 6 penetrate the surface of the treated fluid during their lifetime (which does not exceed 10 ns). the use of jets of “cold” plasma for surface treatment of biologically and chemically active solutions is often limited to the small working surface of the plasma jet. therefore, the development of gasdischarge devices to obtain near-surface plasma of a relatively large area, which can be used for treating surfaces of non-metallic liquid media in air to solve microbial and medicine issues that are not able to solve by known sources of uv radiation and plasma, is relevant [11,12] . in previous research [13], the characteristics of the discharge in the air above the water surface are given. here, a high-voltage tip electrode was used, to which a highvoltage pulse of negative polarity was applied, and a flat, grounded metal electrode was placed in water near the opposite end of an opened discharge cell. the voltage pulse had amplitude of up to 20 kv and duration of 5-1000 μs. the distance between the electrodes reached 20 cm, and the cathode distance from the water surface was 0.1-1 cm. the discharge channel had a width of 0.3-0.5 cm and its main part was parallel to the water surface and separated from it by a fraction of a millimeter. since the plasma channel had a small width, it could not cover a large surface area of water. with a current pulse duration of tens of microseconds 1 ms, heating of the liquid, which is undesirable when working with biosolvents, can be essential. therefore, it is important to study the formation of nanosecond discharges over the surface of non-metallic liquids in air, allowing us to obtain large width discharges in the system of electrodes based on metal blades. for these purposes, the technique of forming plasma electrodes developing on the surface of solid dielectrics (fiberglass laminate, leucosapphire, etc.) can be used. such electrodes have been used in high-pressure lasers pumped with a transverse volume discharge, in particular, on exciplex molecules 308 nm xecl (x-b) [14]. the main disadvantage of such plasma electrodes based on solid dielectrics is their small service life associated with the destruction of a dielectric by a powerful discharge in an aggressive gaseous medium. in our experiments, it was proposed to obtain a nanosecond discharge over the surface of liquid nonmetallic media, which tend to self-recover in the course of operation, which significantly increases their service life. the aim of the work was to develop a gasdischarge reactor based on a surface discharge with a liquid nonmetallic electrode for applications in medicine, biology, agriculture and nanotechnology. this article presents the results of a study of the spatial, electrical, and optical characteristics of a highcurrent nanosecond discharge over the surface of distilled water and a solution of cuso4 salt in distilled water, which was formed in air at pressures of 5–101 kpa. materials and methods technique and conditions of the experiment the use of steel blades in the system for obtaining a high-current nanosecond discharge in air at atmospheric pressure when the discharge in the form of 12 plasma sheets was closed to the surface of distilled water or electrolytes was described in [15–16]. but in this case, the ratio of the area of the overlapped plasma to the total area of the electrode based on the system of steel blades did not exceed 0.3-0.4. the characteristics of the discharge and the system of electrodes, consisting of two steel blades installed under an angle varying during the experiment to each other at some short distance from the surface of non-metallic liquids, were described in [17–18]. in this case, it was also not possible to completely overlap the surface of liquid medium by plasma, which was located under the steel blades. as in the first case, and in the second case, colloidal solutions based on nanoparticles of copper, zinc and iron oxides in a volume of about 10 3 cm 3 were synthesized in such gasdischarge plasma-chemical reactors. therefore, in this study as a basis to create a surface discharge, the design and power supply scheme for the nanosecond surface discharge of plasma electrodes of high-pressure lasers pumped by a transverse volume discharge was chosen [14]. a highcurrent nanosecond discharge at air pressures in the range of 5–101 kpa was ignited in the “blade – electrolyte surface – blade” electrode system in the discharge module, the circuit of which is shown in figure 1. figure 1. 1 discharge chamber made of plexiglass, 2 a cuvette with a non-metallic liquid (diameter 120 mm, height 16 mm), 3 platforms with an electrode system “blade-surface of non-metallic liquid-blade”, 4 grounded metal plate, 5 system adjusting the height of the electrodes above the surface of the liquid (h = 0-15 mm), 6 the system for regulating the distance between steel blades (l = 40-80 mm), 7 the system for pumping out and gases inlet, filling the cuvette with liquid and adjusting the system of electrodes in height, 8 quartz window for recording spectra and radiation from the plasma discharge gap, 9 glass shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 6 window for visual, photographic, video surveillance, 10 window for adjustment and access to the system of adjusting the distance between the blades.u1 -, u2 connection points for the leads from a high-voltage nanosecond source of the discharge ignition. the electrode system was installed in a sealed discharge chamber made of dielectric. the air pressure was varied in the range of 5-101 kpa for a liquid electrode based on a 5% solution of copper sulfate (cuso4) salt in distilled water and was 101 kpa for an electrode based on distilled water. the distance between the surface of the non-metallic liquid and the tips of the thin steel blades was 4 mm, and the distance between the parallel steel blades was 40 mm. the level of the working fluid was kept constant with the help of a special drip system and an additional tank with liquid. the discharge cuvette was made of organic glass and could be pumped out with a vacuum pump to a residual pressure of 10 pa. the overpressure of the gases in the cuvette could reach 200 kpa. the electrodes were made of steel blades, 0.1 mm thick. the discharge cell was installed in the screen of their metal grid, which reduced the influence of the electromagnetic fields of the discharge ignition system on the recording system of its electrical and optical characteristics. the characteristics of a nanosecond surface discharge in the system of electrodes «blade – electrolyte surface – blade» were studied using an experimental setup described in [18–19]. a high-current nanosecond discharge on the surface of the liquid was ignited using a high-voltage source of bipolar nanosecond voltage pulses with a resonant recharge of the storage capacitor with a capacity of 1.54 nf and a hydrogen thyratron switchboard. the amplitude of the voltage pulses of the same polarity on the voltage input of the high-voltage modulator could vary in the range of 10-25 kv. the amplitude of the main maximum of the current pulse reached 100-170 a. the repetition frequency of nanosecond voltage and current pulses was in the range of 35-1000 hz. radiation of surface discharge plasma was recorded in the spectral range λ = 200-665 nm using a spectrometer with a diffraction grating of 1200 lines / mm mdr-2. in the output of the spectrometer, a photomultiplier tube feu-106 connected to a dc amplifier was used to detect radiation. the signal from the amplifier was fed to an analog-to-digital converter and then fed to a personal computer for processing. to estimate the energy contribution to the discharge plasma, current and voltage oscillograms were recorded using a 6-channel wideband oscilloscope 6lor04. the voltage pulses on the electrodes were applied through a capacitive divider to one of the channels of the oscilloscope, and the discharge current pulses were measured using a calibrated rogowski belt on the other channel of the oscilloscope. the synchronization of the pulses of the discharge power supply with the launch of the 6lor-04 oscilloscope was carried out using a g5-15 pulse generator. the disadvantage of the proposed device is the use of static blades as electrodes. during prolonged operation of the reactor, the erosion of steel electrodes occurs and the discharge loses a little in uniformity. in the industrial version of the device, the electrodes must be made of refractory alloys based on tungsten, molybdenum or tantalum, as in high-pressure dischargers in systems for the formation of nanosecond pulses at a current amplitude level of hundreds of amperes. results and discussion spatial, electrical and spectral characteristics of surface discharge in each experiment on the ignition of a surface discharge in the air with a liquid nonmetallic electrode, together with a study of its spectral characteristics, its spatial and electrical characteristics (current and voltage waveforms) were also recorded. using the oscillograms of the voltage across the discharge gap and the discharge current, we determined the pulsed electric power of the discharge and the energy input to the plasma per pulse (figure 2). figure 2. the dependence of the pulsed electric power of the discharge on time, oscillograms of voltage and current of a bipolar nanosecond discharge above the surface of a 5% solution of cuso4 salt in distilled water at the air pressure in the discharge chamber equal to 6.5 kpa. the dependence of the pulsed electric power of the discharge on time was obtained by graphically multiplying the oscillograms of voltage and current, and the magnitude of the energy input to the discharge per pulse was determined by integrating the discharge power of the discharge over time. shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 6 for the discharge of atmospheric pressure over the surface of distilled water, the analysis of the voltage and current waveforms showed that the current pulse has an amplitude of up to 50 a and a duration of about 50 ns and is formed under the action of a voltage pulse with an amplitude of 30 kv, which had a duration of 30 ns. due to the mismatch of the output impedance of the pulsed voltage generator on the voltage oscillograms in the conditions of these experiments, an oscillatory structure was observed. the maximum value of the pulse electric power of the discharge during the first 30 nanoseconds since the moment of ignition of the discharge reached 1 mw. in one discharge pulse, the energy input to the plasma was 30 mj. with a decrease in air pressure in the discharge module to 6.5 kpa, the energy input to the plasma per pulseincreased threefold (figure 2). at the same time, the amplitude of the current pulse reached 150 a for a duration of 70 ns. the voltage pulse had an amplitude of 20 kv and a duration of 25 ns. the pulsed electric power of the discharge reached 3 mw. figures (3 and 4) show images of a nanosecond discharge on the surface of distilled water at an air pressure of -101 kpa and on the surface of a 5% solution of a copper sulfate at air pressure of 6.5 kpa. figure 3. the image of the discharge glow above the surface of distilled water at an air pressure of 101 kpa. figure 4. image of the discharge above the surface of a 5% solution of a cuso4 salt at an air pressure in the discharge chamber of 6.5 kpa. in the first case, the discharge consisted of a fairly uniform set of streamer channels, which were observed against a weak background of a diffuse surface discharge. the nanosecond discharge in air of atmospheric pressure in the system of electrodes "blades surface of distilled water" had a similar appearance [15]. the brightness of the plasma glow slightly decreased in the direction from the tip of the steel blade to the center of the discharge gap, which is probably due to this distribution of the specific electric field strength of the bipolar discharge. when the air pressure in the discharge chamber was reduced to 5–25 kpa (figure 4), the surface discharge acquired a diffuse appearance and completely covered the liquid surface between the steel blades, which were installed parallel to each other. figure 5 shows the emission spectrum of the discharge plasma above the surface of distilled water at an air pressure of 101 kpa. the results of identification of this emission spectrum are presented in table 1. as can be seen from table 1, the bands of the second positive system of the nitrogen molecule prevail in the plasma emission spectrum, and the main part of the radiation was concentrated in the spectral range of 280-390 nm. figure 5. the emission spectrum of the discharge above the surface of distilled water at an air pressure in the discharge chamber equal to 101 kpa. table 1. the results of the interpretation of the emission spectrum of the discharge formed above the surface of distilled water (p = 101 kpa). wavelength, nm molecule v’ v’’ e(v’), ev 297.68 n2 2 0 0,128 315.93 n2 1 0 0,376 337.13 n2 0 0 0,128 357.69 n2 0 1 0,128 371.05 n2 2 4 0,619 375.54 n2 1 3 0,376 380.49 n2 0 2 0,128 394.3 n2 2 5 0,619 399.84 n2 1 4 0,376 405.94 n2 0 3 0,128 shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 6 the absence of hydroxyl radicals bands in the plasma emission spectrum indicates that the density of water vapor in the plasma is low, that is, the plasma heats the water surface a little and can be considered “cold” (not more than 50° c). this is important for biological and medical applications of the investigated discharge. compared with the known plasma jets [10], this source can simultaneously process relatively large surfaces of non-metallic solutions. the characteristic plasma emission spectrum of a nanosecond discharge above the surface of a 5% solution of cuso4 salt in distilled water at an air pressure of 6.5 kpa (figure 6) differs fundamentally from the spectrum for discharge above the water surface by the presence of relatively intense spectral lines of the copper atom that enters the plasma from the solution. figure 6. the emission spectrum of the discharge above the surface of a 5% solution of cuso4 salt in distilled water at an air pressure in the discharge chamber equal to 6.5 kpa. the results of the identification of the spectrum shown in figure 6 in part of the radiation objects entering the discharge from the surface of the solution are summarized in table 2. but even in this case, the bands in the second positive system of the nitrogen molecule, located in the spectral range of 280-390 nm, were major in the spectrum. after treatment the surface of the copper sulfate solution for 2-3 hours at a pulse repetition rate of 100-150 hz, the solution changed its color from blue to green. this indicates the complete transformation of copper cations and anions (so4) in solution into a colloidal solution based on copper oxide nanoparticles (figure 7). figure 7. images of copper sulfate solutions in distilled water before and after plasma treatment of a nanosecond discharge in air at atmospheric pressure. the green color of the solution corresponds to the radiation at the wavelength of the plasmon resonance of copper oxide nanoparticles. this makes it possible to recommend the introduction of small copper sulfate additives in a variety of biologically active solutions in order to enhance the bactericidal effect of uv illumination of the surface of the solution and of copper oxide nanoparticles with a strong bactericidal effect [20, 21]. conclusion thus, the conditions for obtaining a uniform discharge in the air above the surface of non-metallic liquids are established; the maximum pulsed discharge power is in the range (1-3) mw with an energy input to the plasma in one pulse (30-900) mj; in the emission spectra of the plasma, the emission of the second positive system of nitrogen molecule bands in the spectral range of 280-390 nm prevails, but for the plasma on the surface of the copper sulfate solution, a relatively intense emission of copper atoms entering the discharge from the surface of the solution was detected; treatment of the salt of copper sulfate with surfacedischarge plasma allows the synthesis of colloidal solutions based on nanoparticles of copper oxides, promising for use in nanotechnology, microbiology and medicine. table 2. the results of the identification of objects of the radiation of the discharge plasma above the surface of a 5% solution of copper sulfate at air pressure of 6.5 kpa. wavelength, nm atom elow., ev eup., ev lower term upper term 249.215 cu i 0 4,97322 3d 10 4s 2 s1/2 3d 9 ( 2 d)4s4p( 3 p ° ) 4 p3/2 319.41 cu i 1,642133 5,52246 3d 9 4s 2 2 d3/2 3d 9 ( 2 d)4s4p( 3 p°) 4 d 3/2 353.038 cu i 1,642133 5,15286 3d 9 4s 2 2d3/2 3d 9 ( 2 d)4s4p( 3 p ° ) 4f ° 5/2 405.112 cu i 5,724385 8,7842 3d 9 (2d)4s4p( 3 p ° )3/2 3d 9 4s( 3 d)4d2p 3/2 525.20 cu i 5,52246 7,88305 3d 9 ( 2 d)4s4p( 3 p ° ) 4 d°3/2 3d 9 4s( 3 d)5s 4 d 3/2 529.25 cu i 5,394745 7,73659 3d 9 ( 2 d)4s4p( 3 p ° ) 4 d°7/2 3d 9 4s( 3 d)5s 4 d7/2 shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 6 references 1. piskarev im, ivanova ip, samodelkin ag, ivaschenko mn. initiation and investigation of free-radical processes in biological experiments, novgorod. nizhny novgorod state agricultural academy. 2016; p. 140. 2. arkhipova ev, ivanova ip. membrane structural condition and functional activity of peritoneal macrophages after gas discharge exposure. medical technologies in medicine/sovremennye tehnologii v medicine. 2017 sep 1;9(3). 3. piskarev im, astaf'eva ka, ivanova ip. sources of gasdischarge plasma: effect of the absorbed dose and active particle composition on physicochemical transformations in biological substrates. medical technologies in medicine/sovremennye tehnologii v medicine. 2018 jun 1;10(2). 4. vasilyak lm. application of pulsed electrical discharge lamps for bactericidal treatment. surface engineering and applied electrochemistry. 2009 feb 1;45(1):26-34. 5. wasserman al, shandala mg, yuzbashev vg. the use of ultraviolet bactericidal irradiators for disinfecting indoor air. overview of the current state of the problem. epidemiology and hygiene-journal issued by medical alphabet. 2013 3: 24-30. 6. shuaibov ak, shimon ll, dashchenko ai, shevera iv. excimer low pressure emitter for the spectral region of 170-310 nm. instruments and experimental technique. 2002, 1: 104-106. 7. shuaibov a, gritzak r. optical characteristics of uv– vuv lamps on the electronic-vibrational transitions of the hydroxyl radical pumped by a nanosecond capacitive discharge. high voltage. 2017 jul 11;2(2):78-81. 8. shuiabov ok, grabova ia, shevera iv. gas-discharge uv-vuf exciplex and halogen lamps of low pressure. monograph. uzhgorod. uzhhorod national university "hoverla". 2018, p. 260. 9. shuyabov ok, hrytsak rv. ultraviolet lamps on radicals of hydroxyl and exciplex molecules with pumping by barrier nanosecond discharge. monograph, uzhhorod, uzhhorod national university "hoverla", 2018, p. 114. 10. sosnin ea, tarasenko vf, panarin va, skakun vs. apokamp: a new type of plasma jet. horizons in world physics. (ed. a. remer) nova science, 2017, (292) 551. 11. hozák p, scholtz v, khun j, mertová d, vaňková e, julák j. further contribution to the chemistry of plasmaactivated water: influence on bacteria in planktonic and biofilm forms. plasma physics reports. 2018 sep 1;44(9):799-804. 12. piskarev im. effect of spark discharge plasma on water, physiological saline, and hanks’ solution. high energy chemistry. 2018 jul 1;52(4):348-54. 13. vaulin dn, yershov ap, kamenschikov sa, chernikov va. high-voltage pulse discharge propagating along a water surface. high temperature. 2011 jun 1;49(3):356-62. 14. shuaibov ak, neimet ii, sinishin aa, kindrat ss, shevera vs. characteristics of a quasi-stationary xecl excimer laser with plasma electrodes. ukfiz. 1989 apr;34:535-7. 15. shuaibov ak, chuchman mp, mesarosh lv. characteristics of a glow discharge in atmospheric pressure air over the water surface. technical physics. 2014 jun 1;59(6):847-51. 16. shuiabov ok, minya ya o, gomoki zt, shevera iv, danilo vv. method of synthesis of copper oxide nanostructures in nanosecond discharge with electrolytic cathode. patent ua, no. 118235 с2 jul, 2018. 17. shuaibov ak, minya ai, enedi al, shevera iv, gomoki zt, danilo vv. characteristics of a nanosecond discharge with a liquid nonmetallic electrode in the air. surface engineering and applied electrochemistry. 2018 jan 1;54(1):1-5. 18. shuiabov ok, minya oy, gomoki zt, shevera iv, danilo vv. a method of ignition of a spatially homogeneous discharge of atmospheric pressure in a system with a water electrode. patent for utility model. application number: u 2016 05309; date of application: 16.05.2016; the date from which the rights to the utility model are valid: 10.11.2016; publication of information about the issuance of a patent: 10.11.2016, bul. no. 21 19. shuaibov ak, vasil c. parameters of high current nanosecond discharge in air wich electrolytic electrode. international scientic-technical magazine “measuring and computing devices in technological processes”. 2016; 56(3): 45-48. 20. shulgina ta. aqueous solution of copper nanoparticles and its antimicrobial activity. advances in current natural sciences. 2012; 12: 143-143. 21. egorova em. biological effects of metal nanoparticles. medical science. 2014, 350. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202108 research article open access 1 electronic engineering department, federal university of rio de janeiro, rio de janeiro, brazil. 2 department of electrical engineering, hakim sabzevari university, sabzevar, iran. 3 city university health sciences center, federal university of rio de janeiro, biochemistry, rio de janeiro, brazil. 4 department of electrical engineering, azad university, sabzevar, iran. contacts of authors * to whom correspondence should be addressed: h.r.khezri2@gmail.com received: march 23, 2021 accepted: june 15, 2021 published: august 3, 2021 citation: khezri h, farzaneh m, ghasemishahrestani z, moghadam ap . ann-based diagnosis method for skin cancers using dermoscopic images. 2021 aug 3;4:bs202108 copyright: © 2021 khezri et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. ann-based diagnosis method for skin cancers using dermoscopic images hamidreza khezri*1 >< , mojtaba farzaneh2 >< , zeinab ghasemishahrestani3 >< , ali pakizeh moghadam4 >< abstract melanoma is one of the most dangerous skin cancers in the world. it accounts for 55% of all deaths associated with skin cancer. researchers believe that skin cancer increases the risk of other cancers if not diagnosed early. therefore, prompt and timely diagnosis of this disease is very important for the successful treatment of the patient. this system can detect melanoma lethal carcinoma from other skin lesions without the need for surgery, with a low cost, accuracy of about 98.88% and specificity 99%. in this article, a new, intelligent and accurate software (delphi) system has been used to diagnose melanoma skin cancer. to detect malignant melanoma, the abcdt rule, asymmetry (a), boundary (b), color (c), diameter (d) and textural variation (t) of the lesion are calculated and finally, an artificial neural network (ann) is used to obtain an accurate result. the ann with multi-layer perceptron (mlp) contains the five extraction characteristics (abcdt) of lesions is used as inputs, two hidden layers, and two outputs. very good results were obtained using this method. it was observed that for a dataset of 180 dermoscopic lesion images including 80 malignant melanomas, 20 benign melanomas and 80 nevus lesions. due to its automatic recognition and ability to be installed on a computer, this system can be very useful for dermatologists as well as the general public. keywords: melanoma skin cancer, abcdt rule, feature extraction, artificial neural network introduction unfortunately, skin cancer has become very popular these days. melanoma is caused by genetic mutations in the pigment-producing cells called melanocytes [1]. melanoma affects mostly both sexes of the caucasian population [2], and the prognosis of the disease becomes very poor at the metastatic stage [3-4]. there are no effective treatments for metastatic melanoma [5], and has grown rapidly over the past 30 years [6]. according to clinical definitions, malignant lesions are not regularly [6]. figure figure 1 shows the symmetric and asymmetric lesions. diagnosis of melanoma in the early stages of the disease can certainly prevent the death of patients. usually for two reasons skin lesions turn from benign to malignant: first, lack of attention to the skin lesions on body surface. second, high similarity of skin lesions features and inaccessibility to a dermatologist. for example, figure 2 shows two very similar skin lesions, malignant melanoma and clark’s nevus, which is a benign skin lesion. melanoma is the most common skin disease that can lead to death [7], which often begins with malignant pigment cell tumors that cause more than 70% of deaths in patients with skin cancer [8-9]. it should be noted that if skin cancer is not diagnosed in the early stages, it can affect different parts of the body, including the liver, bones, lungs and brain, and makes the treatment process very difficult and complicated. the need for an automatic and accurate device for reduce unnecessary biopsy and rapid detection is quite clear. therefore, a method for early detection of melanoma is very useful and valuable [10-11] so that dermatologists can use this system to diagnose skin lesions with high accuracy. in fact, melanoma is evaluated by clinical imaging. dermoscopy is often used to assess melanoma lesions, a non-invasive type of image analysis. a new approach is shown here, which examines the skin lesion image by a trained neural network to analyze if it is benign or malignant. the article is arranged as follows. highlights in bioscience page 1 of 7 august 2021|volume 4 https://doi.org/10.36462/h.biosci.202108 https://creativecommons.org/licenses/by/4.0/ h.r.khezri2@gmail.com https://orcid.org/0000-0002-5839-3690 mojtaba.farzaneh@gmail.com https://orcid.org/0000-0002-6037-6486 farzaneh.ghasemi.sha@gmail.com https://orcid.org/0000-0002-2482-4296 a.pakizehmoghadam@gmail.com http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images figure 1. a:symmetric and b: asymmetric melanoma lesions. figure 2. clark nevus. the second section describes previous work on melanoma analysis in skin images. in the third section, the new contribution to this article describes the analysis, feature extraction, classification, ann, details, tests and evaluations. section iv presents the conclusions. as a basic step towards computer-aided skin cancers, automatic diagnosis, and image analysis have often been studied in the literature [12-19]. in the last few years, many studies have been done to detect melanoma from skin images with an accuracy of 70% to 96%. telemedicine techniques have been studied as a source for the diagnosis of skin lesions. compared to physicians in-person diagnoses (face to face) and telemedicine diagnoses (remote detection), tests on skin diseases have shown that the use of telecommunications technology diagnosis (teledermatology) is more effective and safer. these techniques include the benefits of easy access, low cost, and quick and accurate access to treatment results [20-22]. melanocytic cutaneous lesions have been reported to be the deadliest among the three skin cancer outbreaks and the second most common among adults aged 15 to 29 years [2]. melanoma is less common in asia, africa and latin america than in australia, europe, north america and new zealand. melanomas sometimes change in appearance, including changes in size, irregular edges, and discoloration, itching or fracture of the skin [23]. in fact, melanoma can rarely occur in the mouth, intestines, or eyes, but is most commonly found on the skin. it is common in men and women in the back and legs, respectively [24]. the automatic detection of asymmetry in digital images was proposed by the stolz technique based on abcd rule [25]. a study on the asymmetry using imaging techniques to identify melanoma skin lesions was presented by ravichandran et al [26]. to date, many researchers are working on image processing, visual techniques, and various melanoma parameters such as size, shape, asymmetry, border, color, and diameter to detect skin cancer [27-31]. one of the known methods is the abcd rule. the algorithm for detection is divided into four steps: asymmetry, border, color, and diameter. abcd is a fast learning, calculation and a reliable way to diagnose melanoma [32-34]. lesion irregularity, borders, colors and diameters can be analyzed and calculated by dividing the image of the lesion into sub-images and extracting the properties of each image [35]. finally, melanoma could be detected by a simple threshold for the values obtained from the extracted features (lesion irregularity, borders, colors and diameter) [36]. these features are used as inputs to the first layer of the ann [37-38]. we’ve developed the novel abcdt rule by improve the abcd, for automatic diagnosis of skin cancer with greater accuracy and precision. in other words, in the current study, the extraction of features is done based on the abcdt rule in dermoscopy. materials and methods this article aims to develop a new, intelligent and accurate software system for skin cancer diagnosis using neural network and abcdt rule. the input of the device is images of skin lesions. this system with pre-processing, abcdt rule, and separation, extract appropriate features from the image. to get the total dermoscopic score (tds), for each of the "asymmetries, boundary, colors, diameters and texture changes", a coefficient is determined by which the tds can be calculated. in other words, to obtain tds (table 1, the score of each "abcdt" is multiplied by a specific weight factor. finally, ushighlights in bioscience page 2 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images ing nn as a smart medical decision-making system based on tds, the type of lesion is determined to be melanoma or benign. in other words, an nn has been used to implement the new automated classification of melanocytic lesions. table 1. the proposed diagnostic criteria. diagnostic criteria score weight factor asymmetric (a) 0-5 1.3 border (b) 0-8 0.5 color variation (c) 1-6 1 diameter (d) 0-1 0.1 textural variation (t) 1-10 0.9 in this work, briefly, after the detection and elimination of noise and hair on the image, abcdt rule, bayes learning algorithm, and the neural network method (feed forward back propagation) were used to detect the lesion is classified as benign or malignant. as shown in figures 3 and 3, the architecture of the skin cancer smart system used in this study consists of the five steps of pre-processing, segmentation, feature extraction, classification and diagnosis as follows: • pre-processing: it involves filtering and contrast enhancement techniques using the retinex algorithm. resize pictures, to analyze and compare data with the bank, all images are transformed into the same size. this size is 450×350 selected. • blurring and segmentation: the purpose of blurring is to reduce noise. if we use edge detection algorithms for highresolution images, we will find many results that we are not interested in. conversely, if we blur the images too much, we will lose data. so, we have to find the amount of blurring we want to use without destroying the desired edges. there are various techniques to achieve blurred effects, but gaussian blur is used with a factor of 2 to remove the noise and the hairs on the skin. so, first, the image is converted to binary format. using filters, detect and eliminate hair and noise from the image. then, the lesion image is completely separated from the background. in other words, the exact location of the lesion on the image is determined by calculating the threshold and statistical characteristics. • feature extraction: abcdt rule examines the characteristics of a lesion. these properties include asymmetry, border, color, diameter, and textural variation. this rule is a development of the known abcd rule [39], commonly used to diagnose melanoma from images. the extracted properties are fed to the first layer of the nn. properties used to characterize the asymmetry of the lesion the asymmetric feature of the lesion is one of the important features in diagnosis. natural moles are usually symmetrical. asymmetry is usually calculated in two ways: entropy and bifold. to calculate the asymmetry score, each lesion is examined by two 90-degree axes, and the ann determines its score. if the lesion is properly symmetrical on both axes, this score is 0, and if it is only on one axis, it will be 2.5. in case of asymmetry in both axes, the score is 5. finally, the asymmetry score must be multiplied by 1.3 as the weight factor. features used for irregular characterization at the lesion border uneven or irregularly shaped margins increase the likelihood of some kind of skin lesion. to calculate boundary irregularities, the lesion is divided into eight sections. if the entire border of the lesion has a severe incision, it is given a maximum score. otherwise the minimum score of 0 is given. the minimum and maximum score of b are defined 0 and 8, respectively with the 0.5 weight factor. features used to characterize lesion color variation color properties are calculated between six colors, and each color represents 1 point. properties used for diameter according to our information, if the lesion diameter is larger than 0.6 mm, the risk of cancer is higher. diameter with a weight coefficient of 0.1 is measured by converting the total number of pixels in the largest diameter to millimeters (mm). characteristics used to characterize the textural variation of the lesion since healthy skin is reddish, and skin lesions have more textural variation and lower pixel intensity than healthy areas of the skin. for this purpose, first, a low pass filter was used to normalize the color and then extracted the correct features [40]. the textural variation values are considered from 1 to 10 with a weight factor of 0.9. classification the different features extracted from the lesion surface have different weights. the weight of each group was drawn based on the experience of dermatologists. table 1 shows the importance of each group. the total dermoscopy score (tds) can be calculated using eq. (1). t ds = 1.3 × a + 0.5 × b + 1 × c + 0.1 × d + 0.9 × t (1) where: a, b, c, d, and t scores are for the asymmetry feature, the border irregularity, the color feature, the diameter size feature, and the textural variation, respectively. highlights in bioscience page 3 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images figure 3. a flowchart illustrating the proposed machine learning system for detecting skin cancers using dermoscopic images. figure 4. clark nevus. diagnosis if individual scores of asymmetry, boundary, color, diameter, and textural variation are multiplied by weightfactor of 1.3, 0.5, 1, 0.1, and 0.9, respectively, a precise distinction can be made between benign and malignant melanocytic lesions. the tds greater than 5.40 means a cancerous lesion, otherwise, it is benign. results and discussion all of these steps and the final decision (benign or malignant) are performed by a trained neural network. various classifications have been performed by researchers for the anns [43-46]. in this work, the ann with a multi-layer perceptron (mlp) utilizes a supervised learning technique and includes features extracted as inputs to the input layer with two hidden layers containing 10 and 7 neurons for each layer (figure 5). the mlp uses a region-oriented hybrid algorithm, a method called elliptical symmetry to determine asymmetry, a gaussian smoothing to measure boundary irregularities, and a threshold method for the lesion segment. therefore, the system designed to diagnose melanoma uses five features of the lesion. the images used in this work were taken from the international skin imaging collaboration (isic) [41]. out of this dataset, 180 dermoscopic lesion images including 80 malignant melanomas, 20 benign melanomas and 80 nevus lesions were extracted and preprocessed for this research. in several studies [42-44] they used abcd rule to detect and analyze pictures, whereas in our study we added one more factor (abcdt) to increase the efficiency and get the better result. we found out the t factor has a huge impact on increasing accuracy. highlights in bioscience page 4 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images figure 5. the neural network diagram of the proposed mlp model. given the weight factor (0.9) and values of the (0-10) change intended for t, the number obtained from t has a great effect on the result, and this is the most important difference between this work and other similar tasks. according to the accuracy (acc) and specificity (spec) obtained and compared with pennisi et al. [47], fan et al. [48], jahanifar et al. [49] and sreelatha et al. [50] can be claimed that the average performance of the technique used in this study is better than previous techniques (table 2). table 2. the average performance evaluation metrics (%). algorithm acc spec pennisi [47] 89.40 97.10 fan [48] 93.60 jahanifar [49] 97.90 98.20 sreelatha [50] 98.64 99.22 proposed mlp 98.88 99.00 the proposed method for the diagnosis of melanoma skin cancer by abcdt method revealed 98.88% accuracy and specificity 99%. this approach is safe, accessible, effective, noninvasive and based on the principles of telemedicine with high accuracy and reasonable price. with this software system, people can make an early diagnosis of their skin lesions without referring to a physician and specialists can use it as an intelligent, fast and accurate assistant. in summary, the abcdt rule, bayes learning algorithm, and neural network method were used to detect the type of carcinogenic or non-cancerous lesions. according to tds, it is a clear fact that the three features of asymmetry, color, and textural variation of the lesion are crucial in the diagnosis of melanoma from benign lesions. a tds value above 5.40 indicates melanoma. in abcdt method, all steps perform by a fully automated neural network. references 1. tolleson wh. human melanocyte biology, toxicology, and pathology. journal of environmental science and health part c. 2005 jul 1;23(2):105-61. 2. ferlay j, soerjomataram i, dikshit r, eser s, mathers c, rebelo m, parkin dm, forman d, bray f. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. international journal of cancer. 2015 mar 1;136(5):e359-86. 3. bombelli fb, webster ca, moncrieff m, sherwood v. the scope of nanoparticle therapies for future metastatic melanoma treatment. the lancet oncology. 2014 jan 1; 15(1):e22-32. highlights in bioscience page 5 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images 4. batus m, waheed s, ruby c, petersen l, bines sd, kaufman hl. optimal management of metastatic melanoma: current strategies and future directions. american journal of clinical dermatology. 2013 jun; 14(3):179-94. 5. kaufman hl, margolin k, sullivan r. management of metastatic melanoma in 2018. jama oncology. 2018 jun 1; 4(6):857-8. 6. balch cm, gershenwald je, soong sj, thompson jf, atkins mb, byrd dr, buzaid ac, cochran aj, coit dg, ding s, eggermont am. final version of 2009 ajcc melanoma staging and classification. journal of clinical oncology. 2009 dec 20;27 (36):6199. 7. celebi me, kingravi ha, uddin b, iyatomi h, aslandogan ya, stoecker wv, moss rh. a methodological approach to the classification of dermoscopy images. computerized medical imaging and graphics. 2007 sep 1;31(6):362-73. 8. lau ht, al-jumaily a. automatically early detection of skin cancer: study based on nueral netwok classification. in2009 international conference of soft computing and pattern recognition 2009 dec 4 (pp. 375-380). ieee. 9. yuan x, yang z, zouridakis g, mullani n. svm-based texture classification and application to early melanoma detection. in2006 international conference of the ieee engineering in medicine and biology society 2006 aug 30 (pp. 47754778). ieee. 10. whited jd. teledermatology research review. international journal of dermatology. 2006 mar;45(3):220-9. 11. ganster h, pinz p, rohrer r, wildling e, binder m, kittler h. automated melanoma recognition. ieee transactions on medical imaging. 2001 mar;20(3):233-9. 12. rose vl. cancer facts and figures. american family physician. 1999 mar 15;59(6):1697. 13. shrestha b, bishop j, kam k, chen x, moss rh, stoecker wv, umbaugh s, stanley rj, celebi me, marghoob aa, argenziano g. detection of atypical texture features in early malignant melanoma. skin research and technology. 2010 feb ;16(1):60-5. 14. sadeghi m, razmara m, lee tk, atkins ms. a novel method for detection of pigment network in dermoscopic images using graphs. computerized medical imaging and graphics. 2011 mar 1;35(2):137-43. 15. sadeghi m, razmara m, wighton p, lee tk, atkins ms. modeling the dermoscopic structure pigment network using a clinically inspired feature set. ininternational workshop on medical imaging and virtual reality 2010 sep 19 (pp. 467-474). springer, berlin, heidelberg. 16. anantha m, moss rh, stoecker wv. detection of pigment network in dermatoscopy images using texture analysis. computerized medical imaging and graphics. 2004 jul 1;28(5):225-34. 17. betta g, di leo g, fabbrocini g, paolillo a, sommella p. dermoscopic image-analysis system: estimation of atypical pigment network and atypical vascular pattern. inieee international workshop on medical measurement and applications, 2006. memea 2006. 2006 apr 20 (pp. 63-67). ieee. 18. celebi me, iyatomi h, stoecker wv, moss rh, rabinovitz hs, argenziano g, soyer hp. automatic detection of blue-white veil and related structures in dermoscopy images. computerized medical imaging and graphics. 2008 dec 1;32(8):670-7. 19. shitara d, nascimento m, ishioka p, carrera c, alos l, malvehy j, puig s. dermoscopy of naevus-associated melanomas. acta dermato-venereologica. 2015 jun 1;95(6):671-5. 20. barata c, marques js, rozeira j. detecting the pigment network in dermoscopy images: a directional approach. in2011 annual international conference of the ieee engineering in medicine and biology society 2011 sep (pp. 5120-5123). ieee. 21. korotkov k, garcia r. computerized analysis of pigmented skin lesions: a review. artificial intelligence in medicine. 2012 oct 1;56(2):69-90. 22. balch cm, buzaid ac, soong sj, atkins mb, cascinelli n, coit dg, fleming id, gershenwald je, houghton jr a, kirkwood jm, mcmasters km. final version of the american joint committee on cancer staging system for cutaneous melanoma. journal of clinical oncology. 2001 aug 15;19(16):3635-48. 23. freedberg ka, geller ac, miller dr, lew ra, koh hk. screening for malignant melanoma: a cost-effectiveness analysis. journal of the american academy of dermatology. 1999 nov 1;41(5) :738-45. 24. rigel ds, friedman rj, kopf aw. the incidence of malignant melanoma in the united states: issues as we approach the 21st century. journal of the american academy of dermatology. 1996 may 1;34(5):839-47. 25. intraocular b. melanoma treatment (pdq): health professional version. pdq cancer information summaries. 2015. 26. wild c, stewart bw. world cancer report 2014. wild cp, stewart bw, editors. geneva, switzerland: world health organization; 2014. 27. cudek p, grzymaa-busse jw, hippe zs. further research on automatic estimation of asymmetry of melanocytic skin lesions. inhumancomputer systems interaction: backgrounds and applications 2 2012 (pp. 125-129). springer, berlin, heidelberg. 28. premaladha j, ravichandran ks. asymmetry analysis of malignant melanoma using image processing: a survey. journal of artificial intelligence. 2014 apr 1;7(2):45. 29. jain s, pise n. computer aided melanoma skin cancer detection using image processing. procedia computer science. 2015 jan 1;48:735-40. 30. ananthi b, balamohan s, hemalatha m. melanoma detection using rgb color model in medical imaging. middle-east journal of scientific research. 2014;21(11):1982-7. 31. iqbal s, sophia m, divyashree j, mundas m, vidya r. implementation of supervised learning for melanoma detection using image processing. international journal of research in engineering and technology. 2015;4(6):325-9. 32. grammatikopoulos g, hatzigaidas a, papastergiou a, lazaridis p, zaharis z, kampitaki d, tryfon g. automated malignant melanoma detection using matlab. inproc. fifth int. conf. on data networks, communications and computers, bucharest, romania 2006 oct 16. 33. iqbal s, sophia m, divyashree ja, mundas m, vidya r. implementation of stolzs algorithm for melanoma detection. international advanced research journal in science, engineering and technology. 2015;2(6):9-12. highlights in bioscience page 6 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images 34. stolz w. abcd rule of dermatoscopy: a new practical method for early recognition of malignant melanoma. eur. j. dermatol.. 1994;4:521-7. 35. ahnlide i, bjellerup m, nilsson f, nielsen k. validity of abcd rule of dermoscopy in clinical practice. acta dermato-venereologica. 2016 mar 1;96(3):367-72. 36. bareiro paniagua lr, leguizamón correa dn, pinto-roa dp, vázquez noguera jl, salgueiro toledo la. computerized medical diagnosis of melanocytic lesions based on the abcd approach. clei electronic journal. 2016 aug;19(2):6-. 37. shih ty. the reversibility of six geometric color spaces. photogrammetric engineering and remote sensing. 1995 oct;61(10) :1223-32. 38. mirzaalian h, lee tk, hamarneh g. learning features for streak detection in dermoscopic color images using localized radial flux of principal intensity curvature. proc. ieee workshop math. methods biomed. image anal, 2012. 39. aswin rb, jaleel ja, salim s. implementation of ann classifier using matlab for skin cancer detection. international journal of computer science and mobile computing. 2013 dec;1002:8794. 40. messadi m, cherifi h, bessaid a. segmentation and abcd rule extraction for skin tumors classification. arxiv preprint arxiv :2106.04372. 2021 jun 8. 41. santiago-montero r, asael d, hernandez g. border and asymmetry measuring of skin lesion for diagnostic of melanoma using a perimeter ratio. asian j comput sci and inf technol. 2016;6(2). 42. jaleel ja, salim s, aswin rb. artificial neural network based detection of skin cancer. international journal of advanced research in electrical, electronics and instrumentation engineering. 2012 sep;1(3). 43. ahmed k, jesmin t, rahman mz. early prevention and detection of skin cancer risk using data mining. international journal of computer applications. 2013 jan 1;62(4). 44. thirumavalavann s, jayaraman s. ann based computer aided diagnosis and classification of skin cancers. power and computing technologies. 2017;12(4).pp1137-1142. 45. alam fi, faruqui ru. optimized calculations of haralick texture features. european journal of scientific research. 2011 mar 1;50(4):543-53. 46. codella nc, gutman d, celebi me, helba b, marchetti ma, dusza sw, kalloo a, liopyris k, mishra n, kittler h, halpern a. skin lesion analysis toward melanoma detection: a challenge at the 2017 international symposium on biomedical imaging (isbi), hosted by the international skin imaging collaboration (isic). in2018 ieee 15th international symposium on biomedical imaging (isbi 2018) 2018 apr 4 (pp. 168-172). ieee. 47. pennisi a, bloisi dd, nardi d, giampetruzzi ar, mondino c, facchiano a. skin lesion image segmentation using delaunay triangulation for melanoma detection. computerized medical imaging and graphics. 2016 sep 1;52:89-103. 48. fan h, xie f, li y, jiang z, liu j. automatic segmentation of dermoscopy images using saliency combined with otsu threshold. computers in biology and medicine. 2017 jun 1;85:75-85. 49. jahanifar m, tajeddin nz, asl bm, gooya a. supervised saliency map driven segmentation of lesions in dermoscopic images. ieee journal of biomedical and health informatics. 2018 may 22; 23(2):509-18. 50. sreelatha t, subramanyam mv, prasad mg. early detection of skin cancer using melanoma segmentation technique. journal of medical systems. 2019 jul;43(7):1-7. highlights in bioscience page 7 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ abstract introduction materials and methods properties used to characterize the asymmetry of the lesion features used for irregular characterization at the lesion border features used to characterize lesion color variation properties used for diameter characteristics used to characterize the textural variation of the lesion classification diagnosis results and discussion references highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 1 of 7 citation: girish k. and dubey s. (2018). higher organism’s molecular databases: an overvie. highlights in bioscience, volume 1.article id 20184, dio:10.36462/ h.biosci.20184 received: march 5, 2018 accepted: april 28, 2018 published: may 10, 2018 copyright:© 2018 girish and dubey. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. eukaryotic molecular biology databases: an overview kumar girish 1 and singh dubey 2 1 department of biotechnology, indian agricultural research institute, india. 2 department of biology, agricultural university, india abstract a biological database is a big, structured body of continuous information, generally connected with computerized software intended to update, query, and recover information elements deposited within the framework. a straightforward database could be a single folder comprising several data, each carrying the same number of data. such famous databases are genbank from the national center for biotechnology information, swissprot from the swiss institute of bioinformatics and pir from the protein information resource. biological databases are bibliotheques of life science data, gathered from science studies, published literature, high-performance experimental technology, and computational analysis. here we brefly described some recently published molecular databases. keywords: biological database, computational analysis, bioinformatics, software. introduction bioinformatics is a steadily increasing area of studies fueled by the need to handle and evaluate the vast amounts of information produced by omics techniques. decades of studies have produced several known multi-omics tools, including genome, proteome and transcriptome (1–3) with the sole objective of understanding every part of biology. here we brefly described recent published database such as phenomicdb, aagmarker, cscd, cr2cancer, activedriverdb, amtdb, hedd, diseaseenhancer, dreambase, dashr, msdd, cancersysdb, mirdip, medreaders, linkedomics, pancan-meqtl, pitdb, qprimerdb, seecancer, unilectin3d, translatomedb, tiss-gdb, and tc3a table (1). review article open access eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 2 of 7 table 1. list of some recently published database. database name link reference database name link reference phenomicdb http://www.phenomicdb.de kahraman et al. (4) mirdip http://ophid.utoronto.ca /mirdip/ tokar et al. (16) aagmarker http://bioinfo.wilmer.jhu.edu/ aagmarker/ pan et al. (5) medreaders http://medreader.org/ wang et al. (17) cscd http://gb.whu.edu.cn/cscd xia et al. (6) linkedomics http://www.linkedomics.org vasaikar et al. (18) cr2cancer http://cis.hku.hk/cr2cancer ru et al. (7) pancan-meqtl http://bioinfo.life.hust.edu.cn/ pancan-meqtl/ gong et al. (19) activedriverdb https://www.activedriverdb.org krassowski et al. (8) pitdb http://pitdb.org saha et al. (20) amtdb https://amtdb.org ehler et al. (9) qprimerdb http://biodb.swu.edu.cn/ qprimerdb lu et al. (21) hedd http://zdzlab.einstein.yu.edu/1/ hedd.php wang et al. (10) seecancer http://biocc.hrbmu.edu.cn/ seecancer zhang et al. (22) diseaseenhancer http://biocc.hrbmu.edu.cn/ diseaseenhancer/ zhang et al. (11) unilectin3d https://www.unilectin.eu/ unilectin3d mariethoz et al. (23) dreambase http://rna.sysu.edu.cn/dreambase zheng et al. (12) translatomedb http://www.translatomedb .net/ liu et al. (24) dashr http://lisanwanglab.org/dashrv2 kuksa et al. (13) tiss-gdb http://zhaobioinfo.org/ tissgdb kim et al. (25) msdd http://tdb.ccmb.res.in/msdb yue et al. (14) tc3a http://tc3a.org feng et al. (26) cancersysdb https://cancersys.uni-koeln.de krempel et al. (15) phenomicdb by combining government genotype/phenotype information from a broad spectrum of model organisms and modern humans, kahraman et al. (4) built a multi-species genotype / phenotype repository. they collected these riches of information into a single embedded database through coarse-grained semiconductor mapping of phenotypic information areas, including prevalent gene codes (ncbi gene) and the use of related orthology. phenomicdb enables researchers to concurrently compare and browse recognized phenotypes for a specified gene or a collection of genes from distinct species with its use-case-oriented interface. it can be found at http://www.phenomicdb.de. aagmarker pan et al. (5) has collected many released raw information sets on proteome microarrays acquired from serum profiling assays and offers a toolbox for mining these information. aagmarker's present edition includes 854 serum samples with 136 092 proteins. for 12 diseases, such as alzheimer's disease, bechet's disease, and parkinson's disease, a total of 7803 (4470 non-redundant) candidate auto virus biomarkers were recognized and gathered. in order to quantitatively evaluate these biomarkers, seven statistical parameters were implemented. users can use fundamental search, sophisticated search and browse to collect, analyze and compare the datasets. these biomarkers can also be downloaded in terms of disease. the aagmarker can be found at http://bioinfo.wilmer.jhu.edu/aagmarker/. cscd xia et al. (6) gathered 228 complete samples of rna or polya(-) rna-seq from both cancer and ordinary cell lines and found 272,152 circr-nas specific to cancer. in ordinary samples only a total of 950 962 circrnas were recognized, and in both tumor and ordinary samples 170 909 circrnas were detected, that could be even farther used as non-tumor context. a cancer-specific circrna databases http://gb.whu. edu.cn/cscd were built. they anticipated the microrna response element locations and rna binding protein positions for each circrna in order to comprehend the functional impacts of circrnas. in addition, prospective open reading frames were anticipated to show translatable circrnas. they also identified the splicing occurrences in linear transcripts of each circrna to comprehend the correlation between linear splicing and back splicing. as the first extensive circrna database specific to cancer. cr2cancer ru et al. (7) introduced cr2cancer, an extensive database for crs in human cancer annotation and visualization built through high-throughput data analysis and mining literature. for more than 400 crs across various kinds of cancer, they gathered and incorporated genomic, transcriptomic, proteomic, clinical and functional data. they also constructed various kinds of cr-related relationships, including dependent and autonomous cancer type relationships. in addition, about 6000 items of aberrant molecular alterations and cr interactions were manually curated from 5007 publications in the development of cancer. cr2cancer offers an easy-to-use online interface to navigate, http://www.phenomicdb.de/ http://bioinfo.wilmer.jhu.edu/%20aagmarker/ http://bioinfo.wilmer.jhu.edu/%20aagmarker/ http://medreader.org/ http://gb.whu.edu.cn/cscd http://www.linkedomics.org/ http://cis.hku.hk/cr2cancer http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ https://www.activedriverdb.org/ http://pitdb.org/ https://amtdb.org/ http://biodb.swu.edu.cn/%20qprimerdb http://biodb.swu.edu.cn/%20qprimerdb http://zdzlab.einstein.yu.edu/1/%20hedd.php http://zdzlab.einstein.yu.edu/1/%20hedd.php http://biocc.hrbmu.edu.cn/%20seecancer http://biocc.hrbmu.edu.cn/%20seecancer http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ https://www.unilectin.eu/%20unilectin3d https://www.unilectin.eu/%20unilectin3d http://rna.sysu.edu.cn/dreambase http://lisanwanglab.org/dashrv2 http://zhaobioinfo.org/%20tissgdb http://zhaobioinfo.org/%20tissgdb http://tdb.ccmb.res.in/msdb http://tc3a.org/ https://cancersys.uni-koeln.de/ http://www.phenomicdb.de/ http://bioinfo.wilmer.jhu.edu/aagmarker/ eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 3 of 7 search and download interesting information conveniently. http://cis.hku.hk/cr2cancer, is readily accessible. activedriverdb activedriverdb is a comprehensive database of human proteogenomics which annotate disorder mutations and variants of the population via the ptm lens. more than 385,000 released ptm locations with 3.6 million replacements from the cancer genome atlas (tcga), the clinvar disease gene database, and human genome mapping projects were incorporated (8). the database involves sitespecific protein interaction networks, upstream enzymes like kinases, and those enzyme-targeted drugs. also, by evaluating advantages and losses of kinase-bound gene motifs, anticipated network-rewiring effect of mutations. for researching ptm-associated mutations, activedriverdb offers comprehensive visualization, processing, navigating, and search opportunities. users can interactively submit mutation datasets and use the coding tool in pipelines. integrative assessment of mutations and ptms, as illustrated by case studies of tp53, brca2 and vhl, can assist decipher molecular processes of disease and phenotypes. it can be found at https://www.activedriverdb.org. amtdb amtdb, the first database of ancient human mitochondrial genomes, is presented by ehler et al. (9). release edition includes 1107 hand-cured ancient samples, freely available for download, along with personal descriptors including geographic place, radiocarbon dating, and association to archeological culture. an interactive map for sample position visualization is also available in the database. on https://amtdb.org, amtdb is a main platform for ancestral population genetic research. hedd wang et al. (10) founded the human enhancer disease database (hedd) to promote enhancers research and their prospective functions in complicated human disorders. for some 2,8 million human enhancers recognized by en-code, fantom5 and roadmap, hedd presently offers extensive genomic data with disease connection results based on enhancer-gene and gene-disease links. it also offers webbased analytical tools to conceptualize enhancer networks and rank enhancers in a particular gene network given a collection of targeted genes. at http://zdzlab.einstein.yu.edu/ 1/hedd.php, hedd is readily available. diseaseenhancer zhang et al. (11) developed diseaseenhancer for disease-associated enhancers, a manually curated database. as of july 2017, in 143 human illnesses, diseaseenhancer involves 847 disease-associated enhancers. database attributes include fundamental data about enhancers; varieties of diseases; related variations of enhancers and their phenotypes mediated. they also include a website function for exporting any request outcomes to a folder and downloading the complete database. diseaseenhancer offers scientists with a successful opportunity for knowing enhancer deregulation in pathogenesis of disease and identifying new biomarkers for diagnosis and treatment of disorder. you can easily access diseaseenhancer at http://biocc.hrbmu.edu.cn/ diseaseenhancer/. dreambase zheng et al. (12) established dreambase to enable research into dna alteration, rna regulation and protein interacting from multidimensional high-throughput sequencing information of probable expressed pseudogenes. based on a series of data sets chip-seq and dnase-seq, genome-wide binding patterns of different transcriptionassociated variables were recognized around pseudo-gene loci. by incorporating some 18 000 rna-seq information, they evaluated pseudogene expression profiles and examined their modelsof co-expressionin 32 cancers and 31 ordinary tissues with their parent genes.they revealed complicated post-transcription regulation networks comprising 275 micrornas and 1201 pseudo-genes by connecting binding sites with microrna. they researched transcriptome-wide relationships based on 458 clip-seq datasets between rna binding enzymes (rbps) and pseudo-genes. they also linked 1039 rna alteration locations to 635 pseudo-genes in combination with epi-transcriptome sequencing information. this database would provide perspectives into pseudo-genes ' transcription regulating, expression, features and mechanisms as well as their functions in biological procedures and illnesses. at http://rna.sysu.edu.cn/dreambase, dreambase is readily available. dashr kuksa et al. (13) created a comprehensive catalog of annotation, expression, handling, conservation, tissue specificity and other biological characteristics for all human sncrna genes and mature products obtained from all main rna groups. the dashr (small human non-coding rnas database) database is the first to incorporate human sncrna gene as well as mature product profiles from numerous rna-seq protocols. for both grch38/hg38 and grch37/hg19 assemblies, 185 tissue / cell forms and sncrna annotations and > 800 curated tests from encode and geo / sra are incorporated into dashr. in addition, dashr is the first to comprise both recognized and novel http://cis.hku.hk/cr2cancer https://www.activedriverdb.org/ https://amtdb.org/ http://zdzlab.einstein.yu.edu/%201/hedd.php http://zdzlab.einstein.yu.edu/%201/hedd.php http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ http://rna.sysu.edu.cn/dreambase eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 4 of 7 sncrna loci detected by unsupervised segmentation, earlier unnoted. dashr also contains > 3,200,000 annotations for non-small genes of rna and other genomic characteristics. in addition, an improved user interface, integrated experiment-by-locus table display, sncrna locus sorting and biological feature processing is introduced. on http:// lisanwanglab.org/dashrv2, dashr is readily accessible. msdd yue et al. (14) have been described as msdd, presently documenting 525 associations among 182 human mirnas, 197 snps, 153 genes, and 164 human illnesses through an analysis of over 2000 articles released. each organization includes data on mirnas, snps, mirna target genes and disorder designations, snp places and allies, mirna dysfunctional model, experimental methods, a short functional description, initial reference and extra annotation. msdd offers a user-friendly interface for browsing, retrieving, downloading and submitting new information conveniently. msdd there is free access to http://tdb.ccmb. res.in/msdb. cancersysdb the cancer systems biology database (cancersysdb) was created by krempel et al. (15), a tool for extremely flexible cancer queries and data analysis across various data types and various research. a cancersysdb primary openly accessible instance can be used to achieve extremely versatile queries across various kinds of information as shown in extremely appropriate instances of use. they also show how the cancersysdb can be used in the study network the cancer genome atlas (tcga) to classify predictive cancer based on all-exome information from 9091 individuals. cancersysdb at https://cancersys.uni-koeln.de, it is publicly accessible. mirdip tokar et al. (16) provided mirdip, providing practically 152 million predictions of human microrna targets collected across 30 various resources. they also incorporated an interdisciplinary score, statistically implied from the predictions obtained and appointed to each unique microrna-target activity to provide a unified measure of trust. they show that integration of predictions throughout various assets does not accumulate predictive bias towards biological procedures or pathways. mirdip is accessible free of charge at http://ophid.utoronto.ca/mirdip/. medreaders a total of 731 tfs that could attach to methylated dna sequences in human and mouse research mentioned in the literature were individually selected by wang et al. (17). six human cell lines and one mouse cell line derived from the encode and geo database were used in silico approaches to anticipate methylated and un-methylated patterns of 292 tfs by incorporating whole genome bisulfite sequencing (wgbs) and chip-seq data. the database of medreaders provides an extensive resource for further research and experiment plans to guide. the portal can be found at http://medreader.org/. linkedomics the database of linkedomics (18) includes information on multi-omics and clinical information for 32 kinds of cancer and a total of 11,158 clients from the tcga initiative. it is also the first multi-omics repository to incorporate worldwide proteomics information from chosen tcga tumor samples based on mass spectrometry (ms) produced by the clinical proteomic tumor analysis consortium (cptac). linked omics has over a billion information points in total., to enable a extensive assessment of these data, the web application linked omics developed three analytical modules. the link finder module enables for flexible discovery of connections between a molecular or clinical trait of concern and all other characteristics, offering a chance to evaluate and visualize connections for each cancer cohort between billions of attribute pairs. they show that linked omics offers biologists and clinicians with a distinctive platform for accessing, analyzing and comparing multi-omics cancer information within and across tumor kinds. on http://www.linkedomics.org, linked omics is readily accessible. pancan-meqtl gong et al. (19) provided pancan-meqtl, a repository that integrates genome-wide genotype and dna methylation information to provide meqtls across 23 cancer kinds from the cancer genome atlas. in maximum, 8,028,964 cis-meqtls and 965,050 trans-meqtls were recognized. overall patient survival rates are correlated with 23,432 meqtls. in addition, 2,214 458 meqtls were recognized that overlap with identified loci recognized by genome-wide correlation studies. pancan-meqtl offers an easy-to-use web interface http://bioinfo.life.hust.edu.cn/ pancan-meqtl/ for browsing, scanning and accessing of interesting information. pitdb pitdb (20) is a free repository of translated genomic elements (tges) found in pit (transcriptomics-informed proteomics) studies. in pit, both rna-seq transcriptomics as well as proteomic mass spectrometry are used to evaluate a https://cancersys.uni-koeln.de/ http://ophid.utoronto.ca/mirdip/ http://medreader.org/ http://www.linkedomics.org/ http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 5 of 7 sample. transcripts constructed from rna-seq reads are being used to develop a library of sample-specific amino acid sequences that are searched for the obtained mass spectra, allowing the detection of any tge, not just those in canonical proteome databases. pitdb includes more than 74 000 separate tges from four species at the moment of writing, backed by more than 600 000 matches in the peptide spectrum. the database, available at http://pitdb.org, offers guiding evidence for each tge, often from multiple studies and an indicator of trust in the assessment and form of the tge, varying from recognized protein to multiple protein component kinds including different splice isoforms, to a putative novel molecule. qprimerdb lu et al. (21) established the qprimerdb repository based on the design of an automatic gene-specific qpcr primer and the verification workflow depending on thermodynamics. the qprimerdb database is the most comprehensive qpcr primary database currently available, with a web front-end that provides gene-specific and precomputed primary pairs across 147 major organisms including human, mouse, zebra fish, yeast, thale cress, rice, and corn. this database provides 3331426 of the best priming pairs for each gene, based on priming pair coverage, and 47760359 alternative gene-specific priming pairs, that can be easily downloaded in batch format. for qpcr priming pairs for 66 randomly chosen genes, the specificity and effectiveness was validated in six distinct species through qpcr assays and gel electrophoresis and it is open to the public via http://biodb.swu.edu.cn/qprimerdb. seecancer zhang et al. (22) has constructed a database on seecancer, which serves to show an extensive developmental stage of cancer-specific somatic occurrences and their temporal orders. by manually curing more than 10 000 published papers, 1231 developmental stage-specific genomic occurrences and 5772 temporal orders containing 82 human cancers and 23 tissue origins were gathered and recorded in the seecancer database. each entry includes the somatic occurrence, the developmental phase, the sort of cancer, the identification strategy and the appropriate evidence. seecancer offers an easy-to-use tool for browsing, finding and downloading developmental stage-specific somatic occurrences and temporal associations in multiple cancers.increasing regard to the evolution of cancer genome, the required data in seecancer has facilitated the knowledge of cancer etiology and the growth of developmental therapy, and has helped clinicians to find biomarkers for tumor progression surveillance. it is freely available via http://biocc.hrbmu.edu.cn/seecancer. unilectin3d mariethoz et al. (23) has created a unilectin3d database, a curated database that classifies source and fold lectins, cross-links to literature, many glycoscience databases and functional information such as recognized specificity. the database offers thorough data on lectins, their linked glycan ligands as well as their relationships using the protein–ligand interaction profiler (plip) server. special attention was paid to the depiction of linked glycan ligands using easy graphical depiction and statistical format of crosslinking to other glycol scientific databases.they designed the layout of the database structure and navigation instruments to account for all species, and the search for oligosaccharide epitopes complexed inside selected binding regions. unilectin3d is available link https://www.unilectin.eu/ unilectin3d. translatomedb liu et al. (24) has developed an extensive translatomedb database that offers a set and embedded assessment of published and client-generated translatom sequencing information. the present edition involves 2453 ribo-seq, 10 rnc-seq and 1394 associated mrna-seq datasets for 13 species. in relation to dataset sets, the database emphasizes the evaluation tasks. all datasets were evaluated using a unified, reliable, precise and experimentally verifiable pipeline centered on the fanse3 mapping algorithm and edger for dge analysis. translatomedb also enables clients to upload their own datasets and use the same unified pipeline to evaluate their information. accessible at http://www.translatomedb.net/. tissgdb kim et al. (25) constructed tiss-gdb (tissue specific gene database for cancer) by collecting and manually curated 2461 tissue-specific genes (tissgenes) across 22 tissue classes corresponding to 28 cancers of the cancer genome atlas (tcga) from three different tissue-specific gene cell assets: the human protein atlas (hpa), the tissuespecific gene expression and regulation (tiger) and the genoty map. these 2461 tissgenes also conducted gene expression, somatic mutation, and prognostic marker-based analyzes of 28 kinds of cancer using tcga information. tissgdb sets out seven types of annotations: tissgeneexp,tissgene summary, tissgenemut, and tissgenemirna. it is freely via http://zhaobioinfo.org/tissgdb. http://pitdb.org/ http://biodb.swu.edu.cn/qprimerdb http://biocc.hrbmu.edu.cn/seecancer https://www.unilectin.eu/%20unilectin3d https://www.unilectin.eu/%20unilectin3d http://www.translatomedb.net/ http://zhaobioinfo.org/tissgdb eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 6 of 7 tc3a feng et al. 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2018;46(november 2017):206–12. 25. kim p, park a, han g, sun h, jia p, zhao z. tissgdb : tissue-specific gene database in cancer. nucleic acids res. oxford university press; 2018;46(september 2017):1031–8. 26. feng x, li l, wagner ej, li w. tc3a : the cancer 3 utr atlas. nucleic acids res. oxford university press; 2018;46(october 2017):1027–30. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20194 highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 1 of 11 research article open access 1 mggm lab., agricultural genetic engineering researchinstitute, agricultural research center, egypt. 2 genetics department, faculty of agriculture, mansoura university. 3 faculty of pharmacy, october 6 university, giza, egypt. 4 department of biodiversity and crop improvement, international center for agriculture research in the dry areas (icarda), giza, egypt. * to whom correspondence should be addressed: smahmoud@ageri.sci.eg citation: alsamman a.m, mousa k.h., nassar a.e., shereif g.a., habib p.t. and ibrahim s.d. (2019). genome-wide identification and comprehensive study of chickpea anti-fungal genes. highlights in bioscience, volume 2. article id 20194, dio:10.36462/ h.biosci.20194 received: september 15, 2019 accepted: october 20, 2019 published: november 10, 2019 copyright: © 2019 alsamman et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests genome-wide identification and comprehensive study of anti-fungal genes in chickpea alsamman m. alsamman * 1 , khaled h. mousa 2 , ahmed e. nassar 2 , ghada a. shereif 3 , peter t. habib 4 and shafik d. ibrahim 1 abstract chickpea is an important crop that delivers nutritious food to the increasing global community and it will become increasingly popular as a result of climate change. our objective was to use comprehensive data analysis to locate and identify candidate genes for fungal disease resistance. we used a comprehensive bioinformatics pipeline of sequence alignment, phylogenetic analysis, protein chemical and physical properties assessment and domain structure classification. in order to study gene evolution and genetic diversity, we compared these genes with known anti-fungal genes in different species of plants. a total of 19721 protein sequences belonging to 187 plant species have been downloaded from public databases, including the entire chickpea genome. we have successfully identified 23 potential anti-fungal genes in 10 different chromosomes and genomic scaffolds using sequence alignment and gene annotation. ca2 and ca6 have the highest number of genes followed by ca3 and ca4. anti-fungal chickpea proteins have been identified as cysteine-rich (10), thaumatin (6), pathogenesis (4) and plasmodesmata (3) proteins. analysis of the chemical and physical correlation of anti-fungal proteins revealed a high correlation between different aspects of anti-fungal proteins. five different pattern patterns have been detected in the anti-fungal chickpea proteins identified, including domain families associated with fungal resistance. the maximum likelihood of phylogenetic analysis was successful in distinguishing between anti-fungal chickpea proteins as seen in their protein patterns / domains. keywords: chickpea, fungal resistance, phylogenetic analysis, protein domain, protein property. introduction chickpea (cicer arietinum) is an important crop that delivers nutritious food to the increasing global community and it will become increasingly popular as a result of climate change. production ranked third following beans with an average annual yield of over 11.5 million tons, where india has the highest share. land allocated to chickpea has dramatically increased and is now reported at 14.56 million hectares. more than 2.3 million tons of chickpea reach world markets every year to fund the requirements of countries unable to satisfy domestic consumption (1). alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 2 of 11 chickpea yield is significantly affected by both abiotic as well as biotic stress susceptibility (2,3). fungal infections have been shown to destructive effects on the chickpea production compared to various diseases triggered by a wide range of pathogens. within numerous fungal diseases, the most prevalent foliar and root infections are the diseases caused by ascochyta rabiei (ascochyta blight) and fusarium oxysporum (fusarium wilt), respectively, which cause serious crop yield decline (4). the genome of chickpea (2n = 2x = 16) is estimated to be 738.09 mb in length, where different cultivars whole genomes have been introduced for publicly use with more than 73% of the genomic content has been successfully sequenced. the chickpea gene pool is estimated to be about 29,000 genes, and about half (49.41%) of the chickpea genome consists of transposable elements and unclassified repetitions. on the other hand, in species-specific groups, 4,468 chickpea genes occur, these groups may arise through structural rearrangements, as happens among the disease resistance genes of nucleotide-binding site leucine-rich repeat (nbs-lrr). in this regard, it has been stated that the chickpea genome has been observed as having 187 disease resistance gene homologs (rghs) (5,6). the continuing breakthroughs in genome sequencing and genome-wide association studies have unlocked the ability to scan genomic content of chickpea for genes to control their resistance to multiple infections of the fungal disease. several predicted genes have been reported to have been statistically associated with chickpea fungal resistance, including nbs-lrr receptor-like kinase, wall-associated kinase, zinc finger protein, and serine / threonine protein kinase (7). in addition, it has been reported that chickpea resistance to some fungal diseases, such as ascochyta blight, may be linked to a number of motif families, such as at-hook motif containing nuclear localized (ahl) (8). in addition, bioinformatics methods have been accelerated over the last few years and several genomic and molecular databases have been established (9). such repositories could be used as a central stone in the quest for anti-fungal resistance in different plant species and in the analysis of their specific and special molecular structure (10,11). such bioinformatics tools have been used to study several gene families in chickpea, these families are considerably important in the plant defense system and essential membrane proteins (12,13). genome-wide characterization of anti-fungal genes in chickpea may enable researchers and breeders to overcome different fungal infections and develop new cultivars with high tolerance and better yield. our objective was to use comprehensive data analysis to locate and identify candidate genes for fungal disease resistance. we used a comprehensive bioinformatics pipeline of sequence alignment, phylogenetic analysis, protein chemical and physical properties assessment and domain structure classification. in order to study gene evolution and genetic diversity, we compared these genes with known anti-fungal genes in different species of plants. materials and methods genomic sequences related to anti-fungal resistance have been downloaded from the ncbi database. (14). a total of 19721 sequences of proteins belonging to 187 species of plants were downloaded from ncbi. the chickpea genome sequence was downloaded from the http:/www.cicer.info database (5). using the chickpea genome, the local blast+ (15) kit was used to create sequence database and align all anti-fungal amino acids with tblastn against the chickpea database. the ncbi tblastn online tool was used to annotate recovered sequences from the previous step. meme suite (16) was used to explore patterns of amino acids in chickpea anti-fungal genes. the megax program was used to perform a phylogenetic analysis using a maximum likelihood algorithm(17). in order to assess the chemical and physical characteristics of the amino acids, the pepstat program (18) was used through in-home perl scripts. these chemical properties are, a280 molar extinction coefficients cystine bridges (a280-meccb), a280 molar extinction coefficients reduced (a280-mecr), acidic (ac), aliphatic (aph), aromatic(ar), average residue weight (arw), basic (bs), charge (chr), charged (chrd), improbability of expression in inclusion bodies (ieeb), isoelectric point (ip), molecular weight (mw), non-polar (np), polar (po), residues (re), small (s), tiny (t). the itol online tool was used to visualize phylogenetic trees combined with information on amino acids (19)⁠ . the statistical correlation analysis (pvalue<0.01) was conducted using r packages (20). the circos package was used for displaying the genomic location of genes (21). results and discussion identification of chickpea anti-fungal genes identifying anti-fungal genes in chickpea could provide a useful resource for plant breeding programs by narrowing the pool of targeted genes. we have successfully identified 23 potential anti-fungal genes on 10 different chromosomes and genomic scaffolds (file s1). the total number of amino acids was 7077, ranging from 147 to 866, with an average of 307.7 amino acids. chromosomes ca2 and ca6 have the highest number of genes (4 genes) followed by ca3 and ca4 (3 genes) (figure 1 and table 1). in this regard, the entire genome re-sequencing of chickpea was used to identify 12 chromosomal regions associated with resistance to ascochyta blight, all of which alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 3 of 11 are located on ca4 (7). in addition, 8 quantitative trait loci (qtls) were identified on chromosomes ca2, ca3, ca4, ca5 and ca6 for the resistance of the same disease (22). the chemical and physical properties of chickpea anti-fungal proteins the chemical properties of the chickpea anti-fungal proteins were assessed across 17 different chemical and physical features of the amino acids. the total amino acid mw was 765.4, ranging from 16.0 kda (ca af17) to 97.7 kda (ca af9) with an average of 33.3 kda (figure 2 and table 2). by studying anti-fungal proteins in wheat total mw 1913 kda with an average of 20 kda (10). the amino acids charge ranges from -25 to (ca_af13) to 14 (ca_af9) (figure 2 and table 2).the extinction coefficient is a measure of how much light at a given wavelength a chemical element attenuates. calculating the content of the amino acid is necessary in order to determine the protein's molar extinction coefficient (23). the a280 molar extinction coefficients reduced (a280-mecr) and a280 molar extinction coefficients cystine bridges (a280-meccb) are two separate extinction coefficient measures, where salt bridges are essential motifs of the tertiary protein structure and are mostly associated with the molecular influence force that maintains the protein's stability (24). the a280-mecr and a280-meccb total values are 836030, 864530 m-1cm-1, ranging from 10430 and 11555 m-1cm-1 (ca af11) to 87560 and 90560 m-1cm-1 (ca af9), respectively (figure 2 and table 2). in some anti-fungal wheat proteins, the a280-mecc and a280-mecr minimum scores were recorded as 1740 and 1490, with the highest scores being 104570 and 103820 respectively (10). improbability of expression in inclusion bodies (ieeb) is a type of solubility measurement. in escherichia coli, for example, recombinant protein can be produced either as insoluble in the bodies of inclusion or soluble throughout the cytosol (25). the total ieeb of chickpea anti-fungal protein was 18.33 ranging from 0 (ca_af11) to 0.972 (ca_af16) with a mean of 0.797 (figure 2 and table 2). the ieib of anti-fungal amino acids revealed an average of 0.794 by examining wheat anti-fungal proteins, ranging from 0.504 to 0.977 (10). the average collective weight as per their length for all amino acid sequences is measure though the average residue weight (arw). table 1: the chromosomal location and gene definition for identified anti -fungal genes in chickpea genome. gene code chr. start end gene name gene code chr. start end gene name ca_af1 chr1 13449810 13450553 cysteine-rich ca_af13 chr5 15134097 15134777 pathogenesis-related ca_af2 chr1 39499412 39500080 thaumatin ca_af14 chr5 64978467 64979294 cysteine-rich ca_af3 chr2 15365865 15366560 plasmodesmata-located ca_af15 chr6 11037122 11037844 pathogenesis-related ca_af4 chr2 15365877 15366521 plasmodesmata-located ca_af16 chr6 12421759 12422469 thaumatin ca_af5 chr2 22066674 22068239 cysteine-rich ca_af17 chr6 13070688 13071419 cysteine-rich ca_af6 chr2 29604091 29604798 thaumatin ca_af18 chr6 65201755 65202474 plasmodesmata-located ca_af7 chr3 39833663 39834379 cysteine-rich ca_af19 chr7 24258666 24259331 thaumatin ca_af8 chr3 51987009 51987668 cysteine-rich ca_af20 chr8 18540320 18541342 cysteine-rich ca_af9 chr3 59460263 59461033 cysteine-rich ca_af21 chr8 5747704 5748729 thaumatin ca_af10 chr4 2480078 2480725 cysteine-rich ca_af22 scaffold0585 6485 7156 thaumatin ca_af11 chr4 4601812 4602510 cysteine-rich ca_af23 scaffold4365 250412 251062 pathogenesis-related ca_af12 chr4 56994913 56995608 pathogenesis-related alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 4 of 11 figure 1 : the genomic location and suggested definition of predicated anti-fungal genes, where the width of the internal links indicates similarity percentage of gene sequences. the total arw was 2478.4 da, where ca af13 and ca af14 have the minimum and maximum values of 101.8 da and 116.7 da, with an average of 107.7 da, respectively. the ieib of anti-fungal amino acids revealed an average of 0.794, ranging from 0.504 to 0.977, through examining wheat anti-fungal proteins (10). the isoelectric point (ip) is the ph level with which the net charge of the protein is positive, and is correlated with amino acid composition and protein structure (26). the ip of chickpea anti-fungal proteins range from 3.9 (ca_af13) to 8.9 (ca_af19) with a mean of 6 (figure 2 and table 2). thus, it revealed a collective ip in wheat anti-fungal proteins with an average of 6,402, ranging from 4 to 10.4 in wheat (10). in addition, the folded structure of a protein becomes less desirable in terms of thermodynamics because it decreases the protein disorder or entropy, where non-polar chains tend to squeeze inside the protein while polar chains push outside the molecule (27). the non-polar (np) values ranges from 49.2 (ca_af20) to 62.1 (ca_af16) with an average of 56 (figure 2 and table 2). the non-polar and polar amino acid scores ranged from 48.81 and 30.081 to 69.919 and 51.19 respectively, in wheat anti-fungal proteins (10). basic amino acids have a certain basic group within the chain whereas acidic amino acids have an acidic group within the chain. basic amino acids have high pka while acidic amino acids have low pka. the count of basic and acidic amino acids range from 9.011 and 9.359 to (ca_af13 and ca_af16) to 13.613 and 16.915 (ca_af19 and ca_af11) (figure 2 and table 2). alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 5 of 11 figure 2: the chemical and physical properties of identified anti-fungal proteins in chickpea. alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 6 of 11 table 2: chemical properties of identified anti-fungal proteins in chickpea. protein a280-meccb a280-mecr ac aph ar arw bs chr chrd ieeb ip mw np po re s t ca_af1 54470 52720 11.364 24.621 10.606 109.947 11.364 -2 22.727 0.82 5.8518 58051.77 51.894 48.106 528 55.303 33.712 ca_af2 29420 28420 7.207 21.622 10.811 104.536 8.108 2 15.315 0.951 7.5063 23206.95 56.306 43.694 222 69.369 47.748 ca_af3 40365 38740 10 26 9 105.494 8 -8 18 0.77 4.5636 42197.5 60.5 39.5 400 63 42.5 ca_af4 20120 19370 9.953 25.118 9.479 104.952 9.005 -3 18.957 0.787 4.929 22144.77 58.768 41.232 211 63.981 44.076 ca_af5 59135 57760 8.802 22.005 13.447 113.33 10.024 2 18.826 0.845 6.899 46352.15 49.633 50.367 409 54.279 32.763 ca_af6 40295 39420 7.623 24.215 11.659 109.223 9.417 3.5 17.04 0.949 7.7696 24356.69 57.399 42.601 223 61.435 34.978 ca_af7 12545 11920 10.596 25.166 8.609 106.551 10.596 0 21.192 0.818 6.208 16089.2 56.954 43.046 151 58.278 38.411 ca_af8 31830 30830 10.638 26.596 11.702 108.504 9.574 -5.5 20.213 0.74 4.941 30598.04 53.191 46.809 282 57.092 39.362 ca_af9 90560 87560 10.162 26.328 12.009 112.861 12.009 14 22.171 0.793 7.9722 97737.52 53.118 46.882 866 51.27 30.947 ca_af10 34155 32780 8.122 22.843 12.183 107.791 8.629 0 16.751 0.841 6.4334 42469.67 54.315 45.685 394 58.376 41.117 ca_af11 11555 10430 16.915 22.886 10.448 113.048 8.955 -17 25.871 0 4.082 22722.56 49.751 50.249 201 51.244 32.338 ca_af12 36410 35410 8.072 23.767 11.211 102.971 6.726 -4 14.798 0.843 4.6572 22962.57 61.883 38.117 223 70.404 48.879 ca_af13 58965 56840 9.545 20.455 10.909 101.798 4.545 -25 14.091 0.756 3.9557 44790.92 59.091 40.909 440 71.818 50.909 ca_af14 40545 39420 9.17 21.834 13.1 116.75 12.227 6 21.397 0.817 7.9878 26735.85 53.712 46.288 229 48.908 27.074 ca_af15 32400 31400 10 20.476 10.952 103.8 4.762 -11 14.762 0.7 3.9687 21797.95 59.048 40.952 210 69.048 51.905 ca_af16 33890 32890 5.023 23.288 12.785 106.665 10.502 10.5 15.525 0.972 8.4799 23359.67 62.1 37.9 219 63.927 41.553 ca_af17 18045 17420 10.884 25.17 10.204 108.53 9.524 -2 20.408 0.613 4.8281 15953.84 52.381 47.619 147 59.864 42.857 ca_af18 43345 41720 9.596 27.273 10.606 106.838 8.586 -4 18.182 0.814 4.8941 42307.82 60.606 39.394 396 62.121 39.899 ca_af19 23210 22460 7.33 19.895 9.424 107.128 13.613 11.5 20.942 0.949 8.9491 20461.39 58.115 41.885 191 61.78 39.267 ca_af20 29630 28880 8.333 25.794 9.524 111.225 9.127 1.5 17.46 0.856 7.2867 28028.72 49.206 50.794 252 58.333 34.524 ca_af21 32400 31400 7.895 20.175 11.842 104.278 6.14 -4 14.035 0.927 4.542 23775.41 57.895 42.105 228 67.982 49.561 ca_af22 61820 59820 9.417 18.834 10.762 105.318 6.726 -13 16.143 0.844 4.3753 46971.95 55.605 44.395 446 68.61 43.498 ca_af23 29420 28420 8.612 20.096 11.962 106.915 9.091 1 17.703 0.925 7.1537 22345.18 57.416 42.584 209 65.55 42.584 alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 7 of 11 on the other hand, in addition to the chickpea genes retrieved through this study, we have studied the chemical and physical properties of 1216 anti-fungal proteins identified in different plant species (figure 3 and table s2). the protein mw range from 21 kda (silene latifolia) to 97.7 kda (cicer arietinum) while the protein charge range from -25 (cicer arietinum) to 21 (rosa chinensis) (figure 3 and table s2). the minimum arw was 99.74 kda (striga asiatica) and the maximum 116.75 kda (cicer arietinum) (figure 3 and table s2). the a280-mecr and a280-meccb range from 30193 and 31133 to 87560 and 90560 m-1cm-1, respectively (figure 3 and table s2). the charged amino acids range from 8.108 (cephalotus follicularis) to 26.941 (populus trichocarpa) (figure 3 and table s2). figure 3 : the phylogenetic tree developed using multiple sequence alignment of chickpea anti-fungal proteins and other species, where its chemical and physical properties are plotted. alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 8 of 11 figure 4 :statistical correlation between different chemical and physical properties of anti-fungal proteins; (a) the heatmap of inter-correlation matrix and (b) the correlation networks, where pvalue > 0.01 and r 2 > 0.3. phylogenetic analysis and domain identification of anti-fungal proteins the phylogenetic tree constructed using 1216 anti-fungal proteins of 187 was clustered into 11 clusters (figure 3). the anti-fungal chickpea proteins identified are cysteine-rich (10), thaumatin (6), pathogenesis-related (4) and plasmodesmata (3) proteins (figure 3). a protein sequence motif is a brief pattern that is preserved by nature. for proteins, a motif may relate to the active site of an enzyme or to a functional unit required to properly fold proteins. hence, sequence motifs are among the basic functional components of molecular evolution (16). five specific motifs for amino acids were discovered among the sequences of identified anti-fungal proteins of chickpea alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 9 of 11 (figure 5). the maximum likelihood phylogenetic analysis was successful in distinguishing between anti-fungal chickpea proteins as illustrated by their protein motifs / domains. these motifs are motif1 (elme000385), motif2 (elme000094), motif3 (elme000321), motif4 (elme000003), and motif5 (elme000287) (figure 5). motif1 is very similar to elme000385 (pvalue of 1.60e-03), which functions as mtr4-air2-interaction site. this domain distinguishes the tramp complex, which engages in the nucleus with exosome-mediated degradation of abnormal rnase. oligo-adenylated tails are introduced to substrates of abnormal rna by air2 and mtr4 , thus highlighting them for degradation (28). motif2 has a high significance similarity with integrin binding sites (elme000094) with a pvalue of 1.16e-05. integrins are cell surface receptors which are responsible for cell migration, cell adhesion to extracellular matrix, and cell adhesion to cells (29)⁠. motif3 is similar to caspase cleavage motif (elme000321) with a pvalue of 5.46e-03. proteases caspases-3 and-7 play a major role in programmed cell apoptosis, and non-apoptotic caspases include involvement in immune response (30). similarly, pattern 5 is similar to iap-binding pattern (ibm): (elme000287) that distinguishes apoptosis protein inhibitor (iap) which exhibits several immune functions, mitosis regulation, tnf-receptor signal transduction, and many more (31)⁠. finally, motif4 was in high similar to ww domain ligands (elme000003) motif, which are small but widespread domains are found in various regulatory circumstances (16). the maximum likelihood of phylogenetic analysis was successful in distinguishing between anti-fungal chickpea proteins as seen in their protein patterns/domains. where it cluster chickpea genes into 4 clusters (figure 6). figure 5: the domains/motifs found by the meme tool in chickpea predicated anti-fungal proteins. alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 10 of 11 figure 6: the phylogenetic analysis of chickpea anti-fungal proteins, where the protein motif structures are shown as detected using meme tool. conclusion it was very helpful to predict anti-fungal resistance genes using publicly available repositories and indicated that several chickpea genes could be used to limit genetic research of genes that hold the key to fungal resistance in chickpea. we have successfully identified 23 potential anti-fungal genes on 10 different chromosomes and genomic scaffolds. a high number of anti-fungal chickpea proteins are cysteine-rich (20), thaumatin (9), and pathogenesis-related (8), which could indicate the importance of these gene classes in chickpea resistance to fungal. in addition, the chemical and physical analysis shed light on the uniqueness and consistency of these proteins, where several of these parameters could be used in future research to identify anti-fungal genes in different plant species. moreover, the domain identification analysis identified several potential anti-fungal protein domains such as tramp complex and caspase cleavage motifs. supplementary files the supplementary material for this article can be found online at: https://doi.org/10.36462/h.biosci.20194 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university, egypt contacts of authors * to whom correspondence should be addressed: mostafa essam eissa citation: rashed er, eissa me (2020). longterm quantitative assessment of women survivability from cancer: a unique descriptive analysis. highlights in bioscience volume 3. article id 20208. dio:10.36462/ h.biosci.20208 received: june 4, 2020 accepted: july 3, 2020 published: july 5, 2020 copyright: © 2020 rashed and eissa. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. long-term quantitative assessment of women survivability from cancer: a unique descriptive analysis engy refaat rashed 1 , mostafa essam eissa* 2 abstract statistical process control (spc) methodologies are a set of statistical methods and techniques that were initially designed for industrial processes but could be adopted for non-industrial applications. the current prospective study aimed to provide a unique quantitative investigation of an epidemiological disease using the spc program platform. the selected case herein was a long-term monitoring record of the yearly cancer mortality rates in women worldwide. multidimensional segregation of the dataset into subgroups was conducted to visualize the clustering pattern based on nations (42 countries as boxplot), time and the gaussian mixture model (two-interfering bell-shaped distributions approach). the trend of death rates versus the elapsed years would demonstrate a moderately negative correlation with the time following the theory of splines. construction of control chart based on the fitted weibull distribution showed a gradual steady improvement in survivability rates from malignancy. the greatest variations in the mortality ratios existed within the european countries. keywords: cancer, gaussian mixture model, mortality rate, weibull. introduction malignancy is one of the most devastating diseases that affect humanity in the modern era of civilization. it threatens human lifestyle quality, productivity, financial and economic status of society and country, in addition to morbidity, disability and mortality [1]. notably, women's health and well-being are severely impacted by the rising challenge of cancer disease in the community, which hinders them from serving their crucial role in any rising and developed nations. the burden of cancer among women is high in both high income countries (hics) and low middle income countries (lmics), although the distribution of most common cancers differs. this burden is predicted to extend as populations grow and age and because the prevalence of cancer risk factors increases in some countries, especially in lmics. the costs of cancer are considerable and even catastrophic in hics and lmics alike. however, this burden of disease, loss of life, and economic hardship is not inevitable [2]. all of the foremost common cancers among women worldwide, including lung, breast, cervix, liver, and colorectum, have known means of prevention and/or early detection, which may be used to cut back incidence and mortality. furthermore, carcinoma and cervical cancer, two of the highest four cancers in women worldwide have several proven prevention measures [3]. these two cancers combined represent about 20% of all cancer deaths among women [4]. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 8 many of those deaths may be prevented through effective tobacco control, vaccination, and screening activities. there are varieties of effective cancer control measures available to countries of all resource levels [4]. many of those measures are extremely cost-effective given the lives saved for the cost of the intervention, especially within the case of vaccination. to stop cancer within the future, countries must prioritize policies to cut back known cancer risk factors and make prevention accessible to any or all. for those that have cancer today, effective treatments and palliative care are also needed. additionally, to those needs, cancer surveillance and research for the prevention and treatment are indispensable for the setting of cancer control priorities and for determining the foremost effective interventions and coverings in an exceedingly given context [4]. for lmics, all of those activities may require support and commitment from the worldwide community. while malignant diseases are holding and exhaustive and comprehensive databases that were collected from extensive surveys and gathering centers from national and international organizations, it is the interpretation of the results that would derive useful conclusions and insight through record’s pattern [5, 6]. the application of statistical process control (spc) methodologies was historically started at the end of the first quarter of the 20 th century in the industrial field the monitoring, control and improvement of the manufacturing processes as well as product quality by walter a. shewhart at bell laboratories [7, 8]. in addition to the wide industrial applications, spc techniques have been applied even in the improvement of service fields. moreover, spc techniques were found to be useful in other non-industrial fields for assessment, evaluation and investigation of specific inspection properties or events [9, 10]. some interesting fields of implementation include but not limited to environmental monitoring (em), microbiological water quality, surgical site infection, epidemiological diseases and outbreaks [11-15]. the previous studies embraced control charts, pareto diagrams, box-and-whisker plots, distribution identification, correlation analysis and fitted line curves, in addition to other conventional analysis tools such as descriptive statistics. while epidemiological diseases constitute a continuous threat that strike humanity, it would be useful to develop a quantitative descriptive tool that describes the progression and behavior of these diseases. one of the important indicators that could provide insight into cancer progression is the mortality ratio of the patients that could be found in the database of national and international health organizations. the application of statistical tools and process control methodologies was sought as a unique metric means in the evaluation and comparison of the epidemiological status that could support in the decision-making and cancer management and control. accordingly, the present analysis might reveal new horizons for researchers to a simple, fast and useful way to study other diseases, outbreaks and pandemics. materials and methods spc perspective of long-term global trend of cancer in women cancer mortality rates records of different countries were obtained as a downloadable database file from the websites: https://www.cancer.gov/ and https://www.who.int/ [16, 17] and women data were extracted from the excel file by filtering and arranged chronologically (table s1). three different computer programs were used in data processing combined as statistical software platform viz prism graphpad v6.01, minitab v17.1.0 and excel built-in xlstat v2014.05.03 [18-21]. the prism graphpad v6.01 commercial scientific software was used for the preliminary data description followed by defining the significance estimation of the change of the yearly mortality rates with time. the excel built-in xlstat v2014.05.03, which is a statistical suite add-in, was used for drawing of gaussian mixture model (gmm), which is a probabilistic model to spot typically disseminated subpopulations inside a general populace. blend models, as a rule, do not require prior knowledge of datasets subordination to the subgroup, permitting the model to become familiar with the subpopulations spontaneously [22]. thus, it will be important in this case for viewing the overall data-clustering pattern for the record from 1960 to 2017. on the other hand, minitab as a statistical tool package was assigned for fitting line modeling, box plot creation, histogram drawing and data trending using process behavior chart [23]. two-dimensional data segregation was done based on gmm and individual countries found in the record to identify clustering pattern of data. construction of the mean (x-bar)-variation ( -s) trending chart was executed according to the distribution identification scheme in minitab. shewhart charts show mean of the death rate and the standard deviation along with the event window i.e. upper control limit (ucl) and lower control limit (lcl). results and discussion descriptive statistics and correlation output a preliminary examination of the database for the basic statistical analysis yielded a result in table 1 showing the coinciding countries with the annual values. data are mildly positive or right-skewed [24]. failure to comply with normality distribution was followed by the screening of the closest possible spreading pattern that might fit the record. a weibull distribution hypothesis could not be rejected as a suitable fit for the raw dataset based on the andersondarling test (ad). https://en.wikipedia.org/wiki/walter_a._shewhart https://en.wikipedia.org/wiki/bell_laboratories rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 8 table 1: descriptive statistics of the cancer mortality rates showing the number of deaths per 100000 individuals at ci * 95% and the corresponding countries numerically to the parameters value using prism graphpad v6.01 column statistics mortality rates (per 100000) concerned department minimum 93.30 tur 2009 25% percentile 159.1 zaf 2005 median 183.8 aus 1963, fin 1984, pol 1985 75% percentile 205.4 gbr 1962, fin 1964, chl 1991, nld 1997 maximum 286.4 isl 1960 10% percentile 140.9 grc 1983 90% percentile 227.4 lux 1984, cze 1996 arithmetic mean 183.2 swe 1987 std. deviation 32.85 na std. error of mean 0.7461 na lower 95% ci of mean 181.8 aus 1975, fin 1980, cri 1990 upper 95% ci of mean 184.7 swe 1984, svn 1988, nor 2003, nzl 2010, cze 2012, gbr 2016 lower 95% ci of median 181.8 aus 1975 upper 95% ci of median 185.4 swe 1983 and 1988, ita 1990 shapiro-wilk normality test w 0.9973 p value 0.0017 na data passing normality test (alpha=0.05) no coefficient of variation 17.93% geometric mean 180.2 svk 2006, isl 2010 lower 95% ci of geo. mean 178.7 pol 2011 upper 95% ci of geo. mean 181.7 est 1990 method of outlier detection: rout ε (q = 10.00%) no aberrant record na skewness 0.04484 na kurtosis -0.3441 na * ci = confidence interval ; na = not applicable; ε robust regression and outlier removal no aberrant values could be detected through the overall record. thus, mortality rates showed apparent concatenation in the values with a range of 193.1 deaths/100000 cases with half and four-fifths of data record resides within a range of 46.3 and 86.5 deaths/100000 cases, respectively, suggesting extensive tailing. the mean and the median were almost coincided with the geometric mean very close to the average confirming that there is no extreme value(s) distant from the group collectively [25]. however, the arithmetic mean is slightly greater than that of the geometric mean as expected for the basic rule in mathematics [26]. this gap is reflected similarly on the confidence intervals (cis) which showed the corresponding shift too. the relative standard deviation (rsd) showed significant dispersion of data around the mean of about 18%. in addition, kurtosis value suggests data broadening with shoulders that is a variation from the normal bellshaped spreading if the normal distribution is expected, a sign of possible mixed intervening patterns [24]. this would probably be the result of the impact of annual and regional variations that was reflected in the total record of women cancer mortality. table 2 demonstrates the moderately negative correlation between the women mortality rates and the time in years [27, 28]. accordingly, there was an improvement of women survivability with time passing, which might be partially attributed to the development of effective detection and control measures for the containment of cancer epidemiology [29]. dataset clustering approaches: a multidimensional analysis data stratification using gmm analysis estimated two possible mixed bell-shaped spreading, indicating a global overlapping distribution of mortality rates among the studied nations in figure 1. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 8 table 2: non-parametric correlation of the mortality rates from malignant diseases versus time (in years) at ci 95% with two-tailed p-value using prism graphpad v6.01 spearman (r) time versus death rate interpretation r -0.4110 95% ci * -0.4484 to -0.3721 p (two-tailed) < 0.0001 p value summary **** true or asymptotic p value calculation ** asymptotic significance at α = 0.05 yes number of paired xy 1938 * ci = confidence interval; ** refer to the calculation method of pvalue using either the exact or approximation to the actual distribution; **** p ≤ 0.0001 cumulative distribution function (cdf) and q-q plots showed the convenience of this assumption. the first and the second distributions were called (φ phi) and (ψ psi) with contribution proportions 0.57 and 0.43, respectively (figure 1). however, there was no clustering tendency that could show any heterogeneity according to the normalized entropy criterion (nec), which was greater than one with the selected the integrated completed likelihood (icl) criterion with the standard algorithm (em) expectation maximization [30]. the means and covariances of φ and ψ for malignancy mortality rates in women were 222.7, 241.0 and 1892.4, 909.9, respectively. figure 1: dataset segregation, based on gaussian mixture model (gmm) approach, showing two mixed distribution pattern with low (green) and high (pink) rates of mortality from malignancy, generated using xlstat v2014.05.03. the geographically based data segregation shows that, ascendingly, turkey, the republic of korea, brazil, mexico and greece demonstrated the lowest average mortality rate values could be demonstrated in figure 2. while countries with the ascending order from the lowest order of survivability to the highest means were denmark, hungary, ireland, czechia, gbr and netherlands (figure 2). figure 2: dataset segregation, based on who geographical distribution by countries shown as box plot, indicating the means, data spreading and the aberrant annual mortality rates records generated using minitab v17.1.0. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 8 the highest mortality rates from cancer occurred primarily in the eur region with the domination of economically rich nations suggesting that the overall survivability of women from cancer depends on other factors that seemed to be important to be controlled in addition to the improvement of the healthcare quality. this assumption might be partially in agreement with previous analysis provided by some researchers that had linked the prevalence of some types of cancer in the high-income countries with some adverse lifestyles such as smoking, obesity and alcohol consumption [31, 32]. nevertheless, it should be noted that beforehand rigorous control, awareness and actions taken about cancer detection and diagnosis associated with controlled monitoring might be one of the important reasons for the comprehensiveness of data records of high-income and developed nations if compared with those from the relatively lower income-countries. this hypothesis may be a factor that contributed to a possible underestimation of the actual overall estimation of the mortality rates in the developing and low-income countries. the time-based data segregation shows that, europe who region was the most versatile with divergent mortality rates between nations, as it is rich with country data, in contrast to the african area that includes only south africa. this could also be seen on the extend of the outlier figures (denoted by asterisks) above and below the box plot graphs in figure 3. south africa data record only started in 1993, which is later than the other three regions by 33 years and reaching its peak in 1999 (figure 3). the annual spreading of death cases rates for amr and wpr was less than that of eur. however, the distribution of wpr values was generally more compact than that of amr, where greater pervasion could be observed between annual records. the general profile can be seen in figure 3 with the yearly steady and almost regular decline in death rates started with amr in late 1980s, eur and wpr at almost 1995. figure 3: dataset segregation based on who geographical distribution by time order shown as box plot, indicating data spreading and the aberrant mortality rates records generated using minitab v17.1.0. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 8 fitted line of yearly average death rates from cancer for the affected women populations versus time dataset pattern showed a steady decline in the global mortality rates from cancer after the initial high plateau that could be demonstrated by the quadratic relation with significant correlation (figure 4). the regression equation was expressed through polynomial regression analysis of average mortality rates from cancer for women versus the time (in years). log10 (y) = 10245 + 6222 log10 (t)944.5 log10 (t) 2 …eq.(1) where: y and t are the years and death rates from cancer worldwide were recorded for women, respectively, with s = 0.00577903, r-sq and r-sq(adj) = 97.0%. figure 4 illustrates this relation between death rates and years graphically. figure 4: polynomial regression analysis graph showing quadratic equation modeling, confidence interval (ci) and prediction interval (pi) along with r 2 and the standard error of the regression (s), generated using minitab v17.1.0. trending of global women survivability from malignancy using process-behavior chart in figure 5, histogram showed a pattern of dual hump that could be explained from gmm model. boxplot graph is indicative of the pattern for the studied who regions (figure 5). afr demonstrated the lowest mean and spreading due to the inclusion of zaf only for relatively a short period from the region, in contrast of eur with upper and lower aberrant records from some countries in the european region from island (1960) and turkey (2009, 2010 and 2011). the survivability rate in wpr is better that amr. analysis of the distribution fitting showed the convenience of the weibull distribution based on the probability plot examination as could be observed in figure 5. plots the method mean (xbar chart) and method standard deviation (s chart) over time for variable information in subgroups (figure 5). this dual type of control chart is extensively used to observe the steadiness of procedures in lots of fields. both the xbar and s charts are displayed together due to the need to interpret each chart to decide if the trend is stable [32]. the procedure variation in s chart was almost stable within the control limits and tendency toward lower annual variation since 1979. the xbar chart in figure 5 showed initially intermittent excursions in the first few years followed by a successive shift in the annual mortality means leading to a declining curve with the improvement of survivability over time until exceeding the lower bound, which is a desirable outcome [33]. the last record of 2017 was incomplete during data registration that may explain the apparently sudden rise in mortality ratio due to the inclusion of few countries with high values in the process-behavior chart of figure 5. figure 5: spc analysis tools, (control chart, histogram and box plot) of the global cancer mortality rates in women with weibull probability fitting plot, showing the overall trend of the annual death rates as -s control chart and the pattern of who regions as box and whisker plot, generated using minitab v17.1.0. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 8 conclusion spc methodologies could provide a unique insight into the characteristics and patterns of epidemiological diseases. quantitative assessment of mortality and/or morbidity levels may be a useful indispensable tool in management and future decision-making. further studies are crucial to access the absolute number of deaths since using the ratio record alone may not reflect the real progression of the disease. for instance, the annual increase in the number of cancer patients may counterbalance the efforts to control deaths and the actual yearly mortality number could be a rising challenge to the efforts of cancer healthcare sector due to exacerbation of cancer epidemiology. in addition, the accuracy of the investigation and analysis is largely dependent on the impartialness and comprehensiveness of the database. however, cancer researchers could adopt similar techniques to track different types of malignancies in their countries over time to access the changes in the epidemiological pattern and the effectiveness of the control measures. the application of spc methodologies would be encouraged to be projected in future studies of other diseases such as coronavirus disease (covid-19) global cases. supplementary files the supplementary material for this article can be found online at: https://doi.org/10.36462/h.biosci.20208 supplementary table s1: list of countries whose data have been used in this study and their corresponding abbreviation. references 1. schmidt h. chronic disease prevention and health promotion. inpublic health ethics: cases spanning the globe 2016 (pp. 137-176). springer, cham. 2. boufford ji, cassel ck, bender kw. the future of the public’s health in the 21st century. an institute of medicine report. 2003. 3. nagai h, kim yh. cancer prevention from the perspective of global cancer burden patterns. journal of 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https://doi.org/10.1002/9781119047063.ch4 25. banas t. differences in arithmetic & geometric mean [internet]. sciencing. 2017 [cited 29 may 2020]. available from: https://sciencing.com/differencesarithmetic-geometric-mean-6009565.html 26. steele j. the cauchy-schwarz master class. cambridge: cambridge university press; 2010. 27. mcdonald jh. handbook of biological statistics. baltimore, md: sparky house publishing; 2014 aug. 28. weaver kf, morales vc, dunn sl, godde k, weaver pf. an introduction to statistical analysis in research: with applications in the biological and life sciences. john wiley & sons; 2017 sep 5. 29. american cancer society. cancer mortality continues steady decline, driven by progress against lung cancer: drop of 2.2 percent from 2016 to 2017 is largest ever reported [internet]. sciencedaily. 2020 [cited 30 may 2020]. available from: https://www.sciencedaily.com/ releases/2020/01/200108074809.htm 30. dempster ap, laird nm, rubin db. maximum likelihood from incomplete data via the em algorithm. journal of the royal statistical society: series b (methodological). 1977 sep;39(1):1-22. 31. nagai h, kim yh. cancer prevention from the perspective of global cancer burden patterns. journal of thoracic disease. 2017 mar;9(3):448. 32. western electric company. statistical quality control handbook. western electric company; 1956. 33. siegel rl, miller kd, jemal a. cancer statistics, 2020. ca: a cancer journal for clinicians. 2020 jan; 70(1):730. https://doi.org/10.1002/9781119047063.ch4 https://sciencing.com/differences-arithmetic-geometric-mean-6009565.html https://sciencing.com/differences-arithmetic-geometric-mean-6009565.html https://www.sciencedaily.com/ highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20212 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 7 research article open access 1 zoonotic disease research team, ethiopian public health institute, addis ababa, ethiopia. 2 department of public health, st. paul’s hospital millennium medical college, addis ababa, ethiopia. 3 vaccine and diagnostics production directorate, ethiopian public health institute, addis ababa, ethiopia. 4 veterinary public health directorate, ministry of agriculture, addis ababa, ethiopia. 5 public health emergency management directorate, ethiopian public health institute, addis ababa, ethiopia. 6 public health emergency management directorate, health bureau of snnp, hawassa, ethiopia. 7 deputy director general office/rtt, ethiopian public health institute, addis ababa, ethiopia. contacts of authors * to whom correspondence should be addressed: yimer mulugeta citation: mulugeta y, lombamo f, alemu a, bekele m, assefa z, shibru e, beyene m, kitila g, getahun g, sifer d, aklilu m, regassa f, and deressa a (2020). assessment of the current rabies situation and its management in epidemic areas of southern ethiopia. highlights in bioscience volume 3. article id 20212. dio:10.36462/h.biosci.202 12 received: may 20, 2020 accepted: july 12, 2020 published: july 14, 2020 copyright: © 2020 mulugeta et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. assessment of the current rabies situation and its management in epidemic areas of southern ethiopia yimer mulugeta 1 *, fantu lombamo 2 , anberber alemu 3 , meseret bekele 4 , zewdu assefa 5 , endashaw shibru 6 , mekoro beyene 3 , gutu kitila 1 , garoma getahun 1 , desalegn sifer 1 , mesfin aklilu 1 , feyesa regassa 5 , and asefa deressa 7 abstract rabies infection is almost always a 100% lethal viral disease in all mammals, including humans. although rabies is vaccine preventable disease, more than 60,000 people worldwide, and 2,700 people in ethiopia die of fatal human rabies annually. the aim of this study was to generate strategic information on the extent of the burden of rabies and the existing major response strategies, in order to overcome and bring attention to the management of rabies outbreaks. cross sectional study design was used to assess the current situation of rabies burden in the southern nations, nationalities, and peoples' regional states of ethiopia. data analysis revealed a total of 16 fatal human rabies and 287 suspected rabies exposures. the highest attack rate of 116.3 cases per 100,000 human subjects was reported in tembaro woreda. as a result, 48 animal deaths of rabies have been reported, while cattle (28) have been more affected than other animal species. the lack of dog immunization, lack of community awareness of dog management, and inaccessibility and inefficiency of anti rabies vaccines were major risk factors associated with the spread of rabies exposure between humans and animals. in conclusion, this study has clearly shown that both the existence and the high magnitude of fatal human and animal rabies are significant in the area. multidisciplinary approach interventions to increase community awareness of dog vaccination, effective and immediate treatment of exposed individuals is recommended as a method of disease control and prevention. keywords: animal, anti-rabies, human, outbreak, rabies exposure, snnp. introduction rabies exposure can always be either 100% fatal or 100% preventable double burden viral disease here in ethiopia and around the world. rabies exposure is 100% fatal, once a clinical symptom has been developed, even if it is characterized by a long incubation period of 2–3 months on average, but may vary from 1 week to 1 year depending on factors such as location of entry and viral load, short disease duration and death within 7 to 10 days of onset of clinical symptoms [1]. while rabies exposure is a 100% preventable disease with effective treatment soon after exposure to rabies, and may even be eradicated if government and donor attention is given to developing a control program/strategy and regulatory system to promote control of rabies in dogs as a key strategy for controlling rabies in humans through sustained dog vaccination programs. https://creativecommons.org/licenses/by/4.0/ mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 7 thus, the management and registration of the dog population, community awareness and cooperation of all stakeholders improve the cost effectiveness of vaccination intervention as a key measure [2-4]. globally, the burden of fatal human rabies is that 60,000 people die every year; almost 99% of human rabies exposure is caused by dogs. about 95% of death occurs in developing countries, particularly in africa and asia [5]. in developing countries, successful zoonoses are difficult to prevent and control, some of the main factors being zoonoses such as rabies that are neglected, under-reported, joint multi-sectoral preparedness and response interventions that remain unsatisfactory due to gaps in the legal framework. other factors attributing to rabies exposure include failure of dog immunization, abundantly free roaming dogs, and insufficient government attention, which may be due to underreporting of cases and lack of community awareness. children's and poor rural communities have a significant impact on the risk of rabies exposure across society, as well as a high gap in access and affordability of anti-rabies vaccines [2, 4, 6]. rabies is endemic in ethiopia and places a huge burden on the economy and public health. every year, 2,700 people in the country die of rabies. while there is no regulatory system for the prevention and control of rabies as mitigation, so far there is no legislation for the prevention of rabies. however, the ethiopian public health institute / ephi has played a key role in selected country pilot project areas in collaboration with various government and international partners, as one of the priority activities of the global health security agenda, cdc, osu, fao and other partners have started working together to build an effective national rabies elimination strategy [7, 8]. rabies is underreported in different corners of this country. as a result, its impact is underestimated due to existing challenges such as the lack of rabies diagnostic facilities at least one laboratory at each regional level and the lack of awareness-raising activities for health professionals and the lack of awareness-raising activities for the community. for example, there are different rare reports from the southern nation, nationalities and people's regional state (snnprs) of ethiopia on the occurrence of rabies exposure (mainly from suspected rabid dogs) in humans from different epidemic sites in the regional state. our aim is to (a) assess the outbreak of rabies in both humans and animals, and (b) generate strategic information on the extent of the burden of rabies and major response to informed decisions on the prevention and control of rabies. methods study area description the southern nations, nationalities and peoples' regional state (snnprs) is one of the largest and most ethnically diverse regions of ethiopia. geographically located in the southern part of the country surrounding kenya in the south, the republic of south sudan in the southwest, the state of gambella in the northwest and the state of oromia in the north and east. the total population size of the snnp is estimated to be 20,767,937 in 2018, of which 93.2% of the total population is in rural areas of the state. the region is also well known for having more than 1,5956,100 different livestock populations, 23 species of wild animals and 300 species of birds and water resources that can be used for fishery, irrigation and hydroelectric development. there are seven preserved locations in the forest. forests and bushes cover around 18% of the state [9]. rabies exposures studied were hawassa zuria woreda, mareka woreda, soro woreda, tembaro woreda, tercha town and gena bossa woreda of snnprs. hawassa zuria is located in the sidama zone; tembaro is located in the kembata tembaro zone; soro is located in the hadiya zone and tercha, mareka and gena bossa are located in the dawuro zone of snnprs, ethiopia. these areas are the focus of numerous wildlife, forests and water resources, including the gilgel gibe river basin and the gilgel gibe iii dam. its geographical location is shown in (figure 1). study design cross-sectional study was conducted in selected rabiesepidemic areas of snnprs, ethiopia. human rabies exposure assessment all humans with any animal bite without discrimination of the socio-demographic characteristics of the victims during the investigation period from july 2017 to may 2018. in the case of suspected human rabies exposure assessment, bite/exposure of animal history, bite site, date of bite, date of admission to the health facility, clinical sign and symptom status and outcome of each human exposure were addressed. livestock rabies exposure assessment all animal species with unusual signs or rabies suggestive behavioral changes have been included in this particular investigation without discrimination of species, immunization status and animal bite history. questionnaire survey semi-structured questionnaires were administered by health care providers, primarily those engaged in emergency care management at selected snnprs health facilities in ethiopia. the main focus areas of the interview and observation were all interested emergency health care providers who participated in taking time on the mode of transmission of rabies, methods of control and prevention, and possible ways of managing and diagnosing animal bite cases in their health facilities and at the same time addressing anti-rabies treatment. anti-rabies handling and utilization assessment the anti-rabies vaccine service was assessed by the total amount of vaccine available, the storage and mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 7 figure 1. geographic location of the investigation/study area. management of the cold chain, the proper administration and the proportion of use. anti-rabies handling and utilization assessment the anti-rabies vaccine service was assessed by the total amount of vaccine available, the storage and management of the cold chain, the proper administration and the proportion of use. sampling technique purposive sampling methods have been used for this particular study to address all these rabies-epidemic areas based on disease outbreak reports in the study area. data collectors/study team multidisciplinary team first organized by the ethiopian public health institute (ephi): public health emergency management (phem), zoonotic disease research and antirabies vaccine production directorates, st. paul's hospital millennium medical college and the ministry of agriculture (former ministry of livestock and fishery resources) were deployed in the hot spot areas of southern nations nationalities and peoples region (snnpr). data collection primary and secondary data were collected from public health offices, veterinary offices and health facilities through key informant interviews and interviews with experts and health care providers using standard semi-structured questionnaires. personal observations were made during interviews with health care facilities. the deployed team met with the snnpr, phem directorate. the owner of the phem core process referred to four hot spot areas (dawuro zone, kembata tembaro zone, hadiya and sidama zone) and their prospective woreda areas accordingly. active community and office searches and visits to health facilities were conducted as well as the major associated risk factor, pep utilization, laboratory findings and burden assessments were also addressed. ethical consideration ethical approval was not obtained from the institutional review board, as there were time constraints to respond in a timely manner to public health emergency management issues. however, the purpose of this study was explained to the intended study participants and verbal consent was obtained from them before proceeding with the study/data collection. data analysis descriptive data from this study from human and veterinary health facilities and the community were analyzed and reported on the status of rabies outbreaks and human and animal burden assessments, major risk factors, pep utilization and laboratory findings. results rabies outbreak status and human burden assessment a total of 16 suspected human rabies deaths have been reported from tercha general hospital, mudula primary hospital, bushillo, gimbicho and jacho health centers in public health facilities. analysis of secondary data revealed a total of 287 suspected rabies exposures that had been observed during the follow-up of anti-rabies in these sites during the investigation period. of the 16 suspected deaths of human rabies, 9 were male and 7 were female. the site of the bite / exposure to the human victims was the leg, finger, lip and arm. domestic dogs were responsible for all human exposure, as detailed in (table 1). mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 7 table 1. suspected fatal human rabies case scenario from investigation sites of snnp, ethiopia. s.n age sex woreda/site date of bite site of exposure exposin g animal duration after exposure remark 1 13 female tembaro november 16, 2017 leg dog 3 months 2 28 male tembaro january 20, 2018 finger dog 3 months came to hf with clinical signs 3 7 female tembaro september 27, 2017 leg dog 3 months death after 6 days of onset of illness 4 5 male tembaro february 13, 2018 leg dog 2 months after 7 days of illness 5 60 female tembaro january 23, 2018 finger dog 3 months after 5 days of illness 6 7 male tembaro january 17,2018 lip dog 2 months took 15 doses and died 7 56 male tembaro january 25, 2018 leg dog 2 months came to hf with clinical signs 8 male soro leg dog was referred to mudulla hospital after onset of clinical sign and died 9 male tembaro lip dog died after 12 th dose of ntv 10 14 male mareka december 25, 2017 unknown dog 2 months 11 7 female mareka arm dog 6 cattle died after being bitten by the same dog 12 46 female soro march 18, 2017 leg dog 1 months 13 28 male soro january 8, 2018 leg dog 3 months 14 16 female mareka finger cat 15 27 female hawassa zuria march 12, 2018 leg dog initially diagnosed as typhoid fever and was being managed with ceftriaxone 16 21 male hawassa zuria dog the highest rate of rabies exposure was reported from tembaro woreda (116.3 per 100,000 population at risk); 55.3, 13.6, 12.1, 5.4 per 100,000 population at risk attack rates were reported in hawassa zuria woreda, mareka woreda, soro woreda and gena bossa woreda, respectively. rabies outbreak status and burden assessments in animals during the investigation period, 48 animal rabies deaths were detected and 28 cattle, 12 goats, 5 sheep and 3 equines were identified. death report due to suspected rabies in the dog was not counted as a result of dog elimination activity conducted using strychnine. of these human-exposed dogs, only 10 rabies-suspected samples were submitted to the national rabies laboratory of the ethiopian public health institute (ephi). of the total samples, 80% were positive for rabies virus infection, and dogs were the most biting / exposing animals for animals and human victims during the time and area of investigation, as shown in (figure 2). major contributing risk factors for rabies outbreak in place summary of the major associated risk factors for the occurrence and spread of rabies exposure among humans and animals has been identified. absence of dog immunization and lack of community awareness of dog management in this investigation, the engaged community is once remote from rural areas and keeps the dog on guard for reasons of lack of awareness of the possibility of disease transmission. all respondents responded that they had a single dog (47%) or more than one dog (53%), and their dogs were free to move to the surrounding area (free roaming dog) and no history of deworming and/or vaccination of dogs. the absence of immunization for dogs has also been reported by the livestock sector, which has created a chance for the occurrence and spread of rabies exposure by addressing multiple factors such as community ignorance, unaffordable cost of anti-rabies vaccine for dogs and little government attention. figure 2. rabies exposure laboratory findings and human victims by animal bite during investigation time and area (snnp). mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 7 challenges on anti-rabies vaccine and inadequate human rabies exposure management inaccessibility and unavailability of cell culture antirabies vaccine (modern vaccine by the national pharmaceutical supply agency) and shortage of nerve tissue vaccine (old anti-rabies) human anti-rabies vaccine have been observed (figure 3). although vaccine shortages have occurred, consistent cold chain management problems have also been observed in health facilities. in fact, the nerve tissue (old anti-rabies pep) anti-rabies vaccine production capacity of ephi has so far not met the demand for and severely affected the required response to deadly diseases. figure 3. coverage of anti-rabies vaccine in snnp before the investigation date of the fiscal year. inadequate heath care provider’s knowledge and skill at those health facilities the opinion and level of knowledge and skills of healthcare professionals in the area of rabies exposure management with selected hfs were assessed on the basis of their interest in semi-structured self-governing interviewers. we found that, 46.6 % (14/30) of the respondents had knowledge of the mode of transmission via saliva from suspected animal bites, in particular dog bites (table 2). ecological factors for increased spillover the existence of natural forests, mountains and rivers such as the gilgel gibe river basin has created a suitable environment for the occurrence and spread of rabies exposure , especially the over filling of the gilgel gibe iii dam, which plays a significant role in displacing the normal habitat of wildlife for interaction and co-existence with domestic animals and peoples. dogs, cats, foxes and donkeys were among the most frequently exposed animals found during the investigation. increased interaction of wild life with dogs and high prevalence of free roaming dogs were considered risk factors for increased exposure to rabies. table 2. opinion of healthcare providers and their inadequate knowledge and skill on human rabies exposure management. variables category frequencies proportion sex of respondents male 18 60 female 12 40 respondents hf tercha gh(dawuro) 16 53 gimbichu & jacho hc 11 37 mudula hospital 3 10 is rabies a major problem in your hf yes 24 80 no 6 20 knowledge & skill of health care providers on mode of transmission yes 14 47 no 16 53 knowledge & skill on clinical signs/symptoms in human yes 20 67 no 10 33 throughout the year rabies exposure occurrence yes 25 83 no 5 17 is there any preventing & control guideline/legislation yes 5 17 no 25 83 which control option do you use health education (he) 14 47 anti-rabies vaccination (pep) 12 40 all options he, pep & wound mg’t 4 13 after exposure where they go? health facility 24 80 traditional healer 4 13 other 1 3 what are the major challenges of human rabies exposure lack of regular animal rabies control activity (arc) 6 20 lack of community awareness(ca) 14 47 lack of training(tot) 3 10 combination factors of above 5 17 mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 7 discussion rabies is one of the most neglected tropical and zoonotic diseases with an increasing trend in human burden [10]. this investigation revealed a high rate of rabies and its exposure in humans and animals and a lack of immunization in dogs compared to studies reported in the ethiopian rabies review (1990 to 2000) in which a total of 322 human deaths were reported [11]. there is also another annual estimate of 2700 human deaths in 2017 [7], which has shown an increasing burden on public health and still needs to be addressed in order to achieve the global rabies elimination program 'zero rabies in humans' by 2030 [1,12]. in this study, fatal human and animal rabies of 16 and 48 were found mainly due to rabies-specific symptoms and epidemiological factors, as the standards of rabies diagnostic methods could not be applied. this is due to the lack of standardized laboratory settings and the difficult nature of rabies cases of cns in suspected human rabies exposure. this study found the highest rates of attack in tembaro woreda with 116.3 cases per 100,000 human population, followed by 55.3 cases in hawassa zuria woreda, 13.6 cases in mareka woreda, 12.1 cases in soro woreda, and 5.4 cases in gena bossa woreda per 100,000 population at risk of attack. this finding is considerably higher than that of 2.33 cases per 100,000 human population in northern gondar, ethiopia [13]. overall, human rabies exposure in this investigation has been admitted to the health care facility too late and varies from area to area for an average of 2-3 months from the date of bite or after onset of clinical symptoms as described in (table 1). this was due to the high perceptions of local people in that country and the widespread use of traditional medicine among the urban and rural population of ethiopia could be attributed to cultural acceptability, physical accessibility and economic affordability compared to modern medicine [14]. a high free roaming dog population with a never-vaccination history and a high level of wildlife interaction was identified as an existing fueling factor for rabies outbreaks. this finding is consistent with the findings reported by admassu and mekonnen that, in 2014 in the same country, a large number of dogs in both urban and rural settings and a low vaccination risk of rabies circulation and spread to humans and other domestic animal populations [14]. in order to manage this burden of human rabies exposure in the snnp region, a lack of anti-rabies vaccine was observed during the investigation, and ephi distributed a total of 4305 rabies post-exposure prophylaxis per 10 months until the investigation was conducted to protect the public. the dawuro and kembata tembaro zones reported the highest levels of exposure to rabies and human fatal cases of rabies, but vaccine coverage was 42 and 68, respectively. this result is in agreement with the findings reported by abraham in this country [15]. this sample was tested in a national rabies laboratory for laboratory analysis of 10 suspected animal brain tissue samples, 80 per cent of which were positive for rabies. all of the positive samples were from the dog. samples submitted to the national rabies laboratory were only ten, due to a number of reasons such as lack of regional laboratory facilities in the area; lack of awareness was a key factor due to remote rural communities. the world health organization recommends that at least 70% of the dog population be vaccinated to control and potentially eliminate human-mediated rabies in dogs [16]. in this study, no organized dog vaccination activity was performed, instead of vaccination, by the community in communication with local authorities to overcome dog bite and rabies exposure in humans and animals. the reason for the elimination of dogs was due to inaccessibility and unaffordability of the anti-rabies vaccine and high free roaming dog population. as a limitation, although the main objective of this study is to respond to rabies outbreaks, there are no estimates of the sample size to include all study participants and standard confirmatory tests in human and animal subjects. some suspected human rabies death data have not yet been completed and a documentary system has been recorded. conclusion and recommendation this study team detected cases of human and animal fatal rabies in the community and at selected public health facilities in snnp, ethiopia, in 2018. the team verified the occurrence of an outbreak of rabies exposure. the multisectoral and multi-disciplinary "one heath approach" has been seen as very critical in addressing such burdens in the human and animal domains. poor collaboration between sectors, inaccessibility/shortness of anti-rabies vaccines and community awareness gaps have been identified as major challenges. high free roaming and unvaccinated dog populations as well as high interactions with wildlife were also observed. on the basis of the above conclusion, the following recommendations are forwarded: 1. awareness-creation and social mobilization programs and events related to rabies problems should be undertaken through the use of different media and other tools for the community, health professionals and policy makers. 2. communication and collaboration between the human and animal professions should be encouraged for an effective system of surveillance, control and prevention of rabies exposure. mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 7 3. mass vaccination of dogs, the prevention of free movement of dogs and contact with wild life and the management of dog populations should be carried out in order to achieve zero rabies in humans and animals. 4. the availability and affordability of the anti-rabies vaccine (pep) for humans and dogs should be addressed with national and international partners. 5. quality cell culture anti-rabies vaccine must be available and accessible to the affected community as a whole. acknowledgement we would like to thank the members of our health and veterinary office and health care providers from the respective areas and districts of sidama (hawassa zuria), hadiya (soro), kembata-tembaro (tembaro) and dawuro (mareka and gena bossa) for their valuable support, advice and guidance in boosting this work. we also recognize the contributions of our driver, mr shimeles, and all those individuals who have given up their time to participate. authors' contributions ym was responsible for the study, collection and analysis of the data and wrote the manuscript. fl, aa, my, za, es, mb, contributed to the collection of data, the study methodology and the co-writing of the manuscript. gk, gg, ds and ma have contributed to laboratory work and guidance. fr and ad contributed to the revision and editing of manuscripts. the final manuscript was read and approved by all authors. references 1. who. rabies fact sheet.https://www.who.int/newsroom/fact-sheats/detail/rabies 2019 [cited 2019 may 21]. 2. takahashi-omoe h, omoe k, okabe n. regulatory systems for prevention and control of rabies, japan. emerging infectious diseases. 2008 sep;14(9):1368. 3. fao, developing a stepwise approach for rabies prevention and control. proceedings of the fao/garc workshop, november 2012, rome, italy. fao animal production and health proceedings, 2013. 18. 4. who, who expert consultation on rabies: frist report, in technical report series 2005, who: geneva. 5. undurraga ea, blanton jd, thumbi sm, mwatondo a, muturi m, wallace rm. tool for eliminating dogmediated human rabies through mass dog vaccination campaigns. emerging infectious diseases. 2017 dec;23(12):2114. 6. oyda s, megersa b. a review of rabies in livestock and humans in ethiopia. international journal of research granthaalayah, 2017. 5(6): 561-577. 7. cdc. rabies in ethiopia. world rabies day, prevent rabies around the world 2017. 8. deressa a, ali a, bayene m, selassie bn, yimer e, hussen k. the status of rabies in ethiopia: a retrospective record review. ethiopian journal of health development. 2010;24(2). 9. adugna a, snnpr, southern nations nationalities and peoples demography and health. ethiodemography and health, 2018. 10. fao/oie/who, inter-agency meeting on planning nzds prevention and control. 2011. 11. moges n. rabies in ethiopia: review article. academic journal of animal diseases. 2015;4(2):74-81. 12. wallace rm, undurraga ea, blanton jd, cleaton j, franka r. elimination of dog-mediated human rabies deaths by 2030: needs assessment and alternatives for progress based on dog vaccination. frontiers in veterinary science. 2017 feb 10;4:9. 13. jemberu wt, molla w, almaw g, alemu s. incidence of rabies in humans and domestic animals and people's awareness in north gondar zone, ethiopia. plos negl trop dis. 2013 may 9;7(5):e2216. 14. admasu p, mekonnen y. rabies and its folk drugs remedies in ethiopia: a review. international journal of basic and applied virology. 2014;3(2):22-7. 15. abraham a, daddi j, milliyon w, almaz a, fikir t. national surveillance for human rabies cases. in: impact, limitation and needs in developing countries. ethiopian health and nutrition research institute, 2010. 16. who, who expert consultation on rabies: third report. who technical report series, no. 1012. licence: cc by-nc-sa 3.0 igo, 2018. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20218 highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 7 research article open access department of science technology, akwa ibom state polytechnic, ikot osurua, p.m.b 1200, ikot ekpene, akwa ibom state, nigeria. contacts of authors * to whom correspondence should be addressed: aniefon alphonsus ibuot citation: james ii, ben mg, jones am, akpan ps, eka ii, oruk ae, ibuot aa (2020). characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites. highlights in bioscience volume 3. article id 20218. dio:10.36462/h.biosci.20218 received: june 6, 2020 accepted: august 18, 2020 published: august 31, 2020 copyright: © 2020 james et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites iniobong ime james, mayen godwin ben, agnes monday jones,patience saturday akpan, idorenyin idorenyin eka, albert ema oruk, and aniefon alphonsus ibuot* abstract changes in soil physicochemical properties and bacterial species present in soil contaminated with waste engine oil were evaluated at three auto-mechanical workshops in uyo, nigeria. this work was aimed at isolating and identifying hydrocarbon degrading bacteria from waste engine oil polluted soil, and assessing their hydrocarbon-utilizing ability. waste engine oil pollution affected soils significantly with increases in soil physicochemical properties, and heterotrophic bacterial population counts. eight bacterial species corynebacterium kutscheri, pseudomonas aeruginosa, flavobacterium aquatile, serratia odorifera, micrococcus agilis, staphylococcus aureus, micrococcus luteus and bacillus substilis were isolated by the selective enrichment technique and screened for hydrocarbon utilization capability in mineral salt media with 1% (v/v) waste engine oil as a sole carbon and energy source. the extent of bacterial growth observed was related to the ability of organisms to biodegrade hydrocarbons present in the medium bacterium species, which showed varying hydrocarbon utilization during the 15 days of incubation. growth in hydrocarbon medium was the most efficient in cultures of corynebacterium kutscheri. all isolates also showed variable emulsification ability, with corynebacterium kutscheri, showing the highest ability. these results demonstrate the presence of indigenous bacteria in hydrocarbon-polluted soils and the potential toward the remediation of hydrocarbons. keywords: hydrocarbon-utilizing bacteria, selective enrichment technique, corynebacterium kutscheri introduction petroleum utilization as fuel and petroleum products leads to severe environmental pollution [1]. large-scale accidental spills pose a great threat to the ecosystem [2]. soil pollution by petroleum hydrocarbons has been shown to produce pronounced changes in the physicochemical and microstructure of the oil-contaminated soil [3]. this affects parameters such as soil porosity, bulk density, and adsorption [4-5]. fresh spills and/or high levels of pollutants may often result in the reduction of large sectors of soil microbial population, although soils with lower levels or old pollution may show an increase in numbers and diversity of microorganisms [6-7]. the diversity and the number of microorganisms at polluted soil sites may assist in the characterization of such a site, such as the toxicity of petroleum hydrocarbons to the microbiome, age of the spill and concentration of the pollutant [8]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20218 https://doi.org/10.36462/h.biosci.20218 http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 7 additionally, microorganisms in soils exposed to hydrocarbon pollution usually exhibit a higher potential for biodegradation of such pollutant compounds than others with no history of such exposure. percentages of hydrocarbonoclastic microbes are quite low in soil when there is no oil spill, but may increase 1,000 fold after oil spill [9]. conventional remediation methods do not seem to be able to address this problem, or tends to aggravate the problem [10]. mechanical methods such as incineration, excavation and/or burial in secure land fill, as well as a host of other chemical decomprelocates osition methods are expensive, time consuming and only the pollution [11]. an efficient way of remediating the oil-contaminated sites could be employment of microorganisms, such as bacteria, microscopic algae, and fungi, isolated from polluted environments or enhanced from the organisms already present in the same environment [12-13]. waste engine oil-polluted soils also serve as a source of indigenous bacteria capable of hydrocarbon degradation. the employment of microorganisms in the biodegradation of hydrocarbons over chemical or conventional treatment is preferred for many reasons; end products are comparatively safer and cost-effectiveness [11]. ogunbayo et al., [18] evaluated the effectiveness of bacteria indigenous to soil in remediating engine oil-polluted, soil and isolated bacillus, pseudomonas, flavobacterium, micrococcus and rhodococcus species, with pseudomonas and rhodococcus species giving most favorable degradation effectiveness and efficiencies. this study therefore considered the isolation of indigenous bacterial communities in waste engine oil-polluted soil using selective enrichment technique, and the assessment of hydrocarbon-utilization capability in waste engine oil-augmented mineral salt medium. materials and methods sample collection waste engine oil –contaminated soil samples used in this study were collected from three auto-mechanic workshops within the mechanic village, uyo, akwa ibom state, nigeria. composite soil samples were obtained at each sampling point using a soil auger from 0-10 cm below the soil surface. the soils were labeled “unpolluted” for the unpolluted sample, “ma” for the mechanic workshop 1 sample, “mb” for the mechanic workshop 2 samples, and “mc” for the mechanic workshop 3 samples. this was followed by bulking and transportation to the laboratory in sterile polythene bags within six hours for isolation of organisms. physico-chemical analysis of soil samples the soil ph was measured using hanna instruments model 209 ph meter [14]. moisture content was calculated on the basis of the air dry weight as described by aoac [15]. total organic carbon was calculated by weighing exactly 0.5 g of the soil sample into a flask, and 10 ml of 1.0 m k2cr2o7 was added and swirled to mix. 20 ml conc. h2so4 was added, gently swirled for a minute and allowed to stand for 20 minutes. the suspension was diluted to about 100 ml of distilled water. five drops of o-phenanthroline indicator were added to each sample and was titrated with 0.5 m ferrous ammonium sulfate to a light blue end point. the reagent blank was also run and the titre values recorded, and used to calculate the organic carbon content [15]. the total hydrocarbon content (thc) was determined by first extracting hydrocarbons by acidifying 2 g of representative soil samples using h2so4, and extracting upon addition of 20 ml of toluene in a separatory funnel. the contents of the funnel were shaken, and allowed to settle into two layers. the absorbance of the supernatant (extract) was read at 420 nm with unicam uv/vis spectrophotometer (spectronic 20d). readings were recorded from the spectrophotometer and using the determined curve to obtain the figure [16]. phosphorus was determined using the ascorbic acid method as described by aoac, [15]. 50 ml of the soil dilution was pipetted into 250 ml erlenmeyer flask, and 1 drop of phenolphthalein indicator was added. exactly 5 n h2so4 (148 ml conc. h2so4 in 100ml h20) is added drop-wise to develop a red colour. exactly 8 ml of combined reagents made up of 50 ml of 5 n h2so4, 5 ml potassium antimonyl tartrate solution (1.372 g potassium antimonyl tartrate in 500 ml distilled water); 15 ml ammonium molybdate solution (20 g ammonium molybdate crystal in 500 ml distilled water); were added and thoroughly mixed, and allowed to stand for 20 min. the phosphorus content was determined by measuring the absorbance of the sample at 880 nm. the nitrogen concentration was determined according to the methods of bremmer and mulvaney, [17]. one milliliter of the soil sample was introduced into the standard kjeldahl flask containing 1.5 g cuso4, and 1.5 g na2so4 as catalyst, alongside concentrated h2so4. the flask was gently heated on a heating mantle, taking care to prevent frothing. the solution was transferred after heating to a 100 ml standard flask and made up to the mark with distilled water. a portion of this digest was pipetted into a semi micro-kjeldahl distillation apparatus and treated with 30 ml of 40% naoh solution. the ammonia evolved was steam-distilled into a 100 ml conical flask containing 10 ml solution of saturated boric acid to which 4 drops of tashirus indicator had been previously added. the tip of the condenser was immersed in the boric acid solution and the distillation continued until about two-thirds of the original volume was obtained. the tip of the condenser was finally rinsed with a few milliliters of distilled water. the distillate was then titrated with 0.1n hcl until a purple-pink end point was observed. a blank determination was also carried out in a similar manner without the sample, and the calculation done as follows: nitrogen (%) = (real titre – blank titre) x 0.1 x 0.014 x 100 weight of the sample enumeration of total heterotrophic bacteria (thb) the thb population in the soil samples was enumerated by adopting the standard plate counts technique using the spread plate method as described by ogunbayo et al., [18]. these involved spreading aliquots of a serially http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 7 diluted 0.1 ml of 10-5 dilutions of the soil sample suspension on nutrient agar plates and the plates were incubated at 30 oc for 24 h. similar aliquots were also incubated in minimal salt agar plates containing used engine oil as the sole source of carbon and energy. the plates were all incubated aerobically at 30 oc. the percentages of hydrocarbon-utilizing bacteria (hub) relative to the total heterotrophic counts were noted. enumeration of hydrocarbon utilizing bacteria (hub) oil-utilizing bacteria were isolated from polluted soil samples by enrichment in mineral salt medium (msm) modified from okpokwasili and nwosu, [19] using waste engine oil as a carbon and energy source. the soil samples were sieved using a 2 mm mesh sieve. 10 g of the sieved soil samples was inoculated into 100 ml sterile msm. 1 ml of the waste engine oil was added to the medium as a sole source of carbon and energy, and the culture was incubated on a rotary shaker at 170 rpm for 1 week. the enrichment procedure was repeated for three cycles. at the end of each enrichment cycle, 1 ml of the culture was diluted serially 10-fold down the gradient to 10-5 and plated. pure cultures of the isolates were obtained by plating 1 ml of the 10-5 dilution of the third enrichment cycle onto msm agar plates, and incubating at 30 oc (± 2) for 48 h. pure cultures obtained by this procedure were stored in slants at 4 oc until further identification. characterization and identification of bacteria isolates were identified on the basis of colonial characteristics, gram’s reaction and cell biochemical reactions as described by cheesbrough [20]. identification used the taxonomic schemes of holt et al. [21]. hydrocarbon utilization screening of bacteria to determine the ability of the isolates to utilize engine oil as the sole carbon and energy source, the growth patterns of isolates in mineral salt medium in the presence of 1% (v/v) of the waste engine oil (5.0 ml in 100 ml msm) were determined according to onuoha et al. [22]. waste engine oil-augmented msm was dispensed into 250 ml erlenmeyer, and inoculated with 0.1 ml of 24 h cultures of the bacterial isolates. incubation was done at 30 oc for 15 days. growth patterns were determined monitoring changes in ph, optical density and total viable count at 5-day intervals during the incubation. the ph of the medium was measured using the ph meter (hanna instruments). growth was also monitored by measuring the optical density (od) at 600 nm using the spectrophotometer (spectrumlab). total viable counts of the cultures were obtained by incubation of 0.1 ml of the cultures using the spread plate technique on nutrient agar plates at 30 oc for 24 h. emulsification activity of bacteria the emulsification index (e24) of the isolates was determined according to the methods of ganesh and lin, [23], by adding 1ml of waste engine oil to the same amount of culture media as used for degradation assay, mixing the vortex for 2 min and leaving to stand for 24 h. the percentage of emulsification index was obtained as follows: e24 = height of the emulsified layer x 100 total height of the liquid column results physicochemical properties of soil samples the results of the physicochemical analysis of the different soil sample are shown in table 1. the high amounts of organic carbon (5.32 ± 2.65% in ma, 9.79 ± 0.51% in mb and 7.29 ± 3.09% in mc), and thc (2933.76 ± 404.27 mg/kg in ma, 3122.72 ± 131.00 mg/kg in mb and 3202.61± 675.07 mg/kg in mc), compared to the unpolluted soil sample (3.7 ± 2.43% organic carbon content and 39.97± 13.49 mg/kg thc) is indicative of heavy pollution of the mechanical workshop samples with petroleum hydrocarbons. soil samples from mechanical workshop 1 contained higher amounts of nitrates (0.25 ± 0.03 mg/g), while samples from mechanical workshop 2 contained the highest amounts of phosphates (10.74 ± 0.88 mg/g) and thc (3202.61± 675.07 mg/kg). the ph values of the soil samples indicate all soil samples as moderately acidic to acidic (from ph 5.78 to ph 6.79). bacterial count of soil samples the total heterotrophic bacterial count and hydrocarbon utilizing bacterial count of the original soil samples is shown in table 2. a higher thb count was recorded in polluted soil samples (4.4 ± 1.90 x 107 cfu/gfrom ma sample, 6.0 ± 0.23 x 107 cfu/g from mb sample and 4.5 ± 0.03 x 107 cfu/g from mc sample) than in the unpolluted soil sample (1.9 x 107 cfu/g). higher thb (6.0 ± 0.23 x 107 cfu/g) and hub (5.2 ± 0.25 x 107 cfu/g) counts were observed in the mb sample than in other similar polluted samples indicative of its extent of pollution. hydrocarbon utilising bacterial counts were slightly lower in all samples than the corresponding heterotrophic bacterial counts. characterization and identification of bacteria the identified bacterial isolates were corynebacterium kutscheri, pseudomonas aeruginosa, micrococcus agilis, flavobacterium aquatile, staphylococcus aureus, micrococcus luteus, serratia odorifera and bacillus substilis, as shown in table 3 and 4. hydrocarbon utilization potential of bacteria table 5 shows the changes in ph of msm during growth of bacteria isolates in hydrocarbon. decreases in ph (to <ph 7.00) were observed in medium containing isolates, corynebacterium kutscheri, micrococcus agilis, serratia odorifera and bacillus substilis, (which, however, increased to above ph 7.00). the greatest decrease in ph occurred in cultures of corynebacterium kutscheri (from 7.00 a.m. on day 0 to 5.83 ± 0.34 on day 15). medium containing other isolates showed slight increases in ph over time. the changes in the total viable counts of bacterial isolates during 15 days of growth in waste engine oil-augmented msm are shown in table 6. total viable counts were higher in cultures containing corynebacterium kutscheri (1.94 ± 0.12 x 108 cfu/ml on day 5, 6.73 ± 0.45 x 108 cfu/ml on day 10 and 3.13 ± 0.02 x 108 on day 15). growth of staphylococcus aureus showed the lowest decreases (0.31± 0.11 x 108 on day http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 7 5, 0.44 ± 0.01 x 108 on day 10 and 0.42 ± 0.02 x 108 on day 15). table 7 shows the results of turbidity measurement (od600) of the medium during incubation. the highest increases in turbidity were recorded in cultures with corynebacterium kutscheri, (0.189 ± 0.04 on day 0 to 0.301± 0.11 on day 15), and bacillus substilis, (0.165 ± 0.04 on day 0 to 0.341± 0.02 on day 15). the lowest turbidity was observed with staphylococcus aureus (0.20 ± 0.04 on day 0 to 0.14 ± 0.02 on day 15). emulsification activity of bacteria figure 1 shows the emulsification index (e24) of the bacterial isolates in waste engine oil. corynebacterium kutscheri had the highest emulsification index (52%). the lowest emulsification index was observed for staphylococcus aureus (8%). table 1: physicochemical properties of different soil samples contaminated with waste engine oil. values represent means of triplicate determinations ± sd. ma = mechanic workshop 1, mb = mechanic workshop 2, mc = mechanic workshop 3. thc = total hydrocarbon content. table 2: bacterial count of soil samples contaminated with waste engine oil. values represent means of triplicate determinations ± sd. ma = mechanic workshop 1, mb= mechanic workshop 2, mc= mechanic workshop 3. table 3 : biochemical characteristics of spore (sp), catalase (ca), motility (mo), oxidase (ox), litmus reaction (lr), urease (ur), gelatin (ge), citrate (ci), glucose (gl), maltose (mal), mannitol (man), lactose (la), sucrose (su), arabinose (ar) and xylose (xy) for studied bacterial isolates from soil contaminated with waste engine oil. keys: ag = acid and gas production; a = acid production only; + = positive reaction; = negative. table 4: phenotypic characteristics of bacterial isolates from soil contaminated with waste engine oil. parameters unpolluted (control) polluted ma mb mc ph 6.79 ± 0.24 6.18 ± 0.13 5.78 ± 0.03 6.44 ± 0.07 nitrate (mg/g) 0.14 ± 0.02 0.25 ± 0.03 0.23 ± 0.02 0.20 ± 0.02 phosphate (mg/g) 6.65 ± 1.72 6.92 ± 2.92 10.74 ± 0.88 8.49 ±1.9 moisture content (%) 32.25 ± 9.30 43.09 ± 0.65 45.13 ± 7.02 27.72 ± 9.09 organic carbon content (%) 3.7 ± 2.43 5.32 ± 2.65 9.79 ± 0.51 7.29 ± 3.09 thc (mg/kg) 39.97 ±13.49 2933.76 ± 404..27 3202.61± 675.07 3122.72±131.00 parameter unpolluted (control) polluted ma mb mc thb (cfu/g) 1.9 ± 0.02 x 107 4.4 ±1.90 x 107 6.0 ± 0.23 x 107 4.5 ± 0.03 x 107 hub (cfu/g) 6.5 ± 0.04x 106 3.2 ± 0.05 x 107 5.2 ± 0.25 x 10 7 1.9 ± 0.10 x 106 isolates isolate code sp ca mo ox lr ur ge ci gl mal man la su ar xy 1 ma3, mb1, mc4 + + a a a ag 2 ma2, mb2 + + + + + a a a a a 3 ma1 + a a ag ag 4 ma4 + + + a a a a 5 mb3, mc1, mc2, ma5 + + a 6 ma6 + + a a 7 mc3 + + + + + a a a a a a 8 mb4, mc5 + + + + + + a a a a a ag isolates isolate code gram reaction/cell shape probable bacteria 1 ma3, mb1, mc4 gram-positive rods corynebacterium kurtscheri 2 ma2, mb2 gram-negative rods pseudomonas aeruginosa 3 ma1 gram-positive cocci micrococcus agilis 4 ma4 gram-negative rods flavobacterium aquatile 5 mb3, mc1, mc2, ma5 gram-positive cocci staphylococcus aureus 6 ma6 gram-positive cocci micrococcus luteus 7 mc3 gram-negative rods serratia odorifera 8 mb4, mc5 gram-positive rods bacillus substilis http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 7 table 5 : ph of the medium during the growth of bacterial isolates in hydrocarbon medium. isolates days 0 5 10 15 micrococcus agilis 7.00 6.51 ± 0.01 6.38 ± 0.03 6.04 ± 0.05 pseudomonas aeruginosa 7.00 7.06 ± 0.07 7.78 ± 0.01 7.71 ± 0.06 corynebacterium kutscheri 7.00 6.51± 0.01 6.38 ± 0.00 5.83 ± 0.34 flavobacterium aquatile 7.00 7.31± 0.23 7.50 ± 0.16 7.17 ± 0.28 micrococcus luteus 7.00 6.13± 0.07 7.23 ± 0.03 7.37 ± 0.06 staphylococcus aureus 7.00 7.23± 0.03 7.31± 0.03 7.23 ± 0.07 serratia odorifera 7.00 7.09 ± 0.01 6.73 ± 0.04 6.42 ± 0.07 bacillus substilis 7.00 6.85 ± 0.13 6.78 ± 0.18 7.24 ± 0.02 data represent means of triplicate determinations ± sd. table 6: total viable count (tvc) of bacterial isolates during growth in hydrocarbon medium (x 108 cfu/ml). isolates days 0 5 10 15 micrococcus agilis 0 0.63 ± 0.03 2.46 ± 0.02 0.87 ± 0.06 pseudomonas aeruginosa 0 0.54 ± 0.02 2.13 ± 0.16 0.45 ± 0.02 corynebacterium kutscheri 0 1.94 ± 0.08 5.27 ± 0.07 3.18 ± 0.07 micrococcus luteus 0 0.95 ± 0.01 2.49 ± 0.03 1.43 ± 0.03 flavobacterium aquatile 0 1.94 ± 0.12 6.73 ± 0.45 3.13 ± 0.02 staphylococcus aureus 0 0.31± 0.11 0.44 ± 0.01 0.42 ± 0.02 serratia odorifera 0 0.54 ± 0.03 2.23 ± 0.25 0.48 ± 0.04 bacillus substilis 0 1.39 ± 0.41 4.08 ± 0.65 2.47 ± 0.07 data represent means of triplicate determinations ± sd. table 7: turbidity measurement of bacterial growth in hydrocarbon medium (od600). isolates hours 0 5 10 15 microccoccus agilis 0.97 ± 0.01 0.154 ± 0.01 0.209 ± 0.12 0.201± 0.03 pseudomonas aeruginosa 0.033 ± 0.001 0.035 ± 0.10 0.182 ± 0.10 0.097± 0.05 corynebacterium kutscheri 0.189 ± 0.04 0.252 ± 0.07 0.386 ± 0.04 0.301± 0.11 flavobacterium aquatile 0.085 ± 0.01 0.153 ± 0.08 0.196 ± 0.07 0.227± 0.05 micrococcus luteus 0.040 ± 0.02 0.083 ± 0.06 0.162 ± 0.03 0.183± 0.04 staphylococcus aureus 0.20 ± 0.04 0.053 ± 0.10 0.084 ± 0.05 0.014± 0.02 serratia odorifera 0.40 ± 0.02 0.172 ± 0.11 0.121 ± 0.001 0.096± 0.01 bacillus substilis 0.165 ± 0.05 0.204 ± 0.01 0.413 ± 0.05 0.341± 0.04 data represent means of triplicate determinations ± sd. figure 1 : emulsification activity (e24) of bacterial isolates. discussion this study on the assessment of hydrocarbon utilizing the potential of bacteria isolated from waste engine oil-polluted soil on hydrocarbons reveals the presence of hydrocarbon-utilizing bacteria in waste engine oil-polluted soil environment as well as the potential of one of these isolates corynebacterium kutscheri to degrade hydrocarbons under a variety of experimental conditions. the results of the physicochemical analysis of the soil samples, as presented in table 1 showed higher levels of properties in the polluted soil samples when compared with the unpolluted soil sample. the results support the results of chikere [24], and chikere and ekwuabu [25], which showed high physicochemical parameters in polluted soil samples compared to unpolluted samples determined and indicated previous exposure of polluted samples to hydrocarbon contamination with traces of other organic and inorganic contaminants. the high bacterial counts recorded in polluted soil samples, as presented in table 2, compared with the unpolluted control samples could be attributed to the myriad of nutrients, high organic matter concentration and other ecological factors that influence the survival of soil microorganisms that play important roles in the decomposition and recycling of nutrients [26]. luepromchai et al. [27] has reported an increase in the numbers of hydrocarbon degraders in soil in the presence of pahs, without any impact on the overall bacterial numbers. the difference between thb and hub counts was observed to be minimally insignificant, which suggests that most of the micro-organisms present in various polluted sample sites are http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 7 hydrocarbon degraders that can withstand the concentrations of hydrocarbons and use them as a source of carbon [25]. the investigation of the morphology and type of bacterial colonies obtained from the oil-polluted soil showed seven bacterial species, which are corynebacterium kutscheri, pseudomonas aeruginosa, micrococcus agilis, flavobacterium aquatile, staphylococcus aureus, micrococcus luteus, serratia odorifera, and bacillus substilis. this shows the majority of bacteria isolated as being gram-positive species. some researchers have, however, reported oil-polluted soils as being dominated by gram-negative bacteria [25] the results of the emulsification test as shown in figure 1 demonstrates that the isolates produced emulsifying compounds. a large variety and number of biosurfactant producers have been isolated from hydrocarbon-impacted sites [28], although they have also been identified from soils, which are unconnected to hydrocarbon contamination [29]. corynebacterium kutscheri showed the highest emulsification (52%) at 1% waste engine oil, while the least was staphylococcus aureus (8%). corynebacterium sp has also been reported by onuoha et al. [22] with high emulsification ability among three hydrocarbon-degrading bacterial species isolated from soil. decrease in the ph of the medium (as shown on table 5), increase in total viable counts (table 6), as well as in turbidity of the medium (table 7) were regarded as indicators of degradation. the correlation between the ph of the medium and cell growth with hydrocarbon utilization has been previously reported by patila et al., [30]. the preliminary estimation of the isolate’s degradative ability during growth in hydrocarbon supplemented medium suggested the effectiveness of growth in hydrocarbon medium by corynebacterium kutscheri, and thus its potential as a candidate for further biodegradation studies. conclusion the result of this study indicates that the contaminated environments possess indigenous populations within them, as seen in bacterial count results of microbiological growth of mineral salt cultures with waste engine oil as the sole carbon source. these indigenous bacteria isolated in this study show promising, ability to degrade petroleum hydrocarbons with corynebacterium kutscheri as a potential candidate for further biodegradation efficiency studies. acknowledgments the authors are grateful to the laboratory technologist of microbiology laboratory, michael okpara university, umudike, nigeria for their support. references 1.atlas rm. oil spills: regulation and biotechnologyogy: guest editorial. current opinion in biotechnology. 1995;3(3):220-223. 2.bragg jr, prince rc, harner ej, atlas rm. effectiveness of bioremediation for the exxon valdez oil spill. nature. 1994 mar;368(6470):413-8. 3.uzoije ap, agunwamba jc. physiochemical properties of soil in relation to varying rates of crude oil pollution. international journal of environmental science and technology. 2011;4(3):313-23. 4.benka-coker mo, ekundayo, ja. effects of exposure of arotherodon niloticus and pariocephalus sp. to waste drilling fluid. journal of science resource development. 1995;23(4):122-125. 5.ogundiran oo, afolabi ta. assessment of the physicochemical parameters and heavy metals toxicity of leachates from municipal solid waste open dumpsite. international journal of environmental science and technology. 2008 mar 1;5(2):243-50. 6.bossert i, bartha r. the fate of petroleum in soil ecosystem. in: petroleum microbiology. atlas, r. m. (ed). new york:macmillian;1984.p. 102-110. 7.leahy jg, colwell rr. microbial degradation of hydrocarbons in the environment. microbiology and molecular biology reviews. 1990 sep 1;54(3):305-15. 8.saadoun i, mohammad mj, hameed km, shawaqfah m. microbial populations of crude oil spill polluted soils at the jordan-iraq desert (the badia region). brazilian journal of microbiology. 2008;39:453-456. 9.prince r. the microbiology of marine oil spill bioremediation. in: oliver b, magot m, editors. petroleum microbiology, washington: asm press; 2005.p.153-164. 10.bamforth, sm, singleton i. bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions. journal of chemistry, technology and biotechnology. 2005;80:723–736. 11.vidali m. bioremediation: an overview. pure and applied chemistry. 2001;37(73):1163-1172. 12.malatova k. isolation and characterization of hydrocarbon degrading bacteria from environmental habitats in western new york stat. department of chemistry [thesis]. new york: rochester institute of technology, rochester; 2005.p.76-88. 13.prakash a, bisht s, singh j, teotia p, kela r, kumar v. biodegradation potential of petroleum hydrocarbons by bacteria and mixed bacterial consortium isolated from contaminated sites. turkish journal of engineering and environmental science. 2014;38: 41-50. 14.association of official analytical chemists. official methods of analysis of the association of analytical chemists. association of official analytical chemists; 2003. 15.association of official analytical chemists. official methods of analysis of the association of analytical http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 7 chemists. association of official analytical chemists; 2005. 16.chaîneau ch, morel j, dupont j, bury e, oudot j. comparison of the fuel oil biodegradation potential of hydrocarbon-assimilating microorganisms isolated from a temperate agricultural soil. the science of the total environmental. 2004;227:237-247. 17.bremner jm, mulvaney cs. total nitrogen determination. in: method of soil analysis, page a l, miller rh, keeney dr, editors. madison: american society of agronomy; 1982.p. 595–624. 18.ogunbayo ao, bello ra, nwagbara u. bioremediation of engine oil contaminated site. journal of emerging trends in engineering and applied science. 2012;3(3): 483-489. 19.okpokwasili gc, nwosu ai. degradation of aldrine by bacterial isolates. nigerian journal of technological resources. 1990;2: 1–6. 20.cheesbrough m. district laboratory practice in tropical countries. uk: cambridge university press. 1984;625 p. 21.holt jg, krieg nr, sneath pha, staley jt, williams st. bergey’s manual of determinative bacteriology. 9th edition. baltimore: williams and williams co.; 1994. 22.onuoha sc, olugbue vu, uraku ja, uchendu do. biodegradation potentials of hydrocarbon degraders from waste lubricating oil-spilled soils in ebonyi state, nigeria. international journal of agriculture and biology. 2011;13: 586–590. 23.ganesh a, lin j. diesel degradation and biosurfactant production by gram-positive isolates. african journal biotechnology. 2009;8(21):5847-5854. 24.chikere cb. culture-independent analysis of bacterial community composition during bioremediation of crude oil-polluted soil. british microbiological resources journal. 2012;2(3):187-211. 25.chikere cb, ekwuabu cb. culture-dependent characterization of hydrocarbon utilizing bacteria in selected crude oil-impacted sites in bodo, ogoni land, nigeria. african journal of environmental science and technology. 2014;8(6): 401-406. 26.eze vc, okpokwasili gc. microbial and other related changes in niger delta river sediment receiving industrial effluents. continental journal of microbiology. 2010;4:15-24. 27.luepromchai e, lertthamrongsak w, pinphanichakarn p, thaniyavarn s, pattaragulwanit k, juntongjin k. biodegradation of pahs in petroleum-contaminated soil using tamarind leaves as microbial inoculums. biodegradation. 2007 mar;29(2):516. 28.banat im, makkar rs, cameotra ss. potential commercial applications of microbial surfactants. applied microbiology and biotechnology. 2000;53(5): 495-508. 29.jennings em, tanner rs. biosurfactant-producing bacteria found in contaminated and uncontaminated soils. in: proceedings of the conference on hazardous waste research, oklahoma; 2000.299 p. 30.patila td, pawara s, kamblea pn, thakare sb. bioremediation of complex hydrocarbons using microbial consortium isolated from diesel oil polluted soil. der chemica sinica. 2012;3(4):953-958. 31.kaplan cw, kitts cl. bacterial succession in a petroleum land treatment unit. applied environmental microbiology. 2004;70: 1777–1786. http://bioscience.highlightsin.org/ highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 1 of 10 citation: nassar a.e., mousa k.h., madbouly a.a., ibrahim s.d. and alsamman a.m. (2018). identification of genes for wheat fungal resistance using bioinformatics techniques. highlights in bioscience, volume 1. article id 20185, dio:10.36462/ h.biosci.20185 received: september 5, 2018 accepted: october 25, 2018 published: november 10, 2018 copyright:© 2018 nassar et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests identification of genes for wheat fungal resistance using bioinformatics techniques ahmed e. nassar 1 , khaled h. mousa 1 , ahmed a. madbouly 2 , shafik d. ibrahim 3 and alsamman m. alsamman 3 * 1 genetics department, faculty of agriculture, mansoura university. 2 plant tissue culture department, date palm research institute, agricultural research institute 3 mggm lab., agricultural genetic engineering research institute, agricultural research center, egypt. * to whom correspondence should be addressed: smahmoud@ageri.sci.eg abstract for the majority of world populations, wheat (triticum aestivum l.) would be the first essential and economic cereal grain crop. pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development. multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. in addition the introduction for restriction fragment length polymorphism (rflp) for such genes in the new primer database. approximately 138 sequences of dna were recovered from the wheat genome by aligning 3845 anti-fungal amino acids through tblastn tool. the ncbi blastn online tool used to detect sequences with functional genes, where 92 genes have been detected. the total number of nucleotides was 48385, where the smallest dna sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. the wheat chromosomes 3d, and 4b have the highest number of sequences (9) followed by chromosomes 3b (7) and 3a(6), where wheat genomes a, b and d have 30, 35 and 27 genes, respectively. five different amino acids motifs have been revealed among studied wheat amino acid sequences. the gene annotation tools used to infer studied amino acid gene annotation. amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (stk), thaumatin, and cysteine-rich repeats representing 2, 9, 8, 19, 23 genes respectively, in addition to 31 hypothetical genes. the proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics. keywords: wheat, pathogens, pathways, fungal, phylogenetic, rflp. research article open access nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 2 of 10 introduction for the majority of world populations, wheat (triticum aestivum l.) would be the first essential and economic cereal grain crop. its 29 percent share shows the world economy's significance of wheat production from world's 730 million hectares of crops cultivated lands. depending on fao figures for 2017, this percentage is comparable to 218 million hectares of wheat region (1). in most of the world, wheat is an key ingredient of human diet. worldwide, 735 million tons of wheat were produced in 2015/2016, worth around us$ 145 billion (2). wheat accounts for almost 55% of the carbohydrates and 20% of the world's food calorie intake (3). in egypt, the significance of wheat crops returns to its strategic importance in egyptian dietary commodities as it offers more than one-third of egyptian consumers ' daily calorie intake and 45% of their daily total protein intake (4). wheat is egypt's largest winter grain and is cultivated across the nile delta and along the nile valley length. wheat is usually planted after summer maize, cotton, or rice crops in early november and collected in late april or early may. germination happens after planting at 19 to 22 weeks (5). while egypt's wheat productivity has increased over the past few years, wheat production provides only 45% of its yearly domestic demand. egypt is still one of the largest importing wheat countries. wheat imports were approximately 9.8 million tons in 2011, costing approximately us$ 3.2 billion (6). pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development (7). a key component for trying to meet this challenge is proper management of fungal diseases that may be accountable for yield losses of 15–20 percent per year. the rusts, blotches and head blight/scab are among the major wheat diseases that make a contribution to these losses(2). the rusts, blotches and head blight/scab are among the major wheat diseases that currently contribute to these losses. there are three diseases of wheat rust, namely stem, stripe and leaf rust, all caused by members of the basidiomycete family, genus puccinia, named p. graminis f. sp. tritici, p. striiformis f. sp. tritici (pst) and p. triticina (pt), respectively (2). multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. the resistance is attributable to the additive impacts of multiple resistance metabolites and proteins generated through a network of multiple plant r gene structures. plants recognize pathogenic elicitors or receptors and then stimulate downstream genes to ultimately produce resistance metabolites and proteins that suppress plant pathogen progression (8). following the view of pathogens and microbes, reactive oxygen species (ros) were consistently identified to accumulate in the plant and over the years, ros was postulated as an essential part of the plant's defense response (9). the connection between genetic variability identified using distinct molecular marker assays and genes responsible for morphological and physiological characteristics could be identified through sophisticated bioinformatics analysis tools (10–12). these tools could be used to identify and reveal new genes in wheat related to resistance to fungal diseases. our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. in addition the introduction for restriction fragment length polymorphism (rflp) for such genes in the new primer database. materials and methods anti fungal gene sequences have been downloaded from ncbi database (13). we have downloaded 3845 antifungal protein sequences from ncbi. the draft genome sequence of wheat has been downloaded from ensemble database (14). the local ncbi blast package (15) was used to build sequence databases using the wheat draft genome and blast all anti-fungal genes with tblastn against wheat database. the online ncbi blast tblatn was used to annotate sequences recovered from previous step. meme suite was used to discover amino acid motifs in the sequence (16). megax program was used to construct phylogenetic analysis through maximum likelihood algorithm (17). clustalo (18) tool was used to construct sequence similarity matrix through multiple sequence alignment. clust-vis was applied to create principal component analysis (pca) plots and heatmaps depicting genes similarities. pepstat (19) was used through in-home per scrips to assess proteins chemical and physical characteristics. the perl script restrictiondigest was used to detect restriction enzymes recognition sites in dna sequences (20). batchprimer3 online tool was used for designing pcr primers could be used to target studied wheat genes (21). result and discussion chromosomal distribution of wheat anti-fungal genes approximately 138 sequences of dna were recovered from the wheat genome by aligning 3845 antifungal amino acids through tblastn tool. the ncbi blastn nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 3 of 10 online tool used to detect sequences with functional genes, where 92 genes have been detected (supplementary file 1). the total number of nucleotides was 48385, where the smallest dna sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. the wheat chromosomes 3d, and 4b have the highest number of sequences (9) followed by chromosomes 3b (7) and 3a(6), where wheat genomes a, b and d have 30 , 35 and 27 genes, respectively (figure 1). . figure 1 : the circos diagram for the position of predicated anti-fungal genes and their proposed gene description, where the inner connections represent similar genes on separate chromosomes with sequence similarity over 75%. wheat anti-fungal proteins chemical and physical properties the proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics (figure 2 and table 1). the total molecular weight (mw) of studied fungal amino acid sequences was 1913 kda with an average of 20 kda, where 1a:551827789-551828139 has the minimum mw (10.6) and 5d:59043196-59044206 generated the maximum mw (71.911.68) extinction coefficients are a measure of how much light the protein can be measured at a certain wavelength from the extinction factor. approximation of this component is needed to be able to track the protein within the spectrophotometer. it is necessary to know the amino acid content to assess the molar extinction coefficient of the protein (22). table 1: the highest and lowest proteins characteristics and their corresponding proteins. protein characteristic total mean min. min. protein max. max. protein a280 molar extinction coefficients cystine bridges 2261000 24576.09 1740 3b 716295639-716296412 104570 5d 59043196-59044206 a280 molar extinction coefficients reduced 2214500 24070.65 1490 3b 716295639-716296412 103820 5d 59043196-59044206 improbability of expression in inclusion bodies 65.124 0.794 0.504 7d 45432639-45432947 0.977 3d 462838207-462838962 average residue weight 9829.348 106.841 98.498 7a 164401571-164402272 116.631 3d 550382548-550383990 isoelectric point 588.9429 6.402 4.0084 1a 20888904-20889590 10.3971 7b 40654848-40655408 molecular weight 1913642.24 20800.46 10658.89 1a 551827789-551828139 71911.68 5d 59043196-59044206 non-polar 5471.072 59.468 48.81 3a 719941535-719942353 69.919 5b 461292418-461292831 polar 3728.357 40.526 30.081 5b 461292418-461292831 51.19 3a 719941535-719942353 nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 4 of 10 protein characteristic total mean min. min. protein max. max. protein residues 17857 194.098 101 7d 45432639-45432947 644 5d 59043196-59044206 small 5629.919 61.195 40.488 6a 610941935-610943086 77.027 5b 518810105-518810797 tiny 3673.776 39.932 20.27 2b 634173118-634173630 54.054 5b 518810105-518810797 acidic 858.259 9.329 5.172 3a 20936815-20937576 17.593 4b 41828978-41829304 aliphatic 2702.584 29.376 19.048 5a 14266438-14266881 41.27 3d 8508332-8509102 aromatic 952.739 10.356 3.604 3b 716295639-716296412 16.292 4d 29673605-29674213 basic 932.965 10.141 4.082 7d 634805112-634805630 19.672 3b 176321011-176321379 charge 24.5 0.266 -9.5 3b 690358283-690358846 22 7b 40654848-40655408 charged 1791.221 19.47 10.204 7d 634805112-634805630 32.258 3d 550382548-550383990 the a280 molar extinction coefficients cystine bridges (a280-mecc ) and reduced (a280-mecr) are two different measurements of extinction coefficient, where salt bridges are important motifs of the tertiary protein structure and are mostly correlated with the structural influence force which maintains the stability of the protein. commonly found on the solvent system and particularly vulnerable to solvent-solute interactions typically with water as well as other cosolvents (23). the minimum scores of a280-mecc and a280-mecr were 1740 and 1490, generated by 3b:716295639716296412, while the highest scores were 104570 and 103820, revealed by 5d:59043196-59044206 with a mean of 24576 and 24070, respectively. expression improbability in bodies of inclusion (ieib) is a form of measurement of solubility. for instance, recombinant protein in escherichia coli could be expressed either as soluble throughout the cytosol or as insoluble in the bodies of inclusion (24). the ieib of wheat amino acids showed a mean of 0.794, where it ranges from 0.504 (7d:45432639-45432947) to 0.977 (3d: 462838207-462838962). figure 2: the protein chemical composition and physical properties for the predicated anti-fungal genes. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 5 of 10 the average residue weight (arw) stands for the average collective weight for all amino acid sequences according to its length. the total arw for all amino acid sequences was 9829.3 da, where 7a:164401571-164402272 revealed the minimum arw (98.498 da). 3d:550382548550383990 generated the maximum (116.631 da) and the mean was 106.841 da. the isoelectric point (ip) is the ph level at which protein's net charge is positive and is associated with its production of amino acids and protein conformation (25). the collective wheat fungal genes amino acids was 588.9 with a mean of 6.402, where it ranges from 4 (1a:20888904-20889590) to 10.4 (7b:40654848-40655408). in addition, a protein's folded structure becomes thermodynamically less desirable because it decreases the protein's disorder or entropy. nonpolar (water hating) side chains tend to push themselves inside a protein whereas side chains of polar (water loving) prefer to put themselves outside of the molecule (26). the non-polar and polar amino acid scores were ranged from 48.81 and 30.081 (3a:719941535-719942353 and 5b:461292418-461292831) to 69.919 and 51.19 (5b:461292418-461292831 and 3a:719941535-719942353), respectively. acidic amino acids have an acidic group in side chain while basic amino acids have some basic group in side chain. acidic amino acids have low pka while basic amino acids have high pka. the score of acidic and basic amino acids range from 5.172 and 4.082 (3a:20936815-20937576 and 7d:634805112-634805630 ) to 17.593 and 19.672 (4b:41828978-41829304 and 3b:176321011-176321379 ). the studied fungal amino acids charges ranged from -9.5 (3b:690358283-690358846) to 22 (7b:40654848-40655408) (figure 2 and table 1). detection of protein domain and phylogenetic analysis a motif for the protein sequence is a brief pattern that nature retains. for proteins, a motif can refer to an enzyme's active site or a structural unit required for proper protein folding. therefore, sequence motifs are among molecular evolution's basic functional components (16). five different amino acids motifs have been revealed among studied wheat amino acid sequences (motif-1: yglaqctpdl, motif-2: cylrysnknf, motif-3: pnrvwgitwc, motif-4: ydvs vvdgfn, and motif-5: npgdcgqclq) (figure 3 and 4). figure 3: the identified protein domains / motifs in predicated wheat genes. the motif-1 and motif-3 has a match to elme000249 in motif database with p-value of 9.64e-04. this motif is a trfh domain docking motifs wich are coordinating the telomeres with other proteins. not only do they form homodimers with their trfh domains, they also provide specific protein-binding surfaces for interaction (27). trfhs and other proteins have been proposed to have important implications for the biology and evolution of telomeres. in general, this offers a basis for recognizing and controlling the hierarchical assembly and stoichiometry of telomere subunits during the cell cycle, cell division and senescence (28). motif-2 has an elme000084 match which is ligands of phosphotyrosine bound by domains sh2. src homology 2 (sh2) domains identify tiny patterns comprising a residue of phosphorylated tyrosine. up to four positions after the ptyr was found mainly to assess additional specificity. in plants sh2 dominates new signaling scenarios and participates in metazoan signal transduction, serving as key mediators of controlled protein-protein interactions with tyrosinephosphorylated substrates (29, 30). moreover, the database motif of elme000377 was highly similar to motif-4, this motif codes for pex14 ligand motif, which belongs to peroxisomes. peroxisomes are subcellular organelles present in eukaryotes that are singlemembrane spherical. peroxisomes relate together with glyoxysomes found in plants and glycosomes found in trypanosomes to microbody group of organelles. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 6 of 10 figure 4: the phylogenetic tree developed using multiple sequence alignment of predicted anti-fungal genes as compared to the expected positions of protein domains/motifs. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 7 of 10 import into the peroxisome of peroxisomal matrix enzyme proteins (pts1 cargo) includes the identification of the pts1 cargo by the cytosol pex5 receptor, the docking of the pts1-pex5 complex at the peroxisomal membrane, and the translocation of the pts1 cargo through the peroxisomal membrane into the matrix. this is preceded by recycling the pex5 receptor back into the cytosol for another round of export of pts1 stock. imported into peroxisome are unfolded, folded oligomeric or cofactor-bound proteins (31). additionally, motif-5 was with high similarity with elme000159, which is mapk phosphorylation site. cascades of mitogen-activated protein kinase (mapk) are highly conserved downstream signaling modules of receptors/sensors that transform extracellular stimuli in eukaryotes into intracellular responses. plant mapk cascades play crucial functions against pathogen invasion in signaling plant defense (32). the ncbi-blastx tool used to infer studied amino acid gene annotation. amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (stk), thaumatin, and cysteine-rich repeats representing respectively, 2, 9, 8, 19, 23 genes, in addition to 31 hypothetical genes (figure 1). lectins are non-immune proteins which attach carbohydrates directly and reversibly. the lectin's biochemical functions are really diverse. a common theme arising from the identified functions of many plant and animal lectins is their involvement in communicating with other organisms whether symbiosis or defense, as effectors or regulators (33). new types of nucleocytoplasmic plant lectins have been described and defined over the past decade, especially lectins expressed within the nucleus and the cytoplasm of plant cells, much as part of a particular plant response when exposed to different stressors or shifting environmental conditions (34). lectins include proteins containing at least one non-catalytic domain which helps them to selectively identify and reversibly attach to different glycans that are either freely available or are member of glycoproteins or glycolipids. plants release an overwhelming number of highly complex lectins with various molecular structures and attaching specificities to endogenous (plant) glycans as well as exogenous (non-plant) glycans (35). the role of plant lectins in plant defense againt different pathogens including fungi have been reported in previous researches (36,37). there are several receptor-like kinases among the plant proteins proposed to engage in immunity pathways (38). for example, multiple kinase proteins have been recorded for wheat resistance to fungal diseases, which provides temperature-dependent resistance to wheat stripe rust (39), and fungal resistance in arabidopsis (40). in this regards , various thaumatin-like proteins have been reported to generate tolerance to fungal pathogens arabidopsis (41), potato (42) and grape (43). moreover, cysteine-rich proteins have been informed to confer resistance for fungal diseases such as maize fungal pathogens (44) and legume modules (45-47). using maximum likelihood analysis phylogenetic analysis was effective in differentiating between anti-fungal wheat proteins according to their protein domains. four classes grouped the phylogenetic tree. group a comprises only motif-3 domain protein fragments, group b has motif-3, motif-4 and motif-5 domain proteins. in addition some group c contains genes with motif-1, motif-2 and motif-5 domains, and eventually group d contains genes with all protein domains (figure 4). pcr primers and restriction enzymes analysis about 85 pcr primers pairs have been designed to target most of the predicted anti-fungal genes (supplementary file 2). the pcr predicated product size ranged from 300 bp to 708 pb, where the gc content ranges from 36.4 to 66.7% and the primers annealing temperature ranges from 58.05 oc to 62.55 oc. a total of 40 different restriction enzymes (re) were used to identify potential locations for future rflp tests for genetic polymorphism inside predicted anti-fungal wheat genes. figure (5) demonstrates the site redundancy ratio for re identification within genes. figure (5) shows that some re such as banii, nlaiii and baegi have high probability to produce fragment length polymorphism isnide most of predicated anti-fungal genes. conclusion predicting using available fungal resistance genes in the public database was very helpful and suggested that several wheat genes could be used to restrict genetic research of genes that hold the key to fungal resistance in wheat. most of the identified protein domains clarify the genetic structure of anti-fungal genes and suggest a potential role for the mapk gene family in such pathways. providing restriction enzyme information and gene-specific pcr primers could be useful to wheat scientists and breeders, saving time and effort. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 8 of 10 figure 5: the ratio heat map for redundancy site identification of different restriction enzymes, where red and blue squares represent high and low rate of re digestion sites, respectively. supplementary files the supplementary material for this article can be found online at: http://bioscience.highlightsin.org/2018/20185.php supplementary file 1: the nucleotide sequences of identified fungal resistance genes in wheat genome. supplementary file 2: the information of pcr primers designed to target identified fungal resistance genes in wheat. referances 1. faostat. 2017; available from: http://www.fao.org 2. figueroa m, hammond-kosack ke, solomon ps. a review of wheat diseases—a field perspective. mol plant pathol. 2018;19(6):1523–36. 3. kumar p, yadava rk, gollen b, kumar s, verma rk, yadav s. nutritional contents and medicinal properties of wheat: a review. life sci med res. 2011;22:1–10. 4. farg e, arafat sm, el-wahed ma, el-gindy am. estimation of evapotranspiration etc and crop coefficient kc of wheat, in south nile delta of egypt 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distelfeld a, blechl a, epstein l, chen x, et al. a kinase-start gene confers temperaturedependent resistance to wheat stripe rust. science (80). 2009;323(5919):1357–60. 40. wan j, zhang x-c, neece d, ramonell km, clough s, kim s, et al. a lysm receptor-like kinase plays a critical role in chitin signaling and fungal resistance in arabidopsis. plant cell. 2008;20(2):471–81. 41. misra rc, kamthan m, kumar s, ghosh s, others. a thaumatin-like protein of ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic arabidopsis. sci rep. 2016;6:25340. 42. ali g, hu x, reddy asn. overexpression of the arabidopsis thaumatin-like protein 1 in transgenic potato plants enhances resistance against early and late blights. biorxiv. 2019;621649. 43. marcato r, sella l, lucchetta m, vincenzi s, odorizzi s, curioni a, et al. necrotrophic fungal plant pathogens display different mechanisms to counteract grape chitinase and thaumatin-like protein. physiol mol plant pathol. 2017;99:7–15. 44. patiño b, vázquez c, manning jm, roncero mig, córdoba-cañero d, di pietro a, et al. characterization of a novel cysteine-rich antifungal protein from fusarium graminearum with activity against maize fungal pathogens. int j food microbiol. 2018;283:4551. 45. maróti g, downie ja, kondorosi é. plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules. curr opin plant biol. 2015;26:57–63. 46. mokhtar mm, adawy ss, el-assal se-ds, hussein eha. genic and intergenic ssr database generation, snps determination and pathway annotations, in date palm (phoenix dactylifera l.). plos one. public library of science; 2016;11(7):e0159268. 47. adawy ss, mokhtar mm, alsamman am, sakr mm. development of annotated est-ssr database in olive (olea europaea). int j sci res. 2015;4(9):1063–73. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202201 review article open access 1 biofuel research laboratory, department of microbiology, school of life sciences, central university of tamil nadu, thiruvarur, tamil nadu, india. * to whom correspondence should be addressed: suchitrar@cutn.ac.in editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): alsamman m. alsamman, african genome center, mohammed vi polytechnic university,morocco.. morad m. mokhtar, agricultural genetic engineering research institute, agricultural research center, giza, egypt. received: october 29, 2021 accepted: january 1, 2022 published: january 15, 2022 citation: ray b, rakesh s . phycoremediation of aquaculture wastewater and algal lipid extraction for fuel conversion. 2022 jan 15;5:bs202201 copyright: © 2022 ray and rakesh. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this work is financially supported by serb dst project (eeq/2018/001463). competing interests: the authors declare that they have no competing interests. phycoremediation of aquaculture wastewater and algal lipid extraction for fuel conversion bobita ray1 >< ,suchitra rakesh*1 ><  abstract in this review, it is discussed the prominent effect generated from aquaculture wastewater considered as the major water polluting crisis in the entire world. the cause rose due to intense development and improvement in aquaculture by the aquatic habitat species triggering quite a challenge in the environment. scrutinizing this problem, researchers have found a way to tackle it by cultivating algal species in aquaculture wastewater in order to remove its high content of organic and inorganic pollutants. the theory proves wastewater serves as a nutrient source for algal growth and development such as phosphorous, nitrogen, and other trace elements. besides harvesting the algal biomass from aquaculture wastewater, the extraction of lipid is also processed for biofuel production. hence, the discussion includes conversion of wastewater into organic and inorganic pollutant-free water with low cost-effective method via algal cultivation in wastewater and high lipid yield for biofuel with a carbon-free and sustainable environment. keywords: algae, aquaculture wastewater, harvesting, lipid extraction, transesterification introduction ever increasing global population and continuous dependence of fossil fuels, increased urbanization and industrialization posing a major threat to energy security and environmental concerns to both developed and developing nations. with the accelerated speed of increasing population growth, wastewater treatment is considered as one of the solutions to control the environmental issues. and the additional challenges for water scarcity bring out the crucial problem related to wastewater. hence, much of the emphasis has been given now a days for wastewater treatment [1]. the anthropological activities such as sewage, industries, agriculture, medical, research laboratories etc., are pointed to be the sources of wastewater which have tremendously polluted the water resources. wastewater from various sources comprises both organic and inorganic pollutants. organic pollutants include proteins, carbohydrates, lipids, etc., whereas inorganic mostly have chemicals and solvents [2], and in industrial wastewater even heavy metals or toxic elements are reported [1]. nowadays the aquaculture wastewater is quite prominent globally due to its intense development and improvement in fish, marine species, algae and aquatic plant farming. thus, this rapid increase of aquaculture effluents poses a serious threat to environment [3]. mariculture other name of aquaculture can be seen their progresses in parallel way with high human demands. the impact performances to the environment for food production has been reported as the fastest growth [4]. aquaculture wastewater contains a high number of pollutants and chemicals that lead the residing aquatic flora and fauna to die due to eutrophication. the toxic algal blooms not only disturb the aquatic life but it also interferes with the sustainable marine aquaculture development. the wastewater treatment via biochemical methods is not economical and further removal of those chemicals from water poses another challenge. hence, wastewater treatment by algae is an ecofriendly and cost-effective approach over other physical and chemical methods [5–7]. highlights in bioscience page 1 of 9 january 2022|volume 5 https://doi.org/10.36462/h.biosci.202201 https://creativecommons.org/licenses/by/4.0/ raybobita92@gmail.com suchitrar@cutn.ac.in https://orcid.org/0000-0002-4357-4042 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion earlier aquaculture wastewaters were treated with bulks of antibiotics which later have evolved to antibiotic-resistant [8]. but in an investigation, green microalgae tetraselmis sp. removed nitrogenous and phosphorous compound from aquatic wastewater within 48 hours [9]. removal of nutrients from livestock wastewater was also reported via, desmodesmus sp. a microalga with potential benefactor [10]. the wastewater treatment via microalgae not only removes the pollutants but also shows positive effect towards carbon fixation (1.83 kg co2/kg of biomass), high amount of biomass generation within a short period of time. the microalgal biomass can be further utilized for biofuel and valuable bio-products production and can also act as substrate in bio-refinery. thereby, it provides a sustainable and ecofriendly approach to many of the problems related with wastewater [11]. since the middle time of 20th century, development in aquaculture growth has risen globally in all over countries providing huge profit to commercial hatcheries and farming system. the wastewater discharge from chemical and other industries has high toxicity level, that poses serious environmental issues [12]. the aquaculture production is kept on increasing due to high demand in the market. hence large-scale production has been carrying out enormously [13]. yang et al., [14] has mentioned in his studies the aquaculture effluent treatment with microalgae is highly efficient in absorbing nutrients and value-added biomass generation. alga-aquaculture has led to many advantages such as compared to other plants, algae has proved to be better in nutrient removal. the construction and operational costs are low with consistent to high nutrient removal efficiency. the microorganisms consortia like algal-bacterial consortia are highly efficient in solid and other waste treatment into low molecular weight compounds [15]. the large quantity of algal biomass can be produced from aquaculture, that can be further utilized for high demand valuable product generation spirulina and chlorella cultures are commonly used as aquaculture feed, as both has very minimal toxicity level and helps in preventing algal blooms as well. on the other way, addition of expensive chemicals and antibiotics for industrial effluent treatment are not economical and poses severe threat to the environmental [16]. it has also been reported that excess use of chemicals in aquaculture affects the food safety and quality of meat produced via aquaculture. in most of the research studies, microalgae such as chlorella sp. is found experimenting in every aspect of wastewater treatment. biofilms are another slimy and foul in nature found on the surface of the algae or any solid surface attached. microalgal biofilms mostly have succeeded in reducing the nutrients of phosphorous starting initially from 15 mg l-1 within 24 hours[12]. the biofilm has succeeded in more production of biomass production for biofuel [17]. this review summarizes our efforts towards various aquaculture wastewater treatment via microalgae and use of algae for feed purposes. it further highlights the biofuel and value-added products generation from the algal biomass. micro and macro algae as a nutritive aquaculture feed aquaculture has been rapidly developing in industrial sector resulting large quantity of polluted effluents being discarded into clear water line. remaining solid residues mostly contains hazardous chemicals and metal elements causing severe incurable diseases [1]. huge amount of cost and labor are invested upon various physical and chemical techniques. electro adsorption and electro-reaction coupling process is one of physical technique to clean the wastewater, removing 99% of solid. but it is reported algae is the most efficient and advanced method with low cost benefits in treating wastewater [18]. in aquaculture, many of the microalgae viz., nannochloropsis, chaetoceros, thalassiosira, tetraselmis etc. are known for essential food sources including marine species such as clams, molluscs, oysters and spirulina sp. for providing high protein diet for freshwater fishes and other invertebrate species [17]. treatment of wastewater with microalgae has been guaranteeing good outcome and have led to great advantages without harming the environment. macroalgae universally known as seaweed is easily visible through naked eye. its habitats are mostly native to marine or other river bodies. saccharina latissima also known as kelps are generally found in river depth. macroalgae are well-known for their nutritional and bioactive components. aquaculture with macroalgae production has a strong demand in the market, according to industrial vision. it is a valuable source of aquaculture feed. however, most examples of eutrophication in the marine environment are caused by the deposition of flowing waste in the sea, which includes high nitrogen and phosphorus nutrients. macroalgae also aids in the bioremediation process by preventing wastes from impacting the marine environment in terms of ph change, turbidity, and increased bod content, as well as causing marine life death and encouraging toxic algal blooms [19–21]. according to brakel et al., [22] macroalgae depicts as fastest growing aquaculture development even in poorest coastal regions. nowadays with advanced facilitation and support of genetic resources, seaweeds such as red algal genera eucheuma and kappaphycus proved economically in many tropical countries. microalgae based biorefinery for aquaculture wastewater treatment wastewater treatment is rising as fundamental priority. the removal of nutrients and solids, as well as the acceptance of environmentally friendly remediation techniques, play a significant role in this. the most photosynthetic machinery technique that we can ever expect is phycoremediation, or treating wastewater with algae. it extracts all unwanted parameters from wastewater and improves water quality to meet environmental standards. [15]. compared to conventional wastewater treatment this method is cheap and also has the involvement in biomass production for biorefinery purposes. chlorella sorokiniana is observed as the highlights in bioscience page 2 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion most utilized objective both for phycoremediation and biomass production [23]. open pond system algal cultivation is the basic necessary condition for more quantity of biomass for biofuel production. depending on cost, labor and time vast kinds of techniques are available. open pond is the common system for large scale algal production exposed directly to environment. here generally, algae species are cultivated in an open pond area covering as much acres of land directly under the sunlight due to their phototrophic nature [24]. open pond is named as raceway because it resembles with racetrack. this raceway pond system takes less space of land for growth. it requires continuous movement of paddle wheel in the pond to prevent sedimentation of cultures at the bottom level [25]. generally, paddle wheels depicts the main principle base for the open pond system where the speed of the wheel helps to cover the light intensity for all over the algal growth within the system [26]. nutrient removal efficiency of aquaculture wastewater with microalgae the most efficient process and cost-effective method for culturing algae is via wastewater sources; rather than cultivating in expensive amounts of chemicals. aquaculture wastewater contains required nutrients such as nitrogen, sulphur, phosphorous which alga feeds on for growth. its composition is mentioned in table 1 , pointing its physico-chemical properties such as its ph, chemical oxygen demand (cod), nitrate, chloride, sodium, potassium, magnesium, nitrite, ammonium and phosphorous were depicted in aquaculture wastewater. in table 1, the content found under those properties extremely higher compared to normal i.e. these wastewater has the capability to cause diseases. wastewater cultivation is positively progressing both in bioremediation and biomass production for biofuel. it is either way sustainable to environment as budget friendly way. ulva sp., codium sp., ecklonia sp., saccharina sp., gracilariopsis sp. have experimented in fish seaweed aquaculture waste for bioremediation that have removed high concentration of ammonia and phosphorous within 30-40% nutrient removal converting into less polluting [30]. whereas for microalgae tetradesmus obliquus has removed 99.3% of ammonia and 99.2% of phosphorous concentration from swine manure wastewater [31]. in table 2, various algal species cultivated in different types of aquaculture wastewater are shown. the inoculated culture in the wastewater is mentioned parallel to the algal species name. the remaining columns are about the results of removal of nutrients described in percentage that found after cultivating in aquaculture wastewater. it specifies how algae worked as bio-remediation. ta bl e 1. c om po si ti on of va ri ou s ty pe s of aq ua cu lt ur e w as te w at er . ty pe s of aq ua cu lt ur e w as te w at er ph c o d (m g/ l ) n it ra te (m g/ l ) c hl or id e (m g/ l ) s od iu m (m g/ l ) p ot as si um (m g/ l ) m ag ne si um (m g/ l ) n it ri te (m g/ l ) a m m on iu m (m g/ l ) p ho sp ho ro us (m g/ l ) r ef er en ce f is he ry 7. 86 32 .4 0. 35 n a n a n a n a 24 .7 6. 25 1. 83 [7 ] f is he ry 8. 1 2. 25 n a 19 ,4 00 10 ,7 90 38 7 12 93 n a n a 1. 21 [2 7] s ea w at er m ar in e 7. 75 7. 84 8. 02 n a n a n a n a 0. 25 0. 48 4. 56 [2 8] o re oc hr om is ni lo ti cu s aq ua cu lt ur e 5. 22 64 .3 52 .0 24 .5 28 .5 8. 3 3. 3 0. 01 12 .8 11 .2 [2 3] f is he ry 7. 2 n a 96 .6 0 65 5 54 0 21 69 0. 00 6 0. 01 0 1. 98 [2 9] n a n ot a va il ab le highlights in bioscience page 3 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion table 2. nutrient removal in aquaculture wastewater with different algal species types of aquaculture wastewater algae species type of algae amount of inoculation (g/l) time of treatment (days) removal compounds reference cod (%) nh4 (%) nitrate (%) nitrite (%) phosphorous (%) total nitrogen (%) fishery p. kessleri ty microalgae 10 3 94.4 96.2 94.3 99 96.6 na [7] salmon farming chlorella minutissima microalgae na 10 na na 88.6 74.3 99 88 [27] shrimp culture gracilaria tenuifrons macroalgae 1.75 8 na 35.1 na 71.7 33.2 2.8 [28] fish-seaweed aquaculture codium fragile macroalgae 1000 28 na 0.07 na na 0.22 0.56 [23] ulva pertusa 1000 28 na 0.04 na na 0.15 0.56 ecklonia stolonifera 1000 28 na 0.11 na na 0.26 0.57 gracilariopsis chorda 1000 28 na 0.11 na na 0.23 0.50 saccharina japonica 1000 28 na 0.15 na na 0.21 0.56 oreochromis niloticus aquaculture chlorella sorokiniana microalgae na 14 na 99.9 75.2 na 77 78 [29] na not available recent advances in microalgae harvesting and lipid extraction after cultivation, harvesting which means collecting or gathering of algal cultivation determines as most difficult and important out of all process work. for large scale harvesting of biomass, it requires quite expensive technique, maintenance of time, man power and so on. in case of microalgae harvesting techniques such as centrifugation, sieving, filtration, sedimentation, flotation, flocculation are predominantly utilized [33]. whereas for macroalgae simple technique such as drying and storing is basically preferred but however few techniques from microalgae harvesting techniques are also operated [34]. thermo reversible gel transition [35,36] characterized with either agar or sol gel for harvesting of algae where clustered cells are settled at bottom and collected the biomass. flocculation is another technique of harvesting. nanocellulose is an insoluble substance where bonding of polysaccharide and glucose monomers occurs with the concept of more concentration of nanofibril more increase of flocculation [37]. bacterial cellulose gluconacetobacter xylinus has found to be successfully harvested with 90% of clump formation [38]. pleaurotus ostreatus [39] and scenedesmus obliquus [40] are another flocculating process. according to leite [41], ph modulation through dissolved air flotation can be harvested at higher biomass. magnetic nanoparticles is another better technique for harvesting [42] mostly utilized lipid extraction method is bligh and dyer as said to be quickly approachable to quantification outcome within less timing but more hazardous to environment as well as self-health. but mtbe i.e., methyl-tert-butyl ether is the better method than the previous method with non-hazardous effect and increase in the extracted lipid [43]. for future perspective role such as to study the characterization from extracted algal biomass production, high resolution nuclear magnetic resonance spectroscopy (hr nmr) or mass spectroscopy technique is used to study the changes of various composition kept in different storage conditions were found in the algal sample. such techniques are extremely advanced in analytical process [34]. on the other hand, dimethyl ether is a gas type where the liquefied gas is passed with the help of nitrogen gas and proceeded for algal extracting [44]. super high hydrostatic pressure technique is even utilized for extracting lipid maintaining pressure 100 mpa to 1000 mpa [45]. another process of lipid extraction solvent is ionic liquid that comprises of ion solvents of non-volatile substance including bubbling co2 gas for extraction [46]. in the given table 3, it basically determines the biomass productivity and lipid growth found after algal cultivation in aquaculture wastewater. in the same table various algal species names and according to that in the left column the types of wastewater are given where following that horizontally we can read the biomass and lipid found after cultivating the algae in that same wastewater. within these three table tables (table 1, 2 and 3) it gives the idea about reading the physico chemical properties before algae cultivating and harvesting the biomass, lipid measuring and lastly with the remaining water and can be proceeded with rereading the physico chemical properties determining removal of nutrients from the wastewater. microalgae as a sustainable future biofuel approach developing with rapid high rise of industries by regular use of natural resources are leading us into depletion of fossil fuels sooner creating havoc in environment. it surges carbon dioxide till peak point making possibly prone to global warming similarly threatening wastewater globally. at the bright side, the microalgae have several unique features like ability to fix co2 and convert it into valuable components via photosynthesis, robust growth with high lipid contents. the microalgae harvesting, qualitative and quantitative estimation of lipid has been reviewed [43,47]. the availability of molecular approaches to increase lipid accumulation and recovery has been extensively discussed [48]. biofuel is the breakthrough for solution. among generation after generation there has been change into biofuel production. initially beginning with edible plants such as soybean, maize, brassica comes under first generation and had a great deal with alternative fuel. the lipid yield was good but in case of highlights in bioscience page 4 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion ta bl e 3. n ut ri en tr em ov al in aq ua cu lt ur e w as te w at er w it h di ff er en ta lg al sp ec ie s a qu ac ul tu re w as te w at er a lg al sp ec ie s ty pe of al ga e to ta ld ur at io n da ys of tr ea tm en tm et ho d ( m g/ l ) b io m as s co nc en tr at io n /p ro du ct iv it y (% ) l ip id gr ow th (% ) r ef er en ce f is he ry p. ke ss le ri t y m ic ro al ga e 5 26 n a [7 ] s ea br ea m fa ct or y te tr as el m is su ec ic a m ic ro al ga e 10 68 25 [2 7] s al m on fa rm in g c hl or el la m in ut is si m a m ic ro al ga e 10 55 46 .3 7 [1 3] n a n ot a va il ab le other matters like production of biodiesel from food crops during the time of world war period was a huge downfall. in second generation jatropha plant being the non-edible is another alternative fuel production which is a good source compared to first one [49]. the life cycle, production in large scale, huge mass of land for cultivation is the major demerit. third generation i.e., microalgae is currently the most successfully running lipid yield production out of all. cultivation of microalgae is only 14 days where this microorganism can be grown in even a small tub or artificial huge ponds. the biomass with high production of yield can be grown in any suitable environment with different stress conditions changing physiological condition. monoraphidium sp.is cultivated in bg 11 media and transferred into high ammonia content wastewater with 44% of stress condition present [50]. transesterification, in case of biodiesel it can be termed as conversion of a 3-methyl glyceride when it reacts with methanol in presence of catalyst to form fatty acid methyl esters to form ethanol, likewise shown in figure 1. for conversion into biofuel after harvesting method and weighing dry biomass, lipid extraction process is followed. it basically consists of two types mechanical and non-mechanical, the previous type usually relates with solvents and the later describes extraction through instrumental techniques. triglycerides act as main components, these are fatty acids extracted from algal species and converted into fatty acid methyl esters through direct transesterification method. this method depicts reaction of triglycerides with mono alcohols in presence of catalyst were analyzing solvent as hexane with better results compared to chloroform and methanol where pointing a strong line selection of solvents affects in lipid yielding after purification [51]. biodiesel has inherent sustainable transportation fuels for future mostly to reduce increasing pollutants emitted from exhaust cylinder. many modernized machine learning techniques and renewable feedstock are emerging rapidly for biofuel conversion compared to chemical catalysts. the main source of biodiesel is manufactured basically from renewable oil derived microbes or plants which causes zero-effect in ecosystem accompanying with carbon reduction. enzyme mediated undertakes non-toxic transesterification compared to same old process of chemical utilization [52,53]. from many processing experiments, microbes such as microalgae is the leading aspect, euglena sanguinea due to its presence of superior combustion characteristics were able to produce biodiesel that blends with the regular agricultural diesel engine till 40% by extracting lipid from the algal biomass [27]. a heterogeneous nano-catalyst ca(och3)2, a novel reactive distillation column is experimented for algal biodiesel production optimized by maximizing biodiesel purity by nsga-ii, non-dominated sorting genetic algorithm, designed both for low cost production and co2 emissions [28]. for another substitute yield of biodiesel an experiment conducted between chlorella sorokiniana and monoraphidium sp. where the biomass, fatty acid prohighlights in bioscience page 5 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion figure 1. flowchart diagram for biodiesel production through use of transesterification. highlights in bioscience page 6 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion file studies were compared showing better outcome from chlorella sp. in the mentioned study, lipid analyzing, its thermal efficiency all were recovered higher for biodiesel benefitting with low emissions of co and hc [51]. lipid extraction through microwave assisted in situ transesterification technique for the biodiesel yield was achieved from algae such as ulothrix sp. 88% dry weight (dw), cladophora sp. 80% dw, oedogonium sp. 73% dw and spirogyra sp. 67% dw. this technique for high biodiesel yield utilizes solvent free method [29]. conclusion from this study it reveals aquaculture wastewater is being the significant source for cultivating algae proceeding with sustainable environment in simple, cost effective way with zero waste reassurance. both microalgae and macroalgae plays vital role in aquaculture production and wastewater remediation. the presence of nutrients in wastewater reveals necessary requirements for their growth. the emitted aquaculture effluent contains highly nutritive source for algae that blends into it recommending as bio or phyco remediation in process. addition to that biomass produced from aquaculture wastewater can also be converted into biofuel through transesterification process with recent ideas of harvesting techniques. from the reported articles, it is known not much work have been proceeded in aquaculture wastewater co-related with micro and macroalgae. it still needs to be explored in order to achieve higher biomass and lipid for biofuel production where a solution is required for further research as there is huge gap in laboratory work and large-scale production. hence, this study needs to be taken to further simplified step by investigating more into it. acknowledgement the authors thank the academic writing group and swayam moocs course initiated by the ministry of human resource development, government of india, for providing an open learning platform. references 1. mao m, yan t, shen j, zhang j, zhang d. capacitive removal of heavy metal ions from wastewater via an electro-adsorption and electro-reaction coupling process. environmental science & technology. 2021 feb 19;55(5):3333-40. 2. nagi m, he m, li d, gebreluel t, cheng b, wang c. utilization of tannery wastewater for biofuel production: new insights on microalgae growth and biomass production. scientific reports. 2020 jan 30;10(1):1-4. 3. khan na, ahmed s, farooqi ih, ali i, vambol v, changani f, et al. occurrence, sources and conventional treatment techniques for various antibiotics present in hospital wastewaters: a critical review. trac trends in analytical chemistry [internet]. 2020;129:115921. 4. giangrande a, gravina mf, rossi s, longo c, pierri c. aquaculture and restoration: perspectives from mediterranean sea experiences. water (switzerland). 2021;13(7):1–16. 5. rahmawati ai, saputra rn, hidayatullah a, dwiarto a, junaedi h, cahyadi d, et al. enhancement of penaeus vannamei shrimp growth using nanobubble in indoor raceway pond. aquaculture and fisheries. 2021 may 1;6(3):277–82. 6. ding y, guo z, mei j, liang z, li z, hou x. 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sulfonamides from marine aquaculture wastewater by concentrated and attached cultivation of chlorella vulgaris in an algal biofilm membrane photobioreactor (bf-mpbr). science of the total environment. 2020 jul 10;725:138524. 29. stamenkovic m, steinwall e, wulff a, henley w. cultivation and photophysiological characteristics of desmids in moderately saline aquaculture wastewater. journal of phycology. 2021: jpy-13150. 30. kang yh, kim s, choi sk, lee hj, chung ik, park sr. a comparison of the bioremediation potential of five seaweed species in an integrated fishseaweed aquaculture system: implication for a multispecies seaweed culture. reviews in aquaculture. 2021 jan;13(1):353-64. 31. miyawaki b, mariano ab, vargas jv, balmant w, defrancheschi ac, corrêa do, santos b, selesu nf, ordonez jc, kava vm. microalgae derived biomass and bioenergy production enhancement through biogas purification and wastewater treatment. renewable energy. 2021 jan 1;163:1153-65. 32. do amaral carneiro ma, de jesus resende jf, oliveira sr, de oliveira fernandes f, dos santos borburema hd, barbosa-silva ms, ferreira ab, marinho-soriano e. performance of the agarophyte gracilariopsis tenuifrons in a multi-trophic aquaculture system with litopenaeus vannamei using water recirculation. journal of applied phycology. 2021 feb;33(1):481-90. 33. tan js, lee sy, chew kw, lam mk, lim jw, ho sh, show pl. a review on microalgae cultivation and harvesting, and their biomass extraction processing using ionic liquids. bioengineered. 2020 jan 1;11(1):116-29. 34. chauton ms, forbord s, mäkinen s, sarno a, slizyte r, mozuraityte r, standal ib, skjermo j. sustainable resource production for manufacturing bioactives from microand macroalgae: examples from harvesting and cultivation in the nordic region. physiologia plantarum. 2021 mar 9. 35. estime b, ren d, sureshkumar r. cultivation and energy efficient harvesting of microalgae using thermoreversible sol-gel transition. scientific reports. 2017 jan 19;7(1):1-9. 36. kumar v, nanda m, verma m. application of agar liquid-gel transition in cultivation and harvesting of microalgae for biodiesel production. bioresource technology. 2017 nov 1;243:163-8. 37. yu si, min sk, shin hs. nanocellulose size regulates microalgal flocculation and lipid metabolism. scientific reports. 2016 oct 31;6(1):1-9. 38. chen q, fan q, zhang z, mei y, wang h. effective in situ harvest of microalgae with bacterial cellulose produced by gluconacetobacter xylinus. algal research. 2018 nov 1;35:349-54. 39. luo s, wu x, jiang h, yu m, liu y, min a, li w, ruan r. edible fungi-assisted harvesting system for efficient microalgae bio-flocculation. bioresource technology. 2019 jun 1;282:32530. 40. chen z, qiu s, yu z, li m, ge s. enhanced secretions of algal cell-adhesion molecules and metal ion-binding exoproteins promote self-flocculation of chlorella sp. cultivated in municipal wastewater. environmental science & technology. 2021 aug 23;55(17):11916-24. 41. de souza leite l, dos santos pr, daniel la. microalgae harvesting from wastewater by ph modulation and flotation: assessing and optimizing operational parameters. journal of environmental management. 2020 jan 15;254:109825. 42. markeb aa, llimós-turet j, ferrer i, blánquez p, alonso a, sánchez a, moral-vico j, font x. the use of magnetic iron oxide based nanoparticles to improve microalgae harvesting in real wastewater. water research. 2019 aug 1;159:490-500. 43. rakesh s, tharunkumar j, sri b, jothibasu k, karthikeyan s. sustainable cost-effective microalgae harvesting strategies for the production of biofuel and oleochemicals. highlights in bioscience. 2020 jul 7;3. 44. wang q, oshita k, nitta t, takaoka m. evaluation of a sludgetreatment process comprising lipid extraction and drying using liquefied dimethyl ether. environmental technology. 2021 sep 19;42(21):3369-78. 45. abera g. review on high-pressure processing of foods. cogent food agriculture. 2019 jan 1;5(1):1568725. highlights in bioscience page 8 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion 46. tang w, row kh. evaluation of co2-induced azole-based switchable ionic liquid with hydrophobic/hydrophilic reversible transition as single solvent system for coupling lipid extraction and separation from wet microalgae. bioresource technology. 2020 jan 1;296:122309. 47. arathi s, kumar jt, jothibasu k, karthikeyan s, suchitra r. qualitative and quantitative estimation of algal lipids for biofuel production. 2020;8(4):2451–9. 48. jothibasu k, dhar dw, rakesh s. recent developments in microalgal genome editing for enhancing lipid accumulation and biofuel recovery. biomass and bioenergy. 2021 jul 1;150:106093. 49. mathew gm, raina d, narisetty v, kumar v, saran s, pugazhendi a, et al. recent advances in biodiesel production: challenges and solutions. science of the total environment. 2021 nov 10;794:148751. 50. aghbashlo m, peng w, tabatabaei m, kalogirou sa, soltanian s, hosseinzadeh-bandbafha h, et al. machine learning technology in biodiesel research: a review. progress in energy and combustion science. 2021 jul 1;85:100904. 51. papu nh, lingfa p, dash sk. an experimental investigation on the combustion characteristics of a direct injection diesel engine fuelled with an algal biodiesel and its diesel blends. vol. 23, clean technologies and environmental policy. 2021. p. 1769– 83. 52. mondal b, parhi ss, rangaiah gp, jana ak. nano-catalytic heterogeneous reactive distillation for algal biodiesel production: multi-objective optimization and heat integration. energy conversion and management. 2021 aug 1;241:114298. 53. hasnain m, abideen z, naz s, roessner u, munir n. biodiesel production from new algal sources using response surface methodology and microwave application. biomass conversion and biorefinery. 2021 may 15:1-6. highlights in bioscience page 9 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ abstract introduction micro and macro algae as a nutritive aquaculture feed microalgae based biorefinery for aquaculture wastewater treatment open pond system nutrient removal efficiency of aquaculture wastewater with microalgae recent advances in microalgae harvesting and lipid extraction microalgae as a sustainable future biofuel approach conclusion acknowledgement references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202101 research article open access 1 faculty of sciences of tunis, biology department, research unit of physiology and aquatic environment, university of tunis el manar, 2092 tunis, tunisia. 2 aquatic environment exploitation resources unit, higher institute fishing and fish farming of bizerte, tunisia. contacts of authors * to whom correspondence should be addressed: imene chetoui received: september 24, 2020 accepted: january 12, 2021 published: january 20, 2021 citation: chetoui i, ghribi f, bejaoui s, ghalghaa m, el cafsi m, soudani n . assessment of stress biomarkers responses in mantle and adductor muscles of mactra stultorum following lead exposure. 2021 jan 20;4:bs202101 copyright: © 2021 chetoui et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. assessment of stress biomarkers responses in mantle and adductor muscles of mactra stultorum following lead exposure imene chetoui*1, feriel ghribi1, safa bejaoui1, mohamed ghalghaa2, m'hamed el cafsi 1, nejla soudani1 abstract the objective of the present work is to evaluate the possible toxic effect engendered by graded doses of lead chloride (pbcl2) on mactra stultorum mantle and adductor muscles through a battery of biomarkers responses. m. stultorum were divided into 4 groups and exposed to three concentrations of pbcl2 (d1:1mg/l, d2: 2.5 mg/l and d3: 5 mg/l) with control during five days. our findings showed decreases of lipid contents in both organs following pbcl2 exposure, while, proteins declined only in the adductor muscles of the treated m. stultorum. during our experiment, the pbcl2 exposure induced the levels of metallothionein (mts), malondialdehyde (mda) and advanced oxidation protein products (aopp) in both organs as compared to the control. these biomarkers responses are distinctly different between mantle and adductor muscles. keywords: lead chloride, mactra stultorum, mantle, adductor muscles, biomarkers responses. introduction the contamination of aquatic ecosystems by several environmental pollutants has become a worldwide problem in the last years [1]. the presence of heavy metals in those environments and their accumulation in marine organisms has been largely investigated during the last decades because of their harmful effects and persistence [2]. for the global environmental health, lead (pb) is considered to be a major hazard. this non-essential and toxic heavy metal is the most abundant metal in the aquatic system. in nature, it is present as a divalent cation and principally forming stable complexes with sulfur. it has a natural origin or it is realized from many industrials discharges such as lead ore mining and smelting, refining, alkyl-lead petroleum combustion, batteries and cement manufacture [3]. at the national level, lead is one of metals contaminating the tunisian coasts because of its highest concentrations and has been considered a major source of pollution in tunisian waters [4,5]. thus, high pb levels which are exceeding the permissible limit (1 mg/kg), [6] have been recorded in tunisian bivalves tissues (values comprised between 5 and 9 mg/kg dw) [5,7]. moreover, their accumulation in aquatic ecosystems can become dangerous to all kinds of organisms including bivalves, fishes, aquatic plants and human life, causing many toxic effects [8]. mactra stultorum is considered as ecologically important components of marine environments and an important edible marine bivalve due to their richness of protein and essentials fatty acids [9, 10]. it has a wide distribution along the mediterranean and atlantic coasts and estuaries [11]. so, it is abundant specie in the subtidal area and shallow seas along the coast of tunisia [12]. moreover, due to their benthic and sedentary mode of life; suspension-feeding mode and high filtration rate, it is easily exposed to environmental pollution [13]. m. stultorum can accrued a enormous number and high concentration of heavy metals in their tissues [14,15,16]. therefore, similar to other species of mactra, mussels and oysters, m stultorum can be used as a good bioindicator of heavy metal pollution in marine environments [17]. highlights in bioscience page 1 of 8 january 2021|volume 4 https://doi.org/10.36462/h.biosci.202101 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure the excessive accumulation of lead in the bivalve tissues can induce oxidative stress through the overproduction of reactive oxygen species (ros) in the cells which affect cellular functions [18,19]. so, when these increases of ros levels get over the scavenging capacity of organisms, the superfluous free radicals may advantage to oxidative damage in basic biological molecules, such as lipid peroxidation, protein oxidation and dna damage [18,19,20]. thus, the lipid peroxidation constitutes an involvement of self-propagating sequence of chemical reactions that occurred in the bulk phase of cell membrane lipid bilayers. malondialdehyde (mda) and 4-hydroxyalkenals are the degradation products of lipid peroxidation and their levels reflect the degree of oxidative damage and constitute a biomarker specific to environmental stresses [21]. moreover, lipid peroxidation and other damages resulting from metal toxicity are modulated by antioxidant systems and stress proteins such as metallothioneins (mt) [22]. these systems of defense play a key role in the alive organisms which can provide for the different cells the protection against environmental toxicity control metabolism homeostasis [22]. metallothionein (mt) is a low-molecular-weight and cysteine-rich protein, identified for the first time in the kidney of the horse [23]. mt’s plays a crucial role in metal metabolism and principally in the detoxification mechanisms as a metal-chelating agent for the excess of metals in the cells [24]. further, it ensure an essential role in immune response [25], antioxidant processes [26,27], and response to estrogenic compounds [28]. in aquatic environments, mt has been implied to be used as a bioindicator for metal contamination because of its possibility to bind to particular metals. even though there are investigations about the impact of pbcl2 on m. stultorum gills and digestive gland [15,16], still information are lacking about the impact of this metal on the metabolism and redox status of other organs. thus, in this study, we focused on identifying the metabolic and redox strategies developed by m. stultorum adductor muscles and mantle to cope with graded pbcl2 concentrations. materials and methods experimental protocol mature clams individuals (shell length (sl): 3.5 ± 0.63 cm and total weight (tw) 8.03±0.47 g) were collected at 1 m depth by scuba divers from the bizerte lagoon. after sampling, clams were acclimated for 7 days in aquaria (20 l). the water was daily renewed and physicochemical parameters were controlled (temperature (18°c), salinity (30 psu), ph (7.4 ± 0.2), and photoperiod (12h/12h)). during the acclimation period, green microalgae isochrysis affinis galbana (t-iso, 2 million cells per ml) was fed regularly to m. stultorum. at the end of the acclimation period, clams were divided in 4 groups of 18 clams. each group was placed in 8 l plastic aquaria and was represented in triplicate (n=6 clams per replicate). clams first group was kept in aquaria1 containing filtered natural seawater (control), while other groups were exposed for 5 days to different concentrations of unmixed pbcl2 metal (lead chloride; pbcl2; sigmaaldrich; powder 98%) which was dissolved in pure water. during metal exposure, clams were exposed to graded concentrations of pbcl2 as follows: aquaria1: control; aquaria2: 1mg/l; aquaria3: 2.5mg/l and aquaria4: 5mg/l with controlled conditions as mentioned above (figure 1) and no added food. half (50%) aquaria water volume was replaced every 24 h in order to maintain the water quality, and concentrations of pbcl2 were reestablished. pbcl2 concentrations were selected based on previous trials achieved on other bivalves [29,30]. during the experimental period, no mortality has been reported. preparation of the samples for biochemical analyses after pbcl2 exposure, clams were sacrificed and the mantle and adductor muscle were quickly removed and rinsed with cold distilled water in order to remove the externally bound pb. 6 replicate of each group tissues (mantle and adductor muscle; n=3 for each replicate) were homogenized in a tris-hcl buffer (20mm; ph=7.4) in cold conditions, then centrifuged at 10.000 × g for 20 min (4°c). tissues supernatants were stored at -80°c for oxidative stress parameters analysis. biochemical analyses for biochemical analysis, chemicals were purchased from local commercial suppliers. except for 5,5-dithio-bis-(2nitrobenzoic acid) (dtnb) and 2-thiobarbituric acid) (tba) they were purchased from sigma chemical co (saint louis, mo 63103, usa). protein quantification based on lowry et al. [31] method, protein content was estimated using folin reagent and bovine serum albumin (bsa) as a standard. 2 ml of a solution mixture (sodium carbonate hydrate dissolved in a solution of naoh (0.1n), copper sulfate and sodium hydrate dissolved in water) were added to 10 µl supernatants. then, 200 µl of folin reagen was added to the formed mixture for reaction activation. after 30 min incubation, protein content was determined at 540nm using the spectrophotometric method. lipid quantification according to the method of goldsworthy et al. [32], lipids were determined and the extraction was carried out following shibko et al. [33] method. 0.5 g of tissues are removed, cut and macerated in 10 ml of trichloroacetic acid (tca, 20%). after grinding and filtration and a first centrifugation 5000× g for 10 min, the pellet is kept in the same tube which we added 1 ml of the mixture ether / chloroform (1 v/ 1v). subsequently, the last volume is submitted to second centrifugation 5000× g for 10 min and 100 μl of the supernatant is taken which we added 1 ml of sulfuric acid. and after stirring, the tubes were placed in a hot water bath (100 °c) for 10 min. then, we added 2.5 ml of the sulfophospho-vanillin mixture (85%) to 200 μl of the extract highlights in bioscience page 2 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure figure 1. experimental conception of mactra stultorum exposed to graded doses of lead chloride (pbcl2). which was incubated for 30 minutes in a darkroom. lipid quantities are determined by the spectrophotometric method at 530 nm. a calibration range was carried out from a stock solution prepared from sunflower oil. the lipid contents are expressed in mg/g wet weight (mg/gww) malondialdehyde (mda) measurement mda level was determined according to draper and hadley [34]. an aliquot of 0.5ml of each tissue supernatant was incubated for 1 hour in heated water (37°c) and mixed with 0.5 ml of trichloroacetic acid (tca 30%). after centrifugation for 10 min (3500× g/4 °c), we added 500µl of thiobarbituric acid (tba 0.67%) to 0.5 ml of supernatant. after incubation for 10min, mda levels were determined by spectrophotometric method at 532 nm and expressed as nmol /mg protein. advanced oxidation protein products level (aopp) measurement the advanced oxidation protein products (aopp) levels were determined following the method of kayali et al. [35]. after protein precipitation in double volumes of phosphate buffer (0.1m; ph = 7.4). then, potassium iodide (1.16m) and absolute acetic acid (200µl) were added to clams supernatants. we used the extinction coefficient of 261 cm-1 mm-1 for aopp quantification. aopp levels were determined at 340 nm and expressed as μmol/ mg of protein. metallothionein (mts) content according to the method of viarengo et al. [36] modified by petrovic et al. (2001), mts were determined. the supernatant of each tissue from each group (500µl) was mixed with ethanol/chloroform solution (95%; 1%). after centrifugation for 6000 x g during 10 min in cold, edta (1mm) and nacl (0.25m) were added to the pellets and mts absorbance was measured at 412nm. mts were expressed as nmol gsh/mg protein. statistical analysis statistica software version 5.0 was used for statistical analysis. the homogeneity and normality of variables were tested using the shapiro-wilcoxon test. differences between variables highlights in bioscience page 3 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure were assessed by one-way anova and deemed significant at p < 0.05. for each parameter, results were expressed as means ± standard deviation (sd). pearson correlation matrix and principal component analysis (pca) were used to discriminate significant correlations between biochemical parameters. results the pbcl2 effects on general behavior of m. stultorum no mortality was noticed in treated clams by pbcl2 different doses. during the experimental period, clams behavior was regularly monitored (e.g. filtration activity, reduced respiration, siphon retraction. . . ). those parameters remained stable even in clams exposed to the high dose of pbcl2 (d3). estimation of total protein contents results showed no significant variation in protein content of clam's mantle tissues after pbcl2 treatment. however, significant decreases in the amount of total protein were revealed in the adductor muscles of m. stultorum following lead exposure (-23%, -24% and -20% in clams exposed to doses d1; d2 and d3 respectively compared to controls (table 1). estimation of lipid contents lipids contents decreased significantly in clams mantle and adductor muscle tissues after 120 hours of exposition to pbcl2. compared to the control, this decline was recorded for the mantle by -37%, -60% and -67% in clams exposed to 1mg/l; 2.5mg/l and 5mg/l respectively) and for the adductor muscles by -32 % and -60% in clams treated by d2 and d3 respectively (table 1). estimation of malondialdehyde (mda) levels results showed that the progressive accumulation of pb in both m. stultorum tissues induced lipid peroxidation in all treated clams by pbcl2 which was revealed by mda levels enhancement. in the mantle tissues and compared to the control, mda levels increased with graded doses (d1, d2 and d3, respectively) by 57%, 150% and 575%. we also noted that mda levels in the adductor muscles increased significantly by 58% and 74%, in clams treated with the highest doses (d1 and d2) (figure 2). estimation of advanced protein oxidation products (aopp) levels significant increases in aopp levels were recorded in the mantle of all pbcl2 treated groups (1, 2.5 and 5 mg/l pbcl2) with a dose dependent manner (+60, +65and +205% respectively). while in the adductor muscles tissue and compared to control, the increase of aopp levels was observed only for clams exposed to 2.5mg /l; (+102%) and 5mg / l; (+132%) (figure 3). estimation of metallothionein (mt’s) levels mt’s levels increased significantly only in clams mantle tissues exposed to the highest dose of pbcl2 (5mg / l; 163%) during 5 days. furthermore, the mt’s levels increased significantly in the adductor muscles with a dose dependent manner (+30, +45 and +268%) with d1, d2 and d3 pbcl2 doses compared to control (figure 4). figure 2. the mda levels in the control and treated m. stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and tissues (n=3 clams). pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). *** p <0.001: pb cl2 groups vs controls for each tissue. ++ <0.01; +++ p <0.001: mantle vs adductor muscles for each condition. figure 3. the aopp levels in the control and treated m. stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and tissues (n=3 clams). pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). ** p <0.01;*** p <0.001: pb cl2 groups vs controls for each tissue. ++p <0.01; +++ p <0.001: mantle vs adductor muscles for each condition. principal component analysis (pca) and correlation matrix correlation matrix and pca were established in order to understand in the first time the effect of lead graded doses on stress biomarkers in mantle and adductor muscles tissues of mactra stultorum and in second time to compare the response of these biomarkers between these tissues (figure 5 and table 2). the first two factorial axes that explain 90.96% of the total variance (figure 5). factor 1 (56.38%) was characterized by high mda and mt’s levels (figure 5). factor 2 (33.37 %) was characterized by aopp levels. results showed that protein and lipid contents are intermediates compounds for f1 and f2. pca results showed that there were two significant separations, the first one between controls of mantle and adductor muscles and the treated groups and the second one between both tissues from all treated groups (figure 5). control mantle and adductor muscles were projected in the positive sides of two factorials axes, highlights in bioscience page 4 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure table 1. protein and lipid contents in the control and treated m. stultorun mantle and adductor muscle with pbcl2 graded doses (d1. d2. d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and each tissues (n=3 clams.). a: nmol/mg protein. b: mg/mg protein. pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). ** p <0.01;*** p <0.001: pb cl2 groups vs controls for each tissue. +++p <0.01; +++ p <0.001: mantle vs adductor muscles for each condition. ct d1 d2 d3 proteina mantle 10.4± 0.67+++ 8.37± 0.76+++ 9.84± 0.64+++ 7.84± 0.69+++ adductor muscle 43.4± 4.91 33.4± 2.02* 32.8± 1.94* 34.7± 5.14* mantle 4.2± 0.92++ 2.62± 0.71*+++ 1.67± 50.27*+++ 1.42± 0.32*++ lipidb adductor muscle 5.92± 0.35 5.89± 0.26 4.02± 0.28* 2.37± 0.83* figure 4. the mt’s levels in the control and treated m. stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and tissues (n=3 clams). pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). ** p <0.01;*** p <0.001: pb cl2 groups vs controls for each tissue. +++ p <0.001: mantle vs adductor muscles for each condition. explaining by the high contents of lipids and protein and minor levels of lipid peroxidation and mt’s. second group was dominated by treated mantle clams by pbcl2 which represented the negative side of f1 and the positive side of f2; revealing important lipid and protein oxidation. the third one including the adductor muscles from m. stultorum from all treated groups was characterized by a minor response of stress biomarkers comparing to mantle tissues and by a remarkable decrease in protein and lipid contents especially for clams exposed to high dose. clearly, biomarkers responses involved in oxidative stress were significantly enhanced in both tissues clams treated with high pbcl2 dose when compared to control groups. table 2. correlation analysis (pearson correlation) between the biochemical parameters in the control and treated m. stultorun mantle and adductor muscle with pb cl2 graded doses (d11.d2. d33 during 5 days. correlation coefficients statistically significant (p <0.05) . ns: not significant (p <0.05). mantle adductor muscles protein lipid mda aopp protein lipid mda aopp lipid ns ns mda ns -0.98 0.98 ns aopp ns ns 0.98 -0.95 -0.96 ns mt ns ns 0.99 0.99 0.97 ns 0.99 ns figure 5. principal analysis component (pca) represented by two factors f1 and f2 and produced by biochemical variables in control and stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. projection of the variables and the cases on the factor-plane (1×2); d1:1mg/l pbcl2; d2:2.5mg/l pbcl2; d3:5mg/l pbcl2; mt: mantle; am: adductor muscles. pearson correlation matrix showed that protein contents in adductor muscles is significantly negatively correlated (p<0.05) with aopp levels. while, lipid contents in mantle showed a negative correlation with mda levels (p<0.05). positive correlation was recorded between aopp, mda and mts levels for mantle tissue. while, negative correlation was observed between aopp levels and lipid contents for adductor muscles tissue. thus, mt’s presents in this tissue positive correlation only with mda and protein levels (table 2). discussion biological responses in native indicator species can give total and pertinent information on the potential impact of metal toxicity on ecosystem dynamics. heavy metals constitute a group among environmental pollutants because of their bioaccumulation and non-degradable property. lead is a non-essential metal, abundant in the oceans and exhibits widely regarded toxic effects associated with the stimulation of radical processes [37]. lead is accumulated by different organisms in aquatic systems and became dangerous to all kinds of organisms, including highlights in bioscience page 5 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure bivalves, fishes, and aquatic plants and finally is transferred to human life [3]. lead in the divalent cation form pb2+ can be transported into the intracellular part through the ca2+ transport systems thereby might altering the calcium homeostasis [38], affecting different cellular functions and enzymatic activities and causing dna damage [39]. to our knowledge, our study is the first to assess the redox status in the mantle and adductor muscles of m. stultorum after pb cl2 exposure. furthermore, among the established mechanisms of lead toxicity is its ability to induce oxidative stress following an overproduction of ros. an imbalance between antioxidants and pro-oxidants is a result of this toxicity and causing an alteration in redox status, lipids peroxidation and protein oxidations [39-40]. malondialdehyde (mda) constitutes the final products of lipid peroxidation indicating the degradation of lipids; it is therefore used as a good biomarker of lipid damage in aquatic organisms [4]. the oxidation of lipids during our experiment was confirmed by significant increases in mda levels in the mantle and adductor muscles in all animals treated groups. the lipid membranes are the primary targets of oxidative damage [42]. so, the membrane integrity and fluidity in the bivalve’s cells is altered by the pro-oxidant effects of pbcl2 which was proved by these raise of mda levels. as well, these high mda levels are accompanied by high and significant decreases of lipid quantities in both tissues mainly for the clams exposed to d2 and d3 which also demonstrated a significant and negative correlation with the lipid contents in both tissues.. moreover, the hypothesis that the inorganic cations as pb2+ can stimulate the lipid peroxidation processes through the oxidation of polyunsatured fatty acids (pufa) is generally suggested [37]. indeed, these pufa are extremely sensitive to oxidation via ros’s attack due to their high number of double bonds per fatty acid molecule [43]. in this context, the richness of mantle and adductor muscles by pufa recorded by chetoui et al. [9]can explain these low levels of lipids content and high mda levels which are necessary the results of the toxic effects of lead [29,44]. additionally, the generation of ros is the main consequence of protein damage [45]. in our experiment, increases of aopp levels are recorded in all m. stultorun treated groups for mantle and in m. stultorun treated with d2 and d3 for adductor muscle suggested that the harmful effects of pb accumulation are leading to excessive protein oxidation. this oxidation of protein in the adductor muscles was associated with a remarkable and significant decrease in the amount of total protein during the treatment which probably due to their richness of total proteins compared to mantle [10]. these declines of protein quantities in am are negatively correlated with the protein contents in this tissue. however, in the mantle, the total protein has not changed during the treatment. previous research has shown that the alteration of protein in other tissues of m. corallina and venus verrcosa are observed following the lead toxicity [16,46]. similar decreases of protein content have been demonstrated in oyster gills and mussel digestive glands after metals exposure [43]. it is widely reported that the induction of mt’s in marine organisms by metals is the result of their use as metal-pollution biomarkers [47]. these sulfhydryl groups (-sh) are involved in the detoxification processes [26]. and by capturing free radicals, these metalloproteins were able to acquire protective activity [48]. our data showed that treatment with high pb concentrations (5mg/l) leads to an increase of mt’s content in the mantle tissue which is highly and positively correlated mda and aopp levels. however, we revealed that total metallothionein (mts) contents in adductor muscle enhanced in a dose-dependent manner which demonstrated a high and positive correlation with proteins and mda levels. these mt‘s inductions reflected the impairment of both tissues functions in m. stultorum. moreover, by comparing to the adductor muscles, this protein (mt's) is more active (or more expressed) in the mantle for each treated group. according to kumari [49], the increase in mt concentrations seems to be a result of the increased transcriptions. and this suggestion has been demonstrated in m. veneriformis following mercury exposure which the basal mvmt mrna expression was according to the ranking of the tissues like the following: digestive gland> mantle> gill>adductor muscle>foot [50]. besides, these high mt’s induction in the mantle than in the adductor muscles in our study can probably be firstly by the possibility of that tissue to accumulate more quantity of metals than the adductor muscle [29,51,52]and secondly by the activation of their main role in metal detoxification when the lipid peroxidation and protein oxidation reached the maximum. our results are therefore in agreement with previous studies carried out on mytilus galloprovincialis mantle and m. corallina tissues after lead exposure [15,16,29]. in the literature, the metals accumulated (as lead) in mantle and adductor muscles tissues seem to be lower than in the digestive gland and gills of venus veruscosa, mytilus galloprovincialis, callista chione, perna viridis and modiolus metcalfei [29, 51,52]. moreover, in our study, the stress biomarker response is clearly and significantly different between the two organs and appears to be more accentuated in the mantle of m. stultorum treated groups. these results can be explained that similar to gills, the mantle is also located in the mantle cavity which can directly interact with marine pollution and consequently can accumulate metals more than adductor muscle [29,51,52]. conclusions in conclusion, the present results demonstrate that pbcl2 exposure (1mg / l; 2.5mg / l and 5mg / l) alters similarly the redox status of m. stultorum mantle and adductor muscles. the toxic effect of lead induces similarly the lipids peroxidation confirmed by the increases of mda levels which they were associated with decreases in lipids contents in both tissues. the alteration of proteins expressed by the elevation of aopp levels in both tissues confirms the harmful effects of lead. they were correlated with a decrease in protein content only in adductor muscles. the capacity of m. stultorum to increase their mt’s highlights in bioscience page 6 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure concentrations in both tissues seems to be an essential cellular adaptive system defending the animal against the lead-induced toxicity. these biomarkers responses in mantle and adductor muscle tissues elucidate the installation of oxidative stress by their increases in pbcl2 treatment as compared to controls. however, they are distinctly different between mantle and adductor muscles. we can then deduce that 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exposure on redox status, dna and histological structures in venus verrucosa gills and digestive gland. chem. ecol. 2020ă; https://doi.org/ 10.1080/0275 7540.2020.1742329 47. amiard jc, amiard-triquet c, barka s et al. metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. aquat. toxicol. 2006; 76(2): 160-202. 48. davis sr, cousins rj. metallothionein expression in animals: a physiological perspective on function. j nutr, 2000; 130:1085 1088 49. kumar d. role of non-enzymatic antioxidants in stimulation of metallothionein against metal toxicity. inter. j. environ. sci. techno. 2012; 2 (3): 15961604. 50. fang y, yang h, liu b. tissue-specific response of metallothionein and superoxide dismutase in the clam mactra veneriformis under sublethal mercury exposure. ecotoxicology. 2012; 21: 1593-1602. 51. chalkiadaki o, lydakis-simantiris n, dassenakis m. lead accumulation and metallothionein induction in different tissues in mussels (m. galloprovincialis) and clams (c. chione) exposed to various pb concentrations. proceedings of the 13th international conference on environmental science and technology athens. 5-7 september 2013, greece. 52. ponnusamy k, sivaperumal p, suresh m. et al. heavy metal concentration from biologically important edible species of bivalves (perna viridis and modiolus metcalfei) from vellar estuary, south east coast of india. j. aqua. res. dev. 2014; 5 (5):1000258-1000263. highlights in bioscience page 8 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods experimental protocol preparation of the samples for biochemical analyses biochemical analyses statistical analysis results the pbcl2 effects on general behavior of m. stultorum estimation of total protein contents estimation of lipid contents estimation of malondialdehyde (mda) levels estimation of advanced protein oxidation products (aopp) levels estimation of metallothionein (mt’s) levels principal component analysis (pca) and correlation matrix discussion conclusions acknowledgments references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20217 highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 8 research article open access 1 department of agrotechnology, faculty of agriculture, halu oleo university, jl. hea mokodompit, kampus bumi tridharma, anduonohu, kendari, southeast sulawesi, indonesia. 2 department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, jl. meranti, darmaga campus 16680, indonesia. 3 department of soil science and land resources, faculty of agriculture, bogor agricultural university, jl. meranti, 16680 campus darmaga, bogor , indonesia. 4 center for research and development of biotechnology and genetic resources (bb-biogen), jl. tentara pelajar no. 3 a cimanggu, bogor, indonesia. contacts of authors * to whom correspondence should be addressed: la ode afa citation: afa l, purwoko bs, junaedi a, haridjaja o, dewi is (2020). growth and yield of new hybrid rice in irrigated and rain-fed lowlands. highlights in bioscience volume 3. article id 20217.dio:10.36462/h.biosci.20217 received: june 6, 2020 accepted: august 20, 2020 published: august 31, 2020 copyright: © 2020 afa et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. growth and yield of new hybrid rice in irrigated and rain-fed lowlands la ode afa1*, bambang s. purwoko2, a. junaedi2, oteng haridjaja3 and iswari s. dewi4 abstract two experiments were conducted at (i) irrigated lowland, village of bojong, cikembar, sukabumi sub-district, indonesia, from november 2011 to february 2012, and (ii) rain-fed lowland, village of sanca, gantar sub-district, indramayu, indonesia, from april to july 2012. the objective of the experiment was to determine the yield of drought-tolerant hybrid rice genotypes. a randomized block design with three replications was used. the treatment consisted of 8 hybrid rice genotypes (bi485a/bp3, bi485a/bp5, bi485a/bp10, bi485a/bp12, bi485a/bp15, bi599a/bp5, bi599a/bp15 and bi665a/bp6) and 2 check varieties (hipa 7 and ciherang) for the experiment in irrigated lowland, 4 hybrid genotypes (bi485a/bp3, bi485a/bp12, bi485a/bp15 and bi599a/bp15) and 3 check varieties (hipa 7, ir64 and limboto) for the experiment in rain-fed lowland. on irrigated lowland, all tested-genotypes produced grain yield per hectare, which was not significantly different from the check varieties ciherang and hipa 7. the yield of genotype bi485a/bp12, bi485a/bp15 and bi559a/bp15 under irrigated lowland showed non-significant different with check varieties ciherang and hipa 7, i.e 5.63, 6.87, and 6.30 tons ha-1, respectively. genotype bi599a/bp15 under severe drought rain-fed lowland yielded 0.90 tons of grain ha-1, whereas hipa 7 (hybrid variety, suitable for rain-fed lowland) and limboto (drought-tolerant check variety) reached 0.34 and 0.29 tons ha-1, respectively. genotype bi599a/bp15 is a new hybrid genotype that potential for advanced development in rain-fed lowland and in irrigated lowland with a high yield potential and quiet early-maturity age. keywords: yield, hybrid rice, irrigated lowland, rain-fed lowland. introduction hybrid rice is an alternative technology to increase rice productivity. research results in several tropical countries showed that hybrid rice had more yield advantages than inbred rice with more than one ton per hectare [1]. one component of the success of hybrid rice development is the availability of excellent varieties that have high heterosis and can increase yield potential by 15-20% [1,2]. experiments on the yield of hybrid rice from 1982 to 2011 showed more advantages than inbred rice in terms of productivity of dry grain and age [2,4,5]. the experiment is continuously conducted to improve rice production by assembling high-yield potential, earlier mature and drought tolerant hybrid rice for marginal land such as the rain-fed lowland. the new hybrid genotypes that potential to be developed in the rain-fed areas have been identified through a series of tests in a controlled environment. tests using peg 6000 and drought stress on nutrients culture in seedling phase and in pot and simulation in the field have successfully identified drought-tolerant hybrid rice genotypes, namely bi485a/bp12, bi485a/bp15, and bi559a/bp15 genotypes [6,7]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20217 https://doi.org/10.36462/h.biosci.20217 http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 8 the genotypes need to be tested on in-situ conditions (field test) to obtain hybrid rice genotypes with high yield traits, both at an optimum and sub-optimum growing environments. testing in rain-fed and irrigated areas is required to obtain good traits according to the conditions of environment where they grow, so that the selection can be performed on high yield potential genotypes, although testing is conducted on a small scale. testing on a small scale is expected to be able to provide information on preliminary yield of drought-tolerant hybrid rice genotypes and potentially be developed in the rain-fed lowland in a wider scale. the genotypes of rice should be able to adapt and steady at any location in various seasons, so that a genotype can be released as a superior cultivar [8-10]. the study aimed to determine the preliminary yield of new rice hybrid in irrigated lowland and the current drought in the rain-fed lowland. materials and methods the experiment growth and yield in irrigated lowland was conducted in november 2011 to february 2012 in irrigated lowland of bojong, district cikembar, sukabumi, west java, indonesia. the climate of the study site was moderate with the average temperature of 18-32 0c and precipitation ranged 1200-2200 mm year-1. the plant materials used were 8 genotypes/varieties of new hybrid rice i.e. bi485a/bp3, bi485a/bp5, bi485a/bp10, bi485a/b p12, bi485a/bp15, bi599a/bp5, bi599a/bp15, bi665a/ bp6, and two check varieties i.e. ciherang and hipa 7. the experiments used a randomized block design with three replications. plot size as the unit of experiments was 2.0 m x 1.5 m, the distance between the treatment plots was 50 cm, and between blocks was 100 cm. seedlings yielded from seedbed were transplanted after at least 21 days, the seedlings were planted with spacing of 25 cm x 25 cm, one seedling per hole. fertilizing used with 300 kg urea, 125 kg sp-36, and 100 kg kcl per hectare. all sp-36 and kcl fertilizers were given at the time of planting; urea was given three times at time of planting, 4 weeks and 7 weeks after planting. the control of pests and diseases was done optimally, while weeding was done manually using a push weeder, three and five weeks after planting. irrigation was adjusted to the character of the rice fields used. irrigation on irrigated lowland was maintained under optimum conditions during the growth phase until harvest. the height of water on the plots was adjusted to the growth phase of plant. the observation was objected to five clumps of sample plants. variables measured were plant height, number of productive shoots, panicle length, number of filled grains, total number of grains, the percentage of filled grain, 1000 grain weight, grain weight per hill, days to 50% flowering and 85% yellow harvesting and grain yield per plot. the experiment growth and yield in rain-fed lowland was conducted from april to july 2012 on rain-fed lowland of sanca village, subdistrict gantar, district indramayu, west java, indonesia. the climate in the study areas was hot climate with average temperature of 32-35 °c and annual rainfall ranged from 1000-2400 mm year-1. the plant materials used were 4 genotypes/varieties of new hybrid rice i.e. bi485a/bp3 (drought-sensitive category), bi485a/bp12, bi485a/bp15, and bi599a/bp15 (drought-tolerant category based on the results of previous studies), and 1 hybrid variety, namely hipa 7 (released-varieties suitable for rain-fed lowland) and two inbred varieties namely ir-64 (drought-sensitive check) and limboto (drought-tolerant check). the experiments used a randomized block design with three replications. the soil sample was taken to analyze its physical and chemical properties, namely the determination of the water content of the soil at field capacity (pf 2.54) and permanent wilting point (pf 4.20) based on the method of pressure plate/membrane apparatus [11]. the experimental plot size of each treatment unit was 3.00 m x 1.75 m, the distance between treatment plots was 50 cm, and inter-groups distance was 100 cm. seedlings yielded from seedbed were transplanted after at least 21 days, the seedlings were planted with spacing of 25 cm x 25 cm, one seedling per hole. the observation was directed to five clumps of sample plants. variables measured were plant height, flag leaf length, number of productive shoots, plant dry weight of the upper part (biomass), 50% flowering-age, panicle length, number of total grains, percentage of filled grains, grain weight per hill and grain yield per plot. data were analyzed using analysis of variance and further test duncan's multiple range test (dmrt) the 0.05 probability level using sas 9.1 software program. results and discussion rainfall and soil moisture conditions the average rainfall in the trial location of irrigated lowland (table 1) was considered to be sufficient for the rice water requirement of > 200 mm/month [12].thus, inadequacy of irrigation water can be supplied with the rainfall. at well-ripening phase, the amount of rainfall was < 200 mm and occurred only for eight days, which was sufficient condition to accelerate the maturation of seeds and harvest time. table 1: average rainfall during the growth of hybrid rice in irrigated lowland of bojong village, cikembar district, sukabumi, indonesia. notes: a = the board of meteorology, climatology and geophysics, geophysical observatory in pelabuhan ratu, sukabumi, indonesia; b = growing phase of rice varieties was 120 days [13]. growing phase b ch(mm) a rain frequency (days) availability planted-primordial (60 days) 492.3 44.0 sufficient floweringseeds filling (30 days) 485.6 26.0 sufficient seeds ripening 218.8 11.0 sufficient well-ripening 166.0 8.0 sufficient http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 8 during the trial in rain-fed lowland, the distribution and frequency of rainfall during the vegetative phase (planting until the primordial) were at insufficient level, only 94.7 mm and rain occurred only 7 days. flowering to grain filling phase was at a critical level of 0.6 mm and rain occurred only 1 day, whereas at well-ripening phase rain did not occur (table 2). the condition caused the plant experienced a severe drought stress, which occurred in 2 weeks after planting. result of recorded soil moisture is presented in figure 1. level of soil moisture in the field capacity was 38.6% and permanent wilting point was 26.9%. table 2: average rainfall during the growth of hybrid rice in the rain-fed lowland of sanca village, district gantar, indramayu, indonesia. notes: nr = no rain; a = direct measurement in the field , b = rice varieties growth phase for 120 days [13]. 0 5 10 15 20 25 30 35 40 45 28 33 36 39 43 53 56 58 61 67 73 79 106 after day plant s o il m o is tu re .. (% v o lu m e) in depth ±10 cm in depth ±20 cm average figure 1: moisture of soil during the trail in rain-fed lowland rice of sanca village, district gantar, indramayu, indonesia, from april to july in 2012. growth and yield results of analysis of variance experiment growth and yield in irrigated lowland showed that variety treatment significantly influenced variables of plant height, 50% flowering age, panicle length, the total grain number, and filled grain, the percentage of empty grains, and 1,000 grain weight, while the variables of number of productive tillers, grain weight per hill and grain yield per hectare were not significantly influenced. results of dmrt test on average growth are presented in table 3 and the average of yield components and yield are presented in table 4. table 3 shows that most genotypes tested had plant height, which was not significantly different from check varieties of ciherang and hipa 7, except for bi599a/bp15, bi599a/bp5, and bi665a/bp6. genotype bi599a/bp15 was the highest, which was not significantly different from bi599a/bp5. plant height is significantly associated with lodging. irri assigned a standard for the height of new rice type with 100 cm [14]. ma et al. [15] confirmed that the ideal crop with lodging resistance ranges from 115 to 120 cm. therefore, this genotype is an ideal genotype to be developed in the rice field because the height character of the plant was within the ideal prototype standard of lodging resistance. table 3: growth components including plant height (ph), number of productive tillers (npt), flowering age at 50% (fa), and harvest age (ha) of hybrid rice genotypes and check varieties in irrigated lowland. notes: figures followed by the same letter in each of the variables were not significantly different by dmrt at α = 0.05. the number of productive tillers among genotypes and the check varieties showed no significant difference. the average number of productive tillers ranged from 12 to 15 (table 3). peng et al. [16] stated that the low capacity of tillers for transplanting were 8-10 productive tillers. furthermore, peng et al. [17] reported that the super hybrid rice in china had 200-250 productive tillers per m2 (12-15 tillers per hill at a distance of 25 cm x 25 cm). genotypes tested in this research had an average of productive tillers in that range. average flowering age among genotypes was not significantly different except for genotype bi665a/bp6 in which the flowering time was 50% longer, but the flowering time of this genotype was not significantly different from that of check varieties, ciherang and hipa 7. generally the tested-genotypes were faster in flowering, i.e. 85.0-87.3 days while flowering age of check varietiess ciherang and hipa7 were 92.3 and 90.3 days, respectively (table 3). age of flowering strongly relates to the efficiency of the utilization of water resources and nutrients because the phase of vegetative growth is shorter. the shorter age of flowering generally has good adaptability to drought by accelerating the maturation of grain [18]. the average harvest age of the tested-genotypes was significantly different from that of check varieties, except for genotype bi485a/bp3 and bi665a/bp6. the average harvest age of the tested-genotypes ranged from 115.0 to 121.0 days. in general, hybrid rice genotypes had earlier-maturing age of 120 days than checks varieties (table 3). differences in environmental factors cause different plant life cycle. yoshida [13] stated that the difference in maturity is mostly determined by the phase length of the vegetative growth because the length of the reproductive phase and ripening is not influenced by the environment. average of panicle length between genotypes and check varieties was not significantly different. panicle length of genotypes ranged from 23.82 to 26.47 cm, while the check growing phase b ch(mm) a rain frequency (days) availability planting-primordial (60 days) 94.7 7.0 insufficient flowering-seeds filling (30 days) 0.6 1.0 severe seeds ripening nr nr severe well-ripening nr nr severe genotype ph(cm) npt fa (days) ha (days) bi485a/bp3 98.60 d 13.3 85.3 de 119.7 a bi485a/bp5 101.80 cd 13.1 85.0 de 117.7 b bi485a/bp10 101.33 d 13.0 86.3 cd 115.3 cd bi485a/bp12 100.40 d 12.1 85.7 de 116.7 bc bi485a/bp15 103.60 cd 14.8 84.7 e 115.0 d bi599a/bp5 110.33 ab 12.9 87.3 c 118.0 b bi599a/bp15 114.33 a 13.1 86.3 cd 117.7 b bi665a/bp6 107.27 bc 11.9 91.7 ab 121.0 a ciherang 100.53 d 13.7 92.3 a 120.0 a hipa 7 100.87 d 12.5 90.3 b 120.3 a http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 8 variety ciherang was 23.7 cm and hipa 7 was 25.35 cm (table 4). panicle length generally correlates with the number of grains per panicle. zang et al. [19] confirmed that large panicles with the huge number of grains per panicle can increase the density of grain. table 4: yield and yield components including panicle length (pl), total grain number (tgn), filled grain number (fgn), filled grain percentage (fgp), 1000 grain weight (gw), grain weight per hill (gwh), and grain yield (gy) of hybrid rice genotypes and check varieties in irrigated lowland. notes: the numbers followed by the same letter for each variable were not significantly different by dmrt at α = 0.05. the grain number per panicle was significantly different between genotypes. the average number of grains was high in genotype bi665a/bp6 (219.2 grains) that was significantly different from that of the check variety ciherang (153.0 grains) (table 4). a relatively high number of total grain on hybrid genotypes is as the implication of the long panicles of hybrid rice genotypes [19]. the number of filled grains was significantly different between genotypes and check varieties. genotype bi665a/bp6 yielded the highest number of filled grains, followed by genotype bi485a/bp12, bi485a/bp15, and bi599a/bp5 of 162.9, 153.6, 147.6, and 142.6 grains, respectively, but was not significantly different from the check variety hipa 7 of 140.3 grains. the number of filled grains of check variety ciherang was only 125.8 grains which was significantly different from genotypes bi665a/bp6, bi485a/bp12, and bi485a/bp15, but not significantly different from the other genotypes. the average number of filled grains of hybrid genotypes ranged between 118.4-162.9 grains, while the check varieties ciherang and hipa 7 had averages of 125.8 grains and 140.3 grains, respectively (table 4). peng and khush [20] stated that the criterion for a higher yield was the number of grains per panicle of about 150 grains. the highest percentage of filled grains obtained in genotype bi485a/bp12 of 88.5%, which was not significantly different from those of bi485a/bp15, bi599a/bp5, and check variety ciherang i.e. 83.7, 82.7, and 82.1%, respectively. genotype with the lowest percentage of filled grains was bi665a/bp6 of 74.0% which was not significantly different from genotypes bi485a/bp3, bi485a/bp10, and check variety hipa 7, i.e. of 74.4, 78.5, and 71.8%, respectively (table 4). the grain filling process is determined by the source in supporting sink or due to the aging process. the limited resources in supporting sink due to the low accumulation of photosynthate or earlier aging process will increase the empty grains percentage. abdullah et al. [21] reported that one of the causes of a void is the imbalance between the sink and the source. dry matter in the leaves started to decrease in 10 days after seed filling, and relatively large leaves play an important role in supplying photosynthate to seeds [22]. the highest 1000 grain weight was obtained in genotype bi599a/bp5 of 30.88 g, followed by genotype bi599a/bp15, which was not significantly different from those of genotype bi485a/bp10 and bi485a/bp12 i.e. 29.19 g and 29.26 g, respectively (table 4). ma et al. [15] stated that trait of ideal plant had panicle with 1000 grain weight of 28-30 g. a new type of rice with grain weight of 28-30 g, panicle length > 26 cm and lots of grains is a fair size for sink to support high grain yield. plants with a lot of grains per panicle need much assimilate for grain filling, so it should be supported by ideal leaf traits i.e. medium leaf width, thick, slightly dropping, not rapidly aging and medium age, as in genotype bi485a/bp12, bi485a/bp15, bi599a/bp5, and bi599a/bp15. the weight of grains per hill between genotypes and check varieties were not significantly different. the weight of grains per hill among genotypes ranged from 33.92-42.92 g, grain weight per hill of check variety ciherang was 38.30 g and 46.81 g for hipa 7. the grain weight of the genotypes when converted to yield ranged from 5.43 to 6.87 ton ha-1. genotype bi485a/bp15 had the highest grain yield of 42.92 g hill-1 or 6.87 tons ha-1 (table 4). virmani et al. [23] suggested that important benchmark in the assembly of hybrid rice is the yield comparison between hybrids and check varieties. genotype bi485a/bp3, bi485a/bp10, bi485a/bp15, and bi599a/bp15, when compared to the check variety ciherang was likely to show an increase i.e. 7.2, 2.8, 12.1, 1.6, and 2.8 percent, respectively (table 4). an effective way to develop a lowland rice cultivar with high yield potential in both the optimum and sub optimum conditions (drought stress) is through direct selection that can combine the selection in irrigated and rain-fed lowlands. therefore, a preliminary yield trial of drought-tolerant hybrid genotypes was performed, which was potential to be developed in the rain-fed areas. results of analysis of variance experiment growth and yield in rain-fed lowland showed that the variety treatment significantly influenced variables of plant height, flag leaf length, weight of biomass, 50% flowering age, panicle length, number of total grains, the percentage of filled grains, grain weight per hill and yield per hectare, but had no significant effect on the variables of productive tillers. results of dmrt test on the growth variables are presented in table 5 and on the yield components and the yield are presented in table 6. table 5 shows that the tallest plant height was obtained on genotype bi599a/bp15, which was not significantly different from check variety limboto, but significantly different from the check varieties ir64 and hipa 7. genotypes bi485a/bp3, bi485a/bp12, and bi485a/bp15 were not significantly different from the check varieties ir64 and hipa 7, but significantly different from the check variety limboto. the low rainfall and rain frequency since the vegetative phase strongly inhibited the growth of plant height, that was only of 54.67 to 82.11 cm because cell division was inhibited [24]. consequently, due to drought genotype pl(cm) tg (grain) fgn (grain) fgp (%) gw (g) gwh (g) gw (ton ha-1) bi485a/bp3 24.88 180.9 bc 134.0 bcd 74.4 cd 28.98 c 41.13 6.5 bi485a/bp5 23.82 149.1 d 118.4d 79.7bc 28.93 cd 37.11 5.93 bi485a/bp10 24.54 161.7 cd 126.9 bcd 78.5bcd 29.19 bc 39.43 6.30 bi485a/bp12 25.15 173.7 bcd 153.6 ab 88.5 a 29.26 bc 35.25 5.63 bi485a/bp15 24.66 175.8 bcd 147.6 abc 83.7 ab 28.53 cde 42.92 6.87 bi599a/bp5 25.31 171.7 bcd 142.6 abcd 82.7 abc 30.88 a 38.94 6.23 bi599a/bp15 24.49 169.5 bcd 135.3 bcd 79.3 bc 29.86 b 39.40 6.30 bi665a/bp6 26.47 219.2 a 162.9 a 74.0 cd 28.59 cd 33.92 5.43 ciherang 23.69 153.0 cd 125.8d 82.1 abc 28.16 de 38.30 6.13 hipa 7 25.35 195.0 ab 140.3 abcd 71.8 d 27.99 e 46.81 7.50 http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 8 stress in rain-fed areas, the average plant height of genotypes was lower than the standard height for a new type rice crop of 100 cm [14], and different with plant height on trial in irrigated lowland. however, drought-tolerant genotypes bi599a/bp15 could still achieve 82.11 cm in height, which was relatively equal to check variety limboto of 81.11 cm. samaullah and darajat [25] stated that the limited supply of water can suppress plant growth by 10-25 cm in a drought-stress growing environment. kumar et al. [26] reported that at severe drought conditions, the decrease in plant height on tolerant strains reached 6-12 cm, and on sensitive strains reached 16-27cm. table 5: growth components including plant height (ph), number of productive tillers (npt), flowering age (fa), and total biomass (tb) of hybrid rice genotypes and check varieties in rain-fed lowland. notes: the numbers followed the same letter for each variable were not significantly different by dmrt test at α = 0.05. the number of productive tillers did not significantly differ among genotypes and check varieties. the average number of productive tillers ranged from 10 to 13 (table 5). this suggests that the formation and growth of tillers were not affected by drought-stress. the genotypes had an average of productive tillers that was suitable for transplanting, i.e. 8-10 [16]. the shortest average flowering -age was obtained in check variety limboto, which was not significantly different with genotypes bi599a/bp15, bi485a/bp12, and hipa 7. the longest average flowering -age was obtained in genotype bi485a/bp3, which was not significantly different from genotypes bi485a/bp15 and check variety ir64 (table 5). flowering age is strongly related to the efficiency of the utilization of water resources and nutrients due to shorter vegetative growth phase. shorter flowering-age generally has good adaptability to drought and is able to accelerate ripening of grains. kumar et al. [26] stated that in conditions of severe drought, tolerant strains accelerated flowering by ± 8 days compared with sensitive strains. samaullah and darajat [25] and ahadiyat et al. [27] reported that the earlier-flowering genotype with a little number of empty grains in a water-stress environment has a drought-tolerant trait. the lateness in flowering of genotypes growing in drought-stress environment will shorten the grain filling phase, which affects the yield. the highest average total biomass (plant dry weight) was obtained on genotype bi599a/bp15, which was not significantly different from genotypes bi485a/bp12 and bi485a/bp15, but significantly different from bi485a/bp3. the genotypes with the highest plant dry weight had relatively equal weights with check variety limboto but significantly different with check variety ir64 (table 5). kumar et al. [26] stated that the total dry substance of late-flowering genotypes gradually declines in drought stress conditions. this indicates that the late-flowering plants may stop early growing phase during the growth cycle causing the dry weight decreases. genotypes bi599a/bp15, bi485a/ bp12, and bi485a/bp15 had a relatively shorter flowering -age, so that produced relatively higher dry weight in severe drought conditions. kumar et al. [28] reported that the tolerant strains have the ability to maintain high biomass production in dry soil and high harvest index. average of panicle length of hybrid genotypes and check varieties hipa 7 and limboto was significantly different from the check variety ir64. the ir64 has the lowest panicle length of 17.9 cm, while the length panicle of genotypes ranged from 20.9-21.8 cm (table 6). severe drought conditions inhibited panicle length of check variety ir64, which was sensitive to drought because the panicles exertion was hampered. this lead some grains to stay in the leaf midrib and become sterile grains, causing the decreasing of grain yield [29]. panicle length generally correlated positively with the number of grains per panicle [19]. total number of grains was fairly high on genotype bi485a/bp3 but was not significantly different from genotype bi599a/bp15, of 137.5 and 123.6 grains, respectively. the number of total grains of the genotypes was still lower than that of the check variety limboto of 153.6 grains, but higher than those of the check varieties hipa 7 and ir64, of 111.7 and 64.7 grains, respectively (table 6). the high total number of grains per panicle should be supported by a sufficient source for better grain filling [30]. table 6: yield components and the yield including panicle length (pl), total grain number of panicle (tgnp), filled grain percentage (fgp), hill grain weight (hgw), and grain yield (gy) of hybrid rice genotypes and check varieties in rain-fed lowland. notes: the numbers followed the same letter for each variable were not significantly different by dmrt test at α = 0.05. in general, the percentage of filled grain of genotypes was very low, ranging from 3.3 to 30.9 percent. genotype bi599a/bp15 had the highest percentage of filled grain of 30.9 percent which was not significantly different from genotypes bi485a/bp12, bi485a/bp15 and hipa 7, but significantly different from genotypes bi485a/bp3, ir64 and limboto. the lowest percentage of filled grain (3.3 percent) was achieved by genotype bi485a/bp3. the percentage of filled grain of check varieties ir64 and limboto was relatively low, of 10.3 and 7.5 percent, respectively (table 6). a severe drought led to the source being interrupted, thus the seed filling stage highly depended on the nature and mechanisms of plants coping with the drought stress. the higher percentage of filled grain on genotype bi599a/bp15 was caused by the earlier-flowering trait. pantuwan et al. [31] stated that drought escape mechanism of early-maturity genotypes with high yield in long drought-stress conditions was possibly due to the pre-anthesis assimilate contribution as a source of assimilates genotype ph(cm) npt fa (days) tb (g) bi485a/bp3 68.33bc 11.1 88a 27.38b bi485a/bp12 67.44bc 11.6 83b 32.95ab bi485a/bp15 64.56c 11.7 86ab 28.53ab bi599a/bp15 82.11a 13.4 83b 34.67a ir64 54.67c 12.9 85ab 19.07c limboto 81.11ab 9.8 82b 34.69a hipa 7 63.56c 10.4 83 b 26.77b genotype pl (cm) tgnp (butir) fgp (%) hgw (g) gy (ton ha-1) bi485a/bp3 21.43a 137.5ab 3.3b 0.54b 0.09 b bi485a/bp12 20.89a 106.2cd 13.1ab 0.97b 0.16 b bi485a/bp15 20.86a 100.5d 13.9ab 1.21b 0.19 b bi599a/bp15 21.80a 123.6bc 30.9a 5.62a 0.90 a ir64 17.78b 64.7e 10.3b 1.16b 0.19 b limboto 20.98a 153.6a 7.5b 1.79b 0.29 b hipa 7 21.16a 111.7cd 14.4ab 2.11ab 0.34 ab http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 8 during the grain filling period. in severe drought conditions, empty grains in ct9993 drought-tolerant strain reached 71 percent, while in ir62266 drought-sensitive strain reached 91 percent [28]. grain weight of genotypes per hill ranged from 0:54 5.62 g per hill. a high percentage of filled grains led to a high yield or grain weight per hill. therefore, genotype bi599a/bp15 had the highest grain weight of 5.62 g per hill or 0.90 tons of grain yield per hectare which was not significantly different from hybrid variety hipa 7 but significantly different from the other genotypes and check varieties ir64 and limboto. hipa 7 was recommended to be planted in rain-fed areas [32]. the lowest grain weight was obtained on genotype bi485a/bp3 of 0:54 g per hill, but was not significantly different from those of the three check varieties and genotypes bi485a/bp12 and bi485a/bp15 (table 6). grain yield obtained in genotype bi599a/bp15 was equal to the grain yield obtained in strain ct9993, which is a drought tolerant genotype when was grown in a severe drought conditions in raipur, northeast region of india. strain ct9993 was only able to produce 0.95 tons per hectare [28]. the difference in yield of genotype bi599a/bp15 and other genotypes may be due to differences in the contribution of pre-anthesis dry matter to seed production. the genotypes had a relatively shorter flowering-age, thus, it produced a higher dry weight in severe drought conditions (table 5). therefore, early-ripening genotype bi599a/bp15 that was able to reallocate reserved assimilates from the stem to the seed under drought condition resulted in a high yield. this mechanism involves rapid phenological development (early flowering and early ripening) and pre-anthesis assimilates remobilization [33]. limboto is a drought-tolerant variety. during the growing in the field in a severe drought conditions, limboto maintained a high level of leaf greenness but grain filling rate was much lower than the genotypes bi599a/bp15 (figure 2 and table 6). fukai and cooper [34] confirmed that there are four trait categories of drought resistance of rain-fed rice i.e. drought escape, drought avoidance, drought tolerance and drought recovery after a drought occurred. fukai et al. [35] stated that the maintenance of leaf greenness in drought conditions is an example of the adaptation mechanisms of drought tolerance of rain-fed rice. drought escape is a effective strategy to reduce the unfavorable effects of drought that adjusts the availability of water with plant phenology [36]. thus, limboto leaf greenness in severe drought conditions in which rain only occurred the first two weeks after planting indicated that limboto variety is a drought tolerant, whereas genotype bi599a/bp15 developed more than one mechanism or adaptation strategies namely drought escape and drought tolerance. in drought conditions, genotype bi599a/bp15 was not only accelerated flowering/harvest (table 5) but also had a low density of stomata and still maintained relative moisture of leaves at a high level in drought conditions [37-39]. mitra [33] confirmed that plants often used more than one mechanism for drought tolerance. thus, genotype bi599a/bp15 was able to produce better grain in drought conditions than other genotypes. therefore the hybrid rice genotype that is potential for further development in the rain-fed lowland and also in irrigated lowland with high yield potential and early-maturity age is genotype bi599a/bp15. genotype bi599a/bp15 that experienced drought stress, in the critical phase was capable of producing 2.72 tons of grain per hectare, whereas ir64 produced only 1:42 tons per hectare of grain [37]. this offers an opportunity that genotype bi599a/bp15 can be planted under conditions of rainfall restriction but not continuous drought during the vegetative to reproductive phase. on the other hand, when an unpredictable severe drought occurred, farmers can still obtain yields even though the productivity was <1 ton per hectare (table 6). these efforts can improve cropping index in the rain-fed lowland. conclusion based on the condition of the field test, the following points can be concluded: 1. on irrigated lowland, all tested-genotypes produced grain weight per hill and grain yield per hectare, which was not significantly different from the check varieties ciherang and hipa 7. 2. drought-tolerant genotypes bi485a/bp12, bi485a /bp15, and bi559a/bp15, in irrigated lowland had equal yields with that of variety ciherang, of 5.63, 6.87 and 6.30 tons per hectare, respectively. 3.genotype bp599a/bp15, at a very severe drought conditions in the rain-fed lowland, generally produced better results than other genotypes for plant height, productive tillers, flag leaf length, total biomass, panicle length, filled grain percentage and grain weight per hill. 4.genotype bp599a/bp15 at a very severe drought conditions in the rain-fed lowland was able to produce 0.90 tons of grain per hectare, while hipa 7 (hybrid variety that had been released to the rain-fed lowland) and limboto (drought-tolerant check variety) produced only 0:34 and 0:29 tons per hectare, respectively. 5.genotype bi599a/bp15 is a new hybrid genotype that potential for advanced development in rain-fed lowland and also in irrigated lowland with a high yield potential and quiet early-maturity age. acknowledgments acknowledgement was submitted to the agency for agricultural research, ministry of agriculture for funding this research through kkp3t in fiscal year 2012 to bambang s. purwoko and team. author contributions l. afa conducted field experimental tests and data analysis. b. s. purwoko, a. junaedi, o. haridjaja and s. s. dewi formulated experimental design, helped in preparing the draft of reports and made some correction on the manuscript. references 1.virmani ss, kumar i. development and use of hybrid rice technology to increase rice productivity in the tropics. international rice research notes. 2004;29(1): 10-19. http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 8 2.huang m, tang q, ao h, zou y. yield potential and stability in super hybrid rice and its production strategies. journal of integrative agriculture. 2017;16(5):1009-1017. 3.yuan l. development of super hybrid rice for food security in china. engineering. 2015;1:13–14. 4.suprihatno b. hybrid rice. in: ismunadji m, syam m, yuswadi, editors. rice book 2. agricultural development and research agency. center for food crops research and development. bogor; 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highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20211 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 9 research article open access 1 uzhgorod national university, st. voloshina, 54, uzhhorod, 88000, ukraine. contacts of authors * to whom correspondence should be addressed: alexander shuaibov citation: shuaibov a, minya a, malinina a, malinin a, gomoki z (2020). synthesis of aluminum oxide nanoparticles in overstressed nanosecond discharge plasma with the ectonic sputtering mechanism of aluminum electrodes. highlights in bioscience volume 3. article id 20211. dio:10.36462/h.biosci.20211 received: may 20, 2020 accepted: july 18, 2020 published: july 27, 2020 copyright: © 2020 shuaibov et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. synthesis of aluminum oxide nanoparticles in overstressed nanosecond discharge plasma with the ectonic sputtering mechanism of aluminum electrodes alexander shuaibov 1 *, alexander minya 1 , antonina malinina 1 , alexander malinin 1 and zoltan gomoki 1 abstract the results of studying the conditions of synthesis and luminescence of aluminum oxide nanoparticles in a plasma of an overstressed nanosecond discharge ignited between aluminum electrodes at an interelectrode distance of 2 mm and air pressure in the range of 50202 kpa are presented. it was shown that the plasma of the investigated discharge is characterized by a wide luminescence band in the spectral range of 300–430 nm, which is associated with the formation of f and f + centers. the research results can be used in micro-nanotechnology, biomedical engineering to obtain nanostructured alumina substrates, on which other nanodevices and films from biomaterials can be placed. the aim of the work was to establish the possibility of detecting small nanoparticles aluminum oxide nuclei by emission spectroscopy methods and the synthesis of nanostructured alumina films under atmospheric conditions (without the use of vacuum technology) over a large area. keywords: luminescence, nanostructures, aluminum oxide, nanosecond overstressed discharge, argon, air. introduction the results of a study of the characteristics and kinetics of processes in a heterogeneous plasma based on mixed flows of a buffer gas -argon, an oxidizing agent (water molecules), and aluminum dustare presented by bityurin et al. [1, 2]. we studied the plasma of glow and pulsed discharges, as well as a combined high-frequency discharge in mixtures of argon, water vapor, and aluminum dust. in such plasma, the gas component, liquid droplets, solids, and plasma simultaneously coexist. these studies are related to the search for effective and cheap hydrogen production technologies based on the plasma-chemical oxidation of aluminum in water vapor. as a result of such a stimulated process, aluminum dioxide is formed on the high-voltage electrode in the form of a developed scaly surface and thermal energy is released [1, 3]. moreover, the price of obtaining a hydrogen molecule does not exceed 1.5 ev/molecule, which is much more economical than the hydrolysis method of producing hydrogen. it is of interest, in order to simplify the design of the reactor, to replace the generator of aluminum dust with a size of tens of microns by producing microdroplets of aluminum by exploding micropoints on the surface of aluminum electrodes in a strong electric field of a nanosecond discharge (ecton formation [4]). the electric and optical characteristics of a spark discharge, the current and voltage of which had an oscillatory shape in the microsecond range, are given in walters et al. [5]. a discharge was ignited between aluminum and graphite electrodes in air and was https://creativecommons.org/licenses/by/4.0/ shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 9 investigated by time-resolved emission spectroscopy. the studies were conducted in the ignition mode of the discharge far from the overstress of the discharge gap. pairs of electrode materials entered the plasma as a result of sputtering under the influence of a spark discharge (the duration of a train of current oscillations was approximately 25 μs, and one full oscillation was 5-6 μs). alumina nanopowders were synthesized by the gasphase method, in which a drop of molten aluminum was held by a high-frequency field [6]. the drop was streamlined by a stream of argon and oxygen, aluminum vapors were carried away into the colder zone, where they condense and oxidize. the resulting alumina powder was collected on a filter. to obtain nanostructured ceramics, powders with particle sizes of 60 and 15 nm were pressed and annealed in air. the luminescence of f , f + centers created by oxygen vacancies in al2o3 and al2o3-c crystals was studied upon excitation by synchrotron radiation with an energy in the range of 3.7 15 ev. at a temperature of 300 k, intense luminescence bands were recorded with maxima at wavelengths of 414 nm (f centers) and 330 nm (f + centers) when crystals were excited by synchrotron radiation with an energy in the range 4.82–8.66 ev. the decay time of the luminescence of the f + centers is 2.0–2.3 ns [7]. sapphire and nanostructured ceramics have high thermal, mechanical, and chemical stability (the melting point of sapphire is 20500 c) [8]. they are also characterized by increased radiation resistance, which is important when using the appropriate substrate microcircuits intended for use in space and nuclear power plants. the luminescence of radiation-induced sapphire defects under the influence of a pulsed beam based on h + / c + ions with an energy of 300 kev was studied. it was found that, along with the f and f + centers, more complex aggregate centers of the f2 type are also observed [9]. using a subnanosecond high-voltage discharge between aluminum electrodes in air, the atmospheric pressure aluminum plasma characteristics were studied with the ecton mechanism for the injection of aluminum vapor into the discharge gap [10]. the production of electrode material vapors in the form of colored plasma jets based on iron and aluminum vapors was most effective in the absence of mismatch of nanosecond voltage pulses and discharge gap resistance. under these conditions, the deposition of structures based on atomized copper electrodes 1-10 in length and 1 μm in diameter was observed on the walls of the discharge chamber. currently, there is practically no work on the synthesis of alumina-based nanostructures using an overstressed nanosecond atmospheric pressure discharge with an ecton mechanism [4] for the injection of aluminum vapor into an oxygen-containing gas plasma. the results of such studies of the synthesis conditions and some characteristics of the nanostructures of copper, zinc, and iron oxides are given in [11-14]. this study presents the results of characteristics and parameters of an overstressed nanosecond atmospheric pressure discharge in air and argon at atmospheric pressure between aluminum electrodes and the luminescence characteristics of aluminum oxide nanostructures that were synthesized under the conditions of this experiment. materials and methods technique and experimental conditions. an overstressed nanosecond discharge in air and argon was ignited in a sealed 3-liter dielectric chamber between aluminum electrodes. a diagram of the discharge module and device for the synthesis of thin films of transition metal oxides is given in previous research [13-14]. the diameter of the cylindrical aluminum electrodes was 5 mm. the radius of the rounded working end of the aluminum electrodes was 3 mm. air and argon pressure varied in the range of 50 202 kpa. the distance between the electrodes was 2 mm. aluminum vapor was introduced into the discharge gap due to microexplosions of the inhomogeneities of the surface of aluminum electrodes in a strong electric field of the discharge and the formation of ectons [4]. to ignite the discharge, high voltage bipolar pulses with a total duration of 50-100 ns and an amplitude of ± (20-40) kv were applied to the electrodes. the discharge was photographed using a digital camera. the distance between the electrodes was used as the scale for determining the plasma volume. at an interelectrode distance of 2 mm, the discharge gap was strongly overstressed. the nanosecond discharge at a pressure of p = 5–202 kpa was fairly uniform [15]. the voltage pulses at the discharge gap and the discharge current were measured using a wide-band capacitive divider, a rogovsky coil, and a 6-lor 04 wideband oscilloscope. the temporal resolution of this recording system was 2-3 ns. the pulse repetition rate varied in the range f = 35-1000 hz. the plasma radiation spectra were recorded using an mdr-2 monochromator, an feu-106 photomultiplier, a direct current amplifier, and an electronic potentiometer. the radiation of the discharge plasma was analyzed in the spectral region of 200-650 nm. the plasma radiation registration system was calibrated by the radiation of a deuterium lamp in the spectral range of 200-400 nm and a gang lamp in the range of 400-650 nm. oscillograms of radiation at the transitions of individual atoms and ions were recorded using an elu 14 fs electronic linear multiplier, the temporal resolution of which was 1 ns. the pulsed electric power of an overstressed nanosecond discharge was determined by graphically multiplying the waveforms of voltage and current pulses. the time integration of the pulsed power made it possible to obtain energy in one electric pulse, which was introduced into the plasma. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 9 thin nanostructured films based on the degradation products of aluminum electrodes and air molecules were deposited during 2-3 hours of operation of the reactor on glass substrates that were installed at a distance of 3 cm from the center of the discharge gap. the resulting films were studied for light transmission in the visible wavelength range. the experimental technique and technique for recording the transmission spectra of synthesized films are described in [16]. results and discussion spatial, electrical and optical characteristics the plasma volume depended on the repetition rate of voltage and current pulses and in the frequency range 10– 150 hz, it increased from 3 to 25 mm 3 . the discharge had a diffuse shape in the form of a ball. the most probable reason for obtaining a spatially homogeneous discharge in atmospheric pressure gases may be the preionization of the discharge gap by a runaway electron beam of about 130-150 ps duration and the accompanying x-ray radiation. it was shown in beloplotov et al. [17] that even when using highvoltage pulses with a leading edge duration of about 200 ns, a runaway electron beam with an intensity of only one order of magnitude lower than even in the same discharge at an air pressure of 130 kpa is formed in a discharge plasma in atmospheric pressure air. the most characteristic waveforms of voltage and current pulses for an overstressed nanosecond discharge between aluminum electrodes in air are shown in (figure 1). figure 1. oscillograms of voltage and current pulses at an air pressure of 101 kpa. due to the mismatch of the output resistance of the pulsed high-voltage modulator and the plasma resistance, the voltage pulse had the form of individual spikes with a duration of 5-10 ns. this mode of ignition of a subnanosecond high-voltage discharge between a metal electrode in the form of a needle and a flat metal plate (or grid), when the total duration of a train of 10 nanosecond voltage pulses was 1–1.5 μs, was used [10] to obtain plasma jets from a material electrodes. it is promising for applying thin metal films to solid substrates of finely dispersed powders based on electrode materials and degradation products of molecules of a gaseous medium. the maximum value of the positive and negative component of the current pulses reached 200 a, voltage amplitudes 30-40 kv. when air was replaced with argon, the form of the oscillograms did not change, the maximum values of the amplitude of the current and voltage of positive and negative polarity decreased, respectively, to 150 a and 15-20 kv. figure (2) presents the pulsed power and energy input into the plasma of an overstressed nanosecond discharge in air per pulse. with an increase in air pressure from 50 to 101 kpa, the maximum value of the pulsed electric discharge power increased from 3 to 6.5 mw, and the maximum energy input increased from 110 to 152.8 mj. as the argon pressure increased from 50 to 101 kpa, the energy deposition into the plasma increased from 225.7 to 441.1 mj. 1 2 figure 2. pulsed power and energy input into a discharge per pulse at an air pressure of 50 kpa (1) and at an argon pressure of 101 kpa (2). the emission spectra of a plasma of an overstressed nanosecond discharge between aluminum electrodes in air and argon are shown in (figures 3 and 4). the experiments with argon, which were carried out in the same pressure range as with air, were performed to demonstrate the absence of emission bands of nanostructures of aluminum oxides in an inert gas plasma (where there are no oxygen carrier molecules). the spectra shown in (figures 3. and 4), are registered under the same conditions for the excitation of the discharge and under the same conditions of registration. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 9 figure 3. plasma emission spectra of an overstressed nanosecond discharge at different air pressures (50.5, 101 and 202 kpa). 1 2 figure 4. plasma emission spectra of an overstressed nanosecond discharge with the interpretation of the most intense spectral lines and bands at an air pressure (1) and argon (2) of 101 kpa. therefore, the intensities of individual spectral lines and bands can be compared with each other. it can be seen from (figure 3 and 4) that the intensity of the glow of the discharge plasma in air at all pressures studied by us exceeds the radiation intensity of spectral lines and discharge bands in argon. in the emission spectra of air plasma with a small admixture of aluminum vapor, radiation was detected at the transitions of the atom and singly charged ions of aluminum ion, nitrogen oxide radicals and nitrogen molecules, as in the emission spectra of a subnanosecond plasma [10]. in an argon-based discharge, radiation was predominantly recorded at transitions of an atom and an aluminum ion. the most intense spectral lines of an atom and a singly charged aluminum ion, which were manifested in the plasma emission spectra, were as follows: (256.8 + 257.5 + 257.5); 265.3; 394.4; 396.2 nm al i; 236.5; (247.5 + 247.6); 286.9; 622.6; (623.1 + 624.3) nm al ii. in the emission spectra of a plasma of an overstressed nanosecond discharge in air mixtures with an admixture of aluminum vapor (figure 4), broad emission bands with peaks in the spectral ranges of 410–420 nm and 300–390 nm were recorded. the highest radiation intensity of these bands was obtained at an air pressure of 202 kpa. in argonbased mixtures, these bands are absent in the discharge emission spectra. since a significant part of nitrogen is present in the air, in principle, in the discharge under study, the formation of inert gas nitride nanostructures is possible. a comparison of the broadband emission spectra of an overstressed nanosecond discharge with the characteristic emission spectra of aluminum nitride nanostructures silvera et al. [18] showed that they do not correlate with each other. egorov et al. [19], characteristic electroluminescence spectra of anodic alumina are presented. spectra in contact with solutions of different electrolytes were recorded at an oxidation current density of 5–15 ma / cm 2 . these spectra had the form of wide luminescence bands in the spectral range with maxima for different electrolytes in the spectral range of 480-550 nm. based on this, the most probable source of broadband plasma radiation of the discharge under study can be aluminum oxide nanostructures. kortov et al. [6] was noted that in the photoluminescence spectrum of anion-defective single crystals and nanostructured ceramics based on aluminum oxide upon excitation of the corresponding samples by radiation with a wavelength of 205 nm, a wide emission band was observed with a maximum at a wavelength of 415 nm. this band coincides with that obtained in the present experiment. it is interpreted as the luminescence band of f centers (the 1s – 3p transition with a maximum of the emission spectrum at an energy of 3.0 ev and a decay time constant of 36–40 ms) [6, 20]. the results of studying the cathodoluminescence spectra of nanostructured alumina ceramics are also presented [6]. cathodoluminescence was excited by a pulsed electron beam with a density of 1 a cm 2 , an energy of 180 kev and a duration of 3 ns. the spectrum of this cathodoluminescence was similar to the spectrum recorded in our experiment at air pressures of 101202 kpa. the main one was the emission band with a maximum at wavelengths of 410–420 nm (quantum energy 3.0 ev), which was adjoined by a wider short-wave band with maximum quantum energies at 3.4, 3.8, and 4.3 ev [6]. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 9 the ultraviolet photo and cathodoluminescence bands of nanostructured alumina ceramics are associated with the emission of f + centers created by oxygen vacancies and have a relatively short decay time (0.6–1.0 μs) [6, 20]. figures (5 and 6) present characteristic oscillograms of plasma radiation at transitions of radicals of nitric oxide and a singly charged nitrogen ion. at an air pressure of 50 kpa, the emission of the nitric oxide radical band appears with a delay of about 50 ns compared with the beginning of the current pulse, since it takes a certain time for the formation of excited no radicals in the plasma. the radiation pulse at the transition of the no radical consisted of two maxima and had a total duration of 120– 130 ns. at large times, it is likely that the discharge turned into a contracted state and no emission of no radicals was observed. with an increase in air pressure to 101 kpa (figure 6), the pulse amplitude and the duration of the radiation pulse at the transition of the nitric oxide radical increased (figure 6). but at the same time, the duration of the first radiation maximum at the transition of the nitric oxide radical decreased and it became more pronounced. the longest radiation duration was recorded at the transition of a singly charged nitrogen ion (approximately 350 ns), which is characteristic of a recombining plasma of nanosecond discharges in air at atmospheric pressure [21]. figure 5. oscillograms of current and radiation of the spectral line of a nitrogen ion 361 nm (n ii) for a discharge in air at a pressure of 50 kpa. transmission spectra in the visible wavelength range of nanostructured films based on copper and aluminum synthesized on a glass substrate are presented in (figure 7). as can be seen from (figure 7) the obtained film is characterized by weak transmission of radiation in the visible wavelength region. according to gasenkova et al. [20], films based on nanostructured alumina ceramics are practically not transparent to the visible region of the spectrum; their transmittance begins to increase in the spectral range of 0.8–2.0 μm from 1-3 to 25%. 1 2 figure 6. oscillograms of the emission of the band 333.5 nm (no) (1) and the spectral line of a singly charged nitrogen ion 361 nm (n ii) (2) in an overstressed nanosecond discharge in air (p 101 kpa). figure 7. the transmission spectra of films based on copper and aluminum in the spectral region of 350-800 nm (band lamp): 1 a pure glass substrate, 2 a nanostructured copper film synthesized in argon at a pressure of 101 kpa, 3 a nanostructured copper oxide film synthesized in air at a pressure of 101 kpa, 4 a film obtained by sputtering aluminum electrodes in air at a pressure of 101 kpa; the repetition rate of voltage and current pulses is 40 hz, and the sputtering time is 3 hours. plasma parameters the discharge plasma parameters for mixtures of aluminum and air vapor and aluminum and argon vapor at atmospheric pressure (component ratio 30 pa: 101 kpa, respectively) were determined numerically and calculated as shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 9 the total integrals of the electron energy distribution function (eedf) in the discharge. eedfs were found numerically by solving the boltzmann kinetic equation in the two-term approximation. eedf calculations were carried out using the program [22]. based on the eedfs obtained, the mean electron energies, electron temperatures, electron drift velocities, electron densities, excitation rate constants of the energy levels of aluminum atoms, and specific discharge losses on elastic and inelastic collisions of electrons with atoms and molecules of both mixtures are determined depending on the magnitude of the reduced electric field (the ratio of the electric field (e) to the total concentration of atoms of aluminum, argon and molecules of nitrogen, oxygen and carbon dioxide for (n)). the variation range of the parameter e/n = 1-1000 td (1 · 10 -17 1 · 10 -14 v · cm 2 ) included the values of the reduced electric field that were realized in the experiment. for gas-vapor mixtures al air and al ar, these reduced electric fields were 820 td and 205 td in the time ranges of 50-100 ns and 100 -480 ns of voltage pulses, respectively (figure 1). the following processes are taken into account in the integral of collisions of electrons with atoms and molecules: elastic scattering of electrons by aluminum atoms, excitation of energy levels of aluminum atoms (threshold energies of 3.1707 ev, 2.9032 ev, 4.1463 ev, 4.2339 ev, 4.1296 ev, 5.1220 ev), ionization of aluminum atoms ( threshold energy 6.0000 ev); elastic electron scattering by argon atoms, excitation of the energy level of argon atoms (threshold energy 11.50 ev), ionization of argon atoms (threshold energy, 15.80 ev); elastic scattering and excitation of energy levels of nitrogen molecules: rotational threshold energy of 0.020 ev, vibrational (threshold energy: 0.290 ev, 0.291 ev, 0.590 ev, 0.880 1.170, 1.470, 1.760, 2.060, 2.350; electronic: 6.170 ev, 7.000, 7.350, 7.360, 7.800, 8.160, 8.400, 8.550, 8.890, 11.03, 11.87, 12.25, 13.00, ionization (threshold energy 15.60 ev); elastic scattering and excitation of energy levels of oxygen molecules: vibrational (threshold energies: 0.190 ev, 0.380 ev , 0.570 ev, 0.750 ev), electronic (threshold energy: 0. 977 ev, 1.627 ev, 4.500 ev, 6.000 ev, 8.400 ev, 9.970 ev, dissociative electron attachment (threshold energy 4.40 ev), ionization (threshold energy 12.06 ev); elastic scattering and excitation of energy levels of carbon dioxide molecules: vibrational (threshold energies: 0.083 ev, 0.167 ev, 0.252 ev, 0.291 ev, 0.339 ev, 0.422 ev, 0.505 ev, 2.5 ev ), electronic (threshold energy: 7.0 ev, 10.5 ev), dissociative electron attachment (threshold energy 3.85 ev), ionization (threshold energy 13.30 ev). data on the absolute values of the effective cross sections of these processes, as well as their dependences on electron energies, were taken from the databases and l.l. shimon [22-25]. figure (8) shows the dependences of the mean electron energy in the plasma of the vapor-gas mixture al: air = 30: 101000 and al: ar = 30: 101000 at a total pressure p = 101.030 kpa on the reduced electric field strength. figure 8. dependences of the mean electron energy in the plasma of a vapor-gas mixture on the reduced electric field strength: 1-al air = 30: 101000, 2-al -ar = 30: 101000 at a total pressure of p = 101.030 kpa. the mean energy of the discharge electrons for the vapor-gas mixture aluminum-air = 30pa 101 kpa almost linearly increases from 0.1364 ev to 16.57 ev (figure 8 (1)), and for the aluminum-argon mixture it also increased from 1.838 ev to 15.07 ev (figure 8 (2)) with an increase in the reduced electric field strength from 1 td to 1000 td. at the same time, a regularity was observed in the increased rate of its change in the ranges of 30–80 td and 1–30 td for mixtures of the first and second, respectively. for the reduced electric field strength range 205 td 820td at which experimental studies of the electric and optical characteristics of the discharge were carried out, the average electron energies varied between 5.323-13.96 ev for the vapor-gas mixture aluminum-air and 7.882 -13.09 for the mixture aluminum-argon. their highest energies corresponded to the values of 62.52 ev 249.0 ev for the first mixture, and for the second mixture 71.14 ev 282.8 ev. table (1) presents the results of modeling the transport characteristics of electrons: mean energies in ε, temperature t k, and drift velocity vdr. and electron concentration for two mixtures of aluminum vapor with air and aluminum vapor with argon. the temperature and electron drift velocity (тable 1) decreases from 161936 k to 61 746.8 k and from 6 ∙ 10 5 m/s to 2 ∙ 10 5 m/s for the first mixture, and for the second mixture from 151844 to 91431.2 and 4 ∙ 10 4 to 1.4 ∙ 10 4 when changing the reduced electric field strength from 820 td to 205 td, respectively. the values of electron concentration increase from 1.1 ∙ 10 20 m -3 to 1.6 ∙ 10 20 m -3 at a current density l. 02 ∙ 10 7 a/m 2 and 5.1 ∙ 10 6 a/m 2 on the surface of the electrode of the radiation source (0.196 · 10 -4 m 2 ) for both mixtures. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 9 тable 1. transport characteristics of electrons for the mixture: al-аir=30 pa – 101 kpa and al ar=30 pa – 101 kpa mixture: al-аir=30 pa – 101 kpa e/n, td ε, ev t 0 k vdr., m/s ne, m -3 1 0.1364 18362 6.2 10 3 5.1∙10 21 97 2.672 30995,2 2.5 10 3 1.3∙10 22 205 5.323 61746,8 2∙10 5 1.6∙10 20 820 13.96 161936 6∙ 10 5 1.1∙10 20 1000 16.57 192212 5.4∙10 4 1.1∙10 21 mixture: al ar=30 pa – 101 kpa e/n, td ε, ev t 0 k vdr., m/s ne, m -3 1 1.838 4431 1.4∙10 5 2.3∙ 0 20 97 6.795 78822 1.7∙10 4 1.9∙10 21 205 7.882 91431,2 1.4∙10 4 1,6∙10 20 820 13.09 151844 4 ∙10 4 1.1∙10 20 1000 15.07 174812 4.8∙10 4 1.3∙ 0 21 figure (9) presents the dependence of the specific power of the discharge losses on both elastic (1, 2) and inelastic (3,4) collisions of electrons with mixture components in a gas-discharge plasma on the reduced electric field strength. an increase in power is observed with increasing values of the reduced electric field, both for elastic processes and for inelastic ones. in addition, higher values of specific power of discharge losses are observed for inelastic collisions of electrons with atoms and molecules in a mixture of aluminum and air vapors (figure 9, table 2). figure 9. the specific discharge power for elastic (1) and inelastic (2) processes per unit of the total concentration of the mixture vs the reduced electric field strength for the aluminum-air mixture and for elastic (3) and inelastic (4) processes for the aluminum-argon mixture. figure (10) and table (3) present the results of numerical simulation of the dependence of the excitation rate constants of the spectral lines of aluminum atoms on the reduced electric field strength in mixtures of aluminum and air and aluminum and argon vapor for the ratio of partial pressures in mixtures of 30 101000 pa at a total pressure of the mixture p = 101030 pa. the rate constants are characterized by a high value, which is associated with the values of the absolute effective cross sections of the corresponding processes. in the range of reduced electric field strength 205 td 820 td, at which experimental studies of the electrical and optical characteristics of the discharge were carried out, they were in the range of k≈10 -16 -10 -15 m 3 /s. тable 2. elastic and inelastic power loss /n (ev m 3 /s) for mixtures al-аir=30 pa – 101 kp and al ar=30 pa – 101 kpa mixture al-аir=30 pa – 101 kp e/n,td elastic, power /n (ev m3/s) inelastic, power /n (ev m3/s) 205 3,448e-17 4,091e-14 820 1,290e-16 3,559e-13 mixture al ar=30 pa – 101 kpa e/n,td elastic, power /n (ev m3/s) inelastic, power /n (ev m3/s) 205 4,995e-17 2,535e-14 820 9,709e-17 2,658e-13 figure 10. dependences of the rate constants of excitation of the spectral lines of aluminum atoms on the reduced electric field strength in a plasma on a mixture of aluminum and air vapors: 30: 101000 pa at a total pressure of the mixture p = 101030 pa and aluminum and argon 30: 101000 pa at the total pressure of the mixture p = 101030 pa : 1λ = 396.15 nm (ethr. = 3.17 ev) for an aluminum-air mixture, 2-λ = 309.27nm (e thr. = 4.23 ev) for an aluminum-argon mixture, 3-λ = 309.27nm (e thr. = 4.23 ev ) for an aluminum-air mixture, 4-λ = 308.21nm (e thr. = 4.13 ev) for an aluminumair mixture. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 9 тable 3. the rate constants of the excitation of the spectral lines of aluminum atoms for the values of the reduced electric field strength in a plasma on a mixture of aluminum and air vapor: 30 101000 pa and aluminum and argon: 30 101000 pa at a total pressure of the mixture p = 101030 pa. mixture al-аir mixture al-аr e/n, td λ = 396.15 nm λ = 309.27 nm λ = 308.21 nm λ = 309.27 nm 205 2.51e-15 1.26e-15 8.52e-16 1.89e-15 820 4.01e-15 1.92e-15 1.55e-15 1.98e-15 limitations methods to reduce the body effects and to obtain better films, in the future it is necessary to switch to the subnosecond mode of operation of the reactor with a total voltage pulse duration of the order of 1 ns. this will contribute to the full implementation of the ectonic mechanism of aluminum atomization and improve the control of the film deposition process. conclusions thus, it was found that a plasma of an overstressed nanosecond discharge between aluminum electrodes at air pressures of 50–202 kpa, a pulsed discharge power of 3–6.5 mw, and an energy input of one pulse of 110–153 mj is a source of electroluminescence of aluminum oxide nanoparticles in the form of a wide band, which is in the spectral range of 300-430 nm; upon deposition of degradation products of electrodes and air molecules in a plasma on a glass substrate, films based on aluminum oxides were obtained, which are characterized by low transparency in the visible region of the spectrum. numerical simulation of plasma parameters in a mixture of aluminum and air vapor established that for the reduced electric field strength of 820 td 205 td, at which experimental studies of the electric and optical characteristics of the discharge were carried out, the mean electron energies varied between 13.96 and 5.323 ev, their highest energies corresponded to the values of 249.0 ev 62.52 ev. in a mixture of aluminum and argon vapor, these values of the discharge parameters varied within 13.09 ev 7.882 ev and 282.8 ev 71.14 ev, respectively. the electron concentration was 1.1 · 10 20 m -3 1.6 · 10 21 m -3 at current density l. 02 ∙ 10 7 a/m 2 and 5.1 ∙ 10 6 a/m 2 on the surface of the aluminum electrode (0.196 · 10 -4 m 2 ). the excitation rate constants of the spectral lines of aluminum atoms =396.15 nm, =309.27 nm, =308.21 nm are in the range of (4.0092.511) ∙ 10 -15 m 3 /s, (1.918 1.259) ∙ 10 -15 m 3 /s, (1.5530. 8.52) ∙ 10 -15 m 3 /s, (1.975– 1.975) ∙ 10 -15 m 3 /s, respectively. the maximum value of the excitation rate constant of the spectral line of aluminum atoms =396.15 nm was (4.009 ∙ 10 -15 ) m 3 s for the reduced electric field strength of 820 td in a mixture of aluminum and air. the specific discharge power loss for inelastic and elastic collisions of electrons with atoms and molecules, which were part of the working mixtures of a gas-discharge plasma, per unit total concentration of the mixture increased with increasing reduced electric field for both inelastic and elastic processes. its maximum value was for inelastic processes in a mixture of aluminum and air vapor was equal to 3.559 ∙ 10 -13 ev m 3 /s for a reduced electric field strength of 820 td. references 1. bityurin va, efimov av, grigorenko av, goryachev sv, klimov ai, chinnov vf. plasma stimulation of aluminum burning in water vapor. modern science. collection of research papers.2011 2(7): 47. 2. bityurin va, grigorenko av, efimov av, klimov ai, korshunov ov, kutuzov ds, chinnov vf. spectral and kinetic analysis of heterogeneous gas discharge plasma in the flow of an al, h 2 o, and ar mixture. high temperature. 2014 jan 1;52(1):1-1. 3. shkolnikov ei, beetle az, bulychev bm, larichev mn, ilyukhina av, vlaskin ms; edited by a.e. scheindlin. oxidation of aluminum with water for efficient power generation. joint institute for high temperatures ras. m .: science. 173 p. 4. mesyats ga. ecton-electron avalanche from metal. uspekhi fizicheskikh nauk. 1995 jun 1;165(6):601-26. 5. walters jp, malmstadt hv. emission characteristics and sensitivity in a high-voltage spark discharge. analytical chemistry. 1965 nov 1;37(12):1484-503. 6. kortov vs, ermakov ae, zatsepin af, uĭmin ma, nikiforov sv, mysik aa, gaviko vs. specific features of luminescence properties of nanostructured aluminum oxide. physics of the solid state. 2008 may 1;50(5):957. 7. zorenko y, fabisiak k, zorenko t, mandowski a, xia q, batentschuk m, friеdrich j, zhusupkalieva g. comparative study of the luminescence of al2o3: c and al2o3 crystals under synchrotron radiation excitation. journal of luminescence. 2013 dec 1;144:41-4. 8. abyzov am. aluminum oxide and alumina ceramics (review). part 1. properties of al 2 o 3 and industrial production of dispersed al 2 o 3. novye ogneupory (new refractories). 2019 apr 26(1):16-23. 9. ananchenko dv, nikiforov sv, ramazanova gr, batalov ri, bayazitov rm, novikov ha. luminescence of f-type defects and their thermal stability in sapphire irradiated by pulsed ion beams. optics and spectroscopy. 2020 feb;128:207-13. 10. beloplotov dv, tarasenko vf, lomaev mi. luminescence of aluminum atoms and ions in a repetitively pulsed discharge initiated by runaway electrons in nitrogen. optics of the atmosphere and ocean 2016 29 (2): 96. 11. shuaibov ak, minya ay, malinina aa, malinin an, danilo vv, sichka my, shevera iv. synthesis of copper oxides nanostructures by an overstressed nanosecond discharge in atmospheric pressure air shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 9 of 9 between copper electrodes. am. j. mech. mater. eng. 2018 may 7;2(8). 12. shuaibov ak, minya ai, gomoki zt, danylo vv, pinzenik pv. characteristics of high-current pulse discharge in air with ectonic mechanism of copper vapor injection into a discharge gap. surface engineering and applied electrochemistry. 2019 jan 1;55(1):65-9. 13. shuaibov ok, malinina ao, malinin om. new gas discharge methods for the production of selective ultraviolet and visible radiation and synthesis of nanostructures of transition metal oxides. monograph. uzhhorod. publishing house of uzhnu “hoverla. 2019. 14. shuaibov ak, minya ay, hrytsak rv, malinina aa, malinin an. characteristics of an overstressed discharge of nanosecond duration between electrodes of chalcopyrite in high pressure nitrogen. advances in nanoscience and nanotechnology 2020 4(1): 1. 15. tarasenko vf. runaway electrons preionized diffuse discharge, new york: nova science publishers inc., 2014. -578 p. 16. holovey vm, popovych kp, prymak mv, birov mm, krasilinets vm, sidey vi. x-ray induced optical absorption in li2b4o7 and li2b4o7: cu single crystals and glasses. physica b: condensed matter. 2014 oct 1;450:34-8. 17. beloplotov dv, tarasenko vf. on the influence of a cathode shape on the parameters of current pulses of runaway electron beams in a gas discharge when applying voltage pulses with a rise time of 200 ns. injournal of physics: conference series 2019 nov (vol. 1393, no. 1, p. 012004). iop publishing. 18. silveira e, freitas jr ja, glembocki oj, slack ga, schowalter lj. excitonic structure of bulk aln from optical reflectivity and cathodoluminescence measurements. physical review b. 2005 jan 7;71(4):041201. 19. egorov ae, chernyshev vv. electroluminescence spectra of anodic alumina in various electrolytes. bulletin of voronezh state university. series. physics mathematics 2005 2:8. 20. gasenkova iv, mukhurov ni, vakhioh yasin mohsin. optical properties of anodized aluminum substrates as a basis for threshold detectors. reports of bsuir 2016 2 (96): 114. 21. van der horst rm, verreycken t, van veldhuizen em, bruggeman pj. time-resolved optical emission spectroscopy of nanosecond pulsed discharges in atmospheric-pressure n2 and n2/h2o mixtures. journal of physics d: applied physics. 2012 aug 10;45(34):345201. 22. http://www.bolsig.laplace.univ-tlse.fr . 23. shimon ll. influence of autoionization states on the population of energy levels of atoms of the aluminum subgroup. scientific bulletin of uzhnu. physics series 2007 20:55-61. 24. http://www.ioffe.ru/es/elastic/data2.html 25. https://physics.nist.gov/cgibin/ionization/ion_data.php?id=ali&ision=i&initial=&t otal=y http://www.ioffe.ru/es/elastic/data2.html https://physics.nist.gov/cgi-bin/ionization/ion_data.php?id=ali&ision=i&initial=&total=y https://physics.nist.gov/cgi-bin/ionization/ion_data.php?id=ali&ision=i&initial=&total=y https://physics.nist.gov/cgi-bin/ionization/ion_data.php?id=ali&ision=i&initial=&total=y highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202105 review article open access 1 department of bioinformatics, colors medical laboratories, cairo, egypt. contacts of authors * to whom correspondence should be addressed: p.habib911@gmail.com received: january 01, 2021 accepted: march 6, 2021 published: march 15, 2021 citation: habib pt . covid-19 symphony: a review of possible music therapy effect in supporting the immune system of covid-19 patient. 2021 mar 15;4:bs202105 copyright: © 2021 habib. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. covid-19 symphony: a review of possible music therapy effect in supporting the immune system of covid-19 patient peter t. habib*1 abstract coronavirus, the current pandemic that takes lives of 1,936,632 until the time of the paper. scientists over the world exhibited all efforts to fight the virus. many supplements developed to fight coronavirus by supporting the immune system which is the strongest worrier against it for now. one of the neglected strategies to enhance immunity is music therapy. music therapy can drive immunity into different conditions. not only able to boost and suppress immunity but also many physiological and psychological diseases are treated with music therapy. it is more than just entertainment, and research shows that music therapy may be helpful and cost-effective to support standard therapy. this systemic review tries to explore the effect of music therapy and arrives at a conclusion suggesting routine procedures to support the patient with music therapy. however, the idea of using music therapy in improving the immune system must undergo clinical and medical confirmation to be used. keywords: psychology, music therapy, immune system, covid-19, coronavirus, pandemic. introduction the epidemic by a new coronavirus, named covid-19, initiated in late 2019 in wuhan, china, is a worldwide public health threat [13]. the virus likely evolved in bat, a zoonotic origin, after amino acid substitution in the spike glycoprotein, as recently suggested, acquired the ability to bind to ace2 receptor and be able to infect humans, which started the new epidemic [4]. currently, there is no efficient treatment against the new coronavirus. therefore, identifying previously known antiviral drugs to fight back is urgently needed. a successful drug research strategy is to check if the current antiviral medicines are efficient in the treatment of coronavirus infections. several drugs, such as ribavirin, ribavirin, chloroquine, and two well-studied broad-spectrum antiviral drugs remdesivir, and favipiravir have been used a long time ago with sars or mers patients, although the efficacy of some drugs remains controversial [5]. till now, the immune system is the only barrier against coronavirus. before existing of an official drug or vaccine for the coronavirus, many individuals recovered due to the immune system with the aid of some vitamins to support the immunity and medications to reduce the inflammation. although, there were some efforts to develop a vaccine [6] and discovering some potent molecules that may inhibit the virus attachment [7]. however, the official protocols aim to enhance immunity with medication. but the neglected fact that not only medication enhances immunity, but music therapy also does the same effect. backing to the 10 th bc century from western the starting from greeks to the eastern including india and china, history proves the curative and healing powers of music therapy. the ancient greeks are thought to have used music therapy to soothe the mentally disabled. western thinkers from pythagorus, aristotle, and plato to schopenhauer and nietzsche provided detailed of their interpretation of music’s therapeutic forces and their confidence in its practical application to offer peace and unity, and thereby to cure mental and physical illnesses [8]. aristotle, who has a strong background in psychotherapy, provides a theory that "after listening to music, those who suffered from uncontrollable feelings would return to their regular state, which brought their souls to happiness" [9,10]. there is also evidence from 4000 bc of using music therapy as a tool of stress relief and is estimated to stretch back as far as palaeolithic times [11]. highlights in bioscience page 1 of 7 march 2021|volume 4 https://doi.org/10.36462/h.biosci.202105 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0001-9178-352x http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient the investigation of music therapy has increasingly delved deeper into the mechanisms influencing music therapy interpretation and production, investigating music psychology [12]and music cognitive neuroscience, also referred to as ’neuro musicology [13] the physiological basis for music-induced emotions [14,15], the neurobiology of some elements of music such as harmony [16] and the neuroanatomy of music performance have been included in this profound study [17]. and the scope of research ranged from the interpretation inside the womb of folk songs [18] to the production of opera on concert platforms [19], to the use of mainstream music in working theatres [20]. music is an emotional language. it is known for improving mental and mood health. any change in emotions affects the hormonal, neurophysiological, and physiological processes; thus, changes in body balance caused by music indicate the efficacy of different musical types [21]. though still not well studied, several studies show that music therapy may promote a decrease in the stress response, a decrease in heart rate, improved tolerance to pain and suffering, change in skin behavior and muscle function, decreased anxiety, and depression. different studies have reported this effect in musically exposed stressed patients [22-24]. on the other hand, noise stress could cause adverse effects on immunity in both human and animal models [25,26]. several studies investigate different stress indicators, including blood pressure, pulse rate, and respiratory rate recorded measuring vital signs. these are the only anatomical measurements being used in nine of these. several research, including [27], used vital sign assessment as proof of a transition from sympathetic to parasympathetic processes, equating a shift in immune function with a wider response to stress. in many trials, calming music was found to decrease blood pressure, pulse rate, and respiration rate. no definitive improvement was observed in just four trials. one of the first music therapy studies reports music as therapy was in 1963 when melzack, weisz, and sprague report when evaluating the effect of the auditory stimulus on behavioral pain due to participants’ fingertips submerged in ice water slow gradually increased discomfort in the group listening to music relative to control group [28]. in 1966, two famous scientists, morosko and simmons tried to shock to the teeth of volunteers and found that music and white noise were effective in decreasing the pain sensation and increase tolerance [29]. in 1974, peretti and swenson used the galvanic skin response (gsr) to assess anxiety caused during a written maze test by negative responses to participants. in this case, music lowered gsr responses dramatically [30]. the effects of music on a specific physiological mechanism such as cardiac output, respiratory rate, and blood pressure were originally reported in many studies including such as in france in 1880, america in 1899, and russia in 1903 [31]. however, the immune system is one of the strongly affected systems in the body that can be manipulated indirectly with music. musically immune stimulation as mentioned, the immune system is strongly associated with mood, psychological condition, and hormonal balance. thus, as a result of negative mood, stress affects the immune system may cause dysregulation leads to a change in the humoral and cellular immunity and increases health risks [32]. bittman [33] presented proof that the drummer's team has improved nk cell response, lymphokine-activated killer cell function, and dehydroepiandrosterone cortisol balance in normal subjects, considered one of the early signs of the immune system-music interaction. wachi [34] found that delightful music modulates immunological responses in adults 65 years of age who are demarcated. important changes were reported in the number of lymphocytes, cd4 + t cells, t cells, and t memory cells as well as in the development of ifn-π and il-6. even music could reverse the immune stress induced by the immune suppressor. not only can affect the immune response but could lead to a reversion of the effect of the medication. music appeared to has a reversal effect as pharmacological interventions (i.e. benzodiazepines, 5 ht agonists) in immunosuppression induced by stress [35-37]. many studies state that music minimized stress-induced hyperactivity of the hypothalamus-pituitary-adrenal [hpa] axis, including acth and corticosteroid production [23,38-44], and modulate norepinephrine, epinephrine, gh, prl, and endorphin secretion [45]. this hormonal and neurotransmitter balance lowers stress and improving the condition of the immune system. other studies show that there a wide range of drugs that suppress the immune system, but one common immune suppressor is the negative mood. on the other hand, music is well studied as an immunity enhancer that re-regulates the immune system, increases nk cell activity, lymphokine-activated killer cell activity, lymphocytes, t cells, cd4+ t cells, and memory t cells [33,34]. music improves the psychological state, re-balance the hormonal and neurotransmitter levels in the body, therefore improve immunity. in the current coronavirus pandemic which began in china, there is an increasing number of infected people every day. after emerging of different vaccines that draw the immune system attention to foreign invaders, the immune system proves that it is the only and strongest barrier against the coronavirus. one promising approach is the plasma transfusion from recovered people to covid-19 patients as many studies find that 200 ml of plasma from recovered people could neutralize the coronavirus and lead to the disappearance of viremia in 7 days [46–49] which shows that the immune system is the only cure for such diseases. the immune system is known to be strongly connected to psychological balance. that is when the individual mood is good which means a high level of dopamine in blood, will optimize and enhance the immune system, and a bad or sad mood will suppress immunity. so, one of the best strategies to support the immune system is to keep the mood in a good state. and some studies support the fact that dopamine is highly liked by the immune system [50,51]. highlights in bioscience page 2 of 7 march 2021|volume 4 http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient the influence of music on leukocytes was investigated in six experiments [33] found that natural killer cells increased, followed by an endocrine reaction. [52] hirokawa and ohira [53] carried out other experiments testing the number of cd4 and cd8 cells. it was observed that inducing recorded music, analyzed by [52], increased plasma cd4+ t cell levels. the impact of music on immunoglobulins was also investigated. the most studied antibody (n = 12) has been immunoglobulin a (iga). some of these studies reported a rise in iga level after a series of musical approaches with a wide range of styles and genres. fancourt introduces a modern model that connects central nervous system (cns), endocrine system, autonomic nervous system (ans), and immune system with them effectively to psychological effects and physiological effects which provides a basis for the creation of a taxonomy of study design variables linked to music and stress and traces the broad paths involved in its impact on the body [54]. gender-dependent response according to the physiological difference between men and women, there is a different response to music as shown in nater et al., the study [55] that reports different interactive patterns in men and women. once met with heavy metal music, according to finger temperature and skin conductance tests, women displayed a significant gradual increase in the sympathetic nervous system (sns) response than men's response. however, men displayed an abnormal autonomic reaction after exposure to heavy metals as demonstrated by increased salivary amylase secretion, which is caused by sympathetic and parasympathetic stimulation [55]. several studies have suggested that the difference in sex-based psycho-physiologic responses to music might have a hormonal basis [56–59]. however, research by trost [60] using functional magnetic resonance imaging (fmri) indicated that cheerful music stimulates the left striatum and insula while nostalgic sad music stimulates the right striatum and orbitofrontal cortex in both genders. criteria of theraputic music it is well known that music is related to mood and can make people feel happy, depressed, alive, or pleasant [61]. listening to techno music (tm) or fast rhythm music[44] could cause increases in plasma norepinephrine, epinephrine, gh, prl, cortisol, and beta-endorphin concentrations with a gradual rise in heart rate, systolic blood pressure, and significant changes in emotional response. in gerra [62] that found that exposure to techno music increases the heart pulse rate and norepinephrine, cortisol, and adrenocorticotropic hormone secretion levels and vice versa such in the white study [63] who showed, in a group of patients in intensive care unite (icu) when they listened to relaxing classical music, a reduction of heart rate and blood pressure. other studies suggested that classical music has been found to improve the mood state caused by tm and increased gastric slow-wave in adults [31], but not to significantly reverse changes in hormonal concentrations [64]. an old study investigated the physiologic effect of listening to no music, fast-rhythm music, and sedative music for athletes and non-athletes during the exercise of varying intensities. under conditions of intensive exercise, runners listening to fast rhythm music had increased plasma cortisol levels [65]. this indicates a possible metabolic advantage for the use of energetic music during exercise, considering the role of cortisol in stimulating the catabolism of energy substrates in fat, adipose, and connective tissues. mccraty's study shows the effects of music in this study was titled 'heart zones'. s-iga increased by 55 percent (p < 0.01) when listening to the heart zones' music alone [40]. criteria of selected music according to [66] should be full of character, slow rate, repeated rhythm, predictable tone dynamics, pleasant harmony with no vocal content. three songs studied by [67] are consist of 70 to 80 beats per minute and have a slow and steady, repeated rhythm as reported in the previous study. the three songs are "canon in d major", "love story", and "dance of the iguana" played by johann pachelbel, richard clayderman, and stevan pasero respectively. valence and arousal in music have also been shown to be major factors. for calm, low tempo songs, [68] compared recorded music with four distinct tempi and moods, but only found a decrease in heart rate. [69] found that while heart rate during a stressful activity was diminished a little by high tempo music, with low tempo music there was a much greater impact. the respiratory rate was lowered only due to low tempo songs [70] found that blood pressure was not changed by soothing music when contrasting six distinct tempo pieces of music, but raised by high tempo music. the most compelling outcomes were provided by experiments that implemented more complicated controls. [71] stated, for instance, that listening to mozart had a stronger influence on vital signs than a progressive relaxation session, and [72] found that listening to relaxing recorded music was as effective as diazepam in decreasing vital signs of anxiety. three specific styles of music were listened to by adults and adolescents in another study : classical, new age, and designer (designed to promote a sense of wellbeing) [73]. the respondents surveyed personal emotions at baseline and directly after listening to each style of song. classical music reduced anxiety. through new age music, relaxation increased dramatically and aggression, mental focus, vigor, and anxiety reduced. the subjects registered significantly more calm, mental focus, vigor, and sympathy after listening to designer music, and significantly decreased aggression, depression, exhaustion, and anxiety. although addressed music should be consist of 70 to 80 beats per minute with a slow and steady, repeated rhythm as reported such as "canon in d major", "love story", and "dance of the iguana", the cultural aspect should be considered when choosing music and mentioned music example is just to clarify the type of therapeutic music. highlights in bioscience page 3 of 7 march 2021|volume 4 http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient music therapy for covid-19 patient covid-19 coronavirus disease, which is an infectious disease, and it was clear that negative feelings were mostly physiologically caused by infectious diseases and that a wide spectrum of health risks could be exacerbated or enhanced as a result of this infection. [74–76] in le roux's study, he reveals that classical music, such as j. s. bach's magnificat in d majorbwv 243 has an effect on emotion, immunity, and endocrine parameters in patients with specific infectious lung diseases, an experimental and control group was randomly distributed to patients. the experimental group received physiotherapy with the preferred music over a 3day duration, while the control group only received physiotherapy. the outcome indicates major changes in parameters of the immune system, such as poms-scale, cd4+:cd8+ ratio, cortisol, and cortisol: dhea ratio. and music's intervention reveals contact between the mind and the body. [77]. such experiment which conducted on a patient with infection disease, similar to coronavirus situation, may be applicable to covid-19 patient. previous studies prove the therapeutic effect of music, which made music a strong candidate to be used along with medication for covid-19 patients. a type of music administered by healthcare professionals for children also has a pain decreasing effect. the songs used consisted of age-appropriate children’s songs. yinger provides a list of music and its purpose [78]. such strategies may be beneficial for decrease the pain resulted from the medical procedures. as a severe case of covid-19 patients should be placed on a respirator, one study demonstrated a significant fell in systolic and diastolic blood pressure fell causing a decrease in anxiety and depression during music therapy sessions for the patients on the respirator, as is shown in paired samples [78]. the qualitative part of the study showed that the patients remembered little of their time on the respirator which may be a good effect to remove the memories about the disease to avoid any psychological complications that may be caused by negative memories. recently, in some mild covid-19 cases, it is recommended for the patient to be isolated in a room, which may increase patient anxiety and depression. the same situation reported before in brodsky's work where music therapy was most effective in isolating hospital experiences, reducing distress, and fearprovoking hallucinations, and helping to have a more positive effect than other therapies[79]. although several reports demonstrate the beneficial impact of classical music, it will be best to encourage the participant, as much as practicable, to select the type of music [31]. although music therapy is deemed free of side effects, the technique should be used with the supervision of medical staff. a patient may react deeply, emotionally to a specific piece of music. others reported a stress reduction and an increase in t-cell proliferation in mice who listened to music. in mice exposed to auditory stress, the nk activity of spleen cells also decreased during 8 days [80–83]. such findings have been supported by previous research on the impact of auditory stress and its negative effect on the immune system [84]. applying music therapy way to the patient should be considered and carefully chosen in order to prevent or at least minimize infection transmission between healthy individuals and patients. a trained music therapist must deliver music interventions; the music therapist often adapts the live music experiences to the patients' needs at the time. this also presents the patient with a highly humanizing and validating experience. furthermore, listening to self-selected pre-recorded music, introduced by the patient himself, may lead to an enhanced sense of autonomy and empowerment in a critical care setting where certain facets of care are beyond the control of the patient. [85,86]. lee study warning from using headphones that may cause crosscontamination and nosocomial infections [87] show that speaker of personal or one-use headphones might be the best way to listen to music in case of covid-19 patient conclusion an epidemic of novel coronavirus diseases (covid-19) in wuhan, china has spread widely throughout the nation. the world focuses all efforts to stop the spreading of the virus using a different drug, and recently by enhancing the immune system using vitamins or plasma transfusion from the recovered person. but, one of the unnoticed supplements for the immune system is music therapy. the results of a recent investigation of music effect for medical procedural support shows that there is an urgent need for more high-quality research on the use of effective music therapy before, during, and after medical procedures, with particular emphasis on the interventions and techniques used by the music therapist [88] for patients and staff [40,89] in treating a patient with infection [77]. many studies state the improving effect of music on the immune system reported in thirteen studies [66,67]. and it seems that slow, repetitive rhythm, predictable dynamics, low tonic register, pleasant harmony such as classic music is the most effective [78] style not only in improving immunity but also in a medical procedure such in isolating room or placing the patient on a respirator [78]. although this idea must undergo clinical trials to be proved, developing a "covid-19 symphony" a music track based on recent research and experiments may be urgently required to be a part of the medical protocols alongside supplements and drugs to improve mood and immunity to help the patient pass such disease with the precaution of addressing the music therapy in a personal way to prevent infection transmission without using any instrument that may transmit the infection [87]. references 1. zhu n, zhang d, wang w, others. china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china, 2019 [published january 24, 2020]. n engl j med. 2. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, highlights in bioscience page 4 of 7 march 2021|volume 4 http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient china. the lancet. 2020;395(10223):497–506. 3. hui ds, azhar ei, madani ta, ntoumi f, kock r, dar o, et al. the continuing 2019-ncov epidemic threat of novel coronaviruses to global health—the latest 2019 novel coronavirus outbreak in wuhan, china. international journal of infectious diseases. 2020;91:264–6. 4. benvenuto d, giovanetti m, ciccozzi a, spoto s, angeletti s, ciccozzi m. the 2019-new coronavirus epidemic: evidence for virus evolution. journal of medical virology. 2020;92(4):455–9. 5. zumla a, chan jfw, azhar ei, hui dsc, yuen k-y. coronavirusesdrug discovery and therapeutic options. nature reviews drug discovery. 2016;15(5):327–47. 6. habib pt. vaccine design, adaptation, and cloning design for multiple epitope-based vaccine derived from sars-cov-2 surface glycoprotein (s), membrane protein (m) and envelope protein (e): in silico approach. 2021; 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paper format highlights in bioscience issn: 2682-4043 highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 1 of 10 citation: alsamman m.a. (2019) the art of bioinformatics learning in our arabic world. highlights in bioscience, volume 2. article id 20193, dio:10.36462/ h.biosci.20193 received: august 19, 2019 accepted: september 13, 2019 published: october 12, 2019 copyright: © 2019 alsamman .this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. the art of bioinformatics learning in our arabic world alsamman m. alsamman * 1 1 molecular genetics and genome mapping lab. , agricultural genetic engineering research institute, agricultural research center, egypt. * to whom correspondence should be addressed: smahmoud@ageri.sci.eg abstract bioinformatics became a significant field in life sciences that, draws a number of researchers and extends into a wide range of biological disciplines. rendering bioinformatics analysis techniques are the most desirable skills in a variety of scholarship programs and academic positions. teaching bioinformatics is very challenging since it is a multidisciplinary field, where most of the undergraduate programs in colleges provide only one area required for bioinformatics. besides the regular education system, few bioinformatics training courses are offered and less are affordable to fresh graduates in countries most of which are categorized as developing countries. the high cost of learning, confusing education systems, and the complexity of bioinformatics science has made it very difficult to be taught and more challenging to be studied in arab countries. this review provides possible solutions to most of these issues and offers the best practice to guide future arab bioinformaticians to learn bioinformatics in a way that fits our social, financial and academic circumstances. moreover, it discusses the key aspects that a bioinformatician needs to be aware of and the basic knowledge that must be gained. on the other side, it will illustrate how to start learning, to address some of these challenges and how to deal with some of the related social issues. keywords: bioinformatics, arab, programming, education, linux, biological data. introduction bioinformatics analysis techniques are the most desirable skills in a wide range of scholarship programs and academic positions. as the study of bioinformatics and computational biology grows and evolves, it is essential to quantify the factors that contribute to the development of professionals in this field (1). bioinformatics is an interdisciplinary research field where computational resources and techniques are used to interpret biological data through mathematical and statistical approaches. perspective review open access the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 2 of 10 the rapid acceleration in computing power and memory storage capacity have given rise to a new golden age in the biological data analysis (2). there is almost no biological sector in which bioinformatics has not yet been incorporated. its techniques are used in microbiology to examine microbial diversity and species occurrence by identifying and quantifying the association of microbial communities among different biological samples (3). throughout the pharmaceutical industry, bioinformatics offers analytical tools that can enhance drug target identification, drug candidate monitoring and drug optimization. in particular, it promotes the recognition of side effects and forecasts drug resistance (4). recently, bioinformatics has proposed the idea of personalized medicine, where treatments will be adapted to the unique genotype of patients. integrating the vast genetic information provided by the genome wide association studies (gwass) is a valuable resource for mapping genetic traits with drug reactions and phenotypes, allowing individual characteristics of each patient to be monitored and their susceptibility to certain diseases to be considered (5). last but not the least, the integration of machine learning in bioinformatics analytical methods has opened a new era in which sample data and past experience could be used to improve the output criterion in computational algorithms, the optimized criteria could be the rating of reliability provided by the statistical model and the significance of the performance (6). machine learning has therefore enabled what appears to be a breakthrough in biological research, where computer programs can solve complex biological problems efficiently and effectively. through cancer diagnosis (7), neurooncology imaging (8) and drug design (9) in medicine and plant physiology (10), forecasting crop yields (11) and livestock production in agriculture (12), machine learning has been used to solve both challenging and basic tasks. the rapid development of life sciences and information technology requires the continuous development of bioinformatics learning programs in order to sustain their significance (1). the quality of the education systems in the arab countries has gradually improved over the last 30 years in the arab world. more work has been done by non-governmental and government agencies and organizations to improve the educational opportunities for arab students, and to promote their independence and integration into their societies and to prepare them for future careers, taking into account current trends in the labor market (13). few leading bioinformatics programs in arab countries have been established over the last decade in egypt, lebanon, ksa, uae, and oman. some of these programs have launched throughout the biotechnology and genetics departments of computer science, science and agriculture colleges in both special and governmental universities (14). so far, in arab countries, neither colleges nor departments have been specifically founded to teach bioinformatics. as multidisciplinary science, bioinformatics is difficult to teach, most of the programs are designed for undergraduate students in colleges that provide only one field required for bioinformatics. these programs address these issues by borrowing a few courses from other colleges, for example, when bioinformatics programs are embraced by computer science colleges, students are expected to pursue a few courses in biology in science or agriculture colleges. these courses could take place in the second or last years, and could be acquired in the worst situations a few weeks before graduation. this system creates a gap in the experience of the participant, a hole that must be filled in the next few years of his life as a researcher or even as an employee. beyond the regular education program, few bioinformatics training courses are offered and less are affordable to fresh graduates in countries most of which are classified as developing countries. the high cost of training, confusing education systems and the complexity of bioinformatics science has made it very difficult to teach and more difficult to study in arab countries. inducing bioinformatics education systems to move slowly toward improving the quality of life of arab residents and addressing crucial food and drug issues. this review is written in order to offer possible solutions to most of these issues and to guide future arab bioinformaticians throughout the best methods to study bioinformatics in a way that fits our social, financial and academic circumstances. it also addresses the key aspects that the bioinformatician needs to know about, such as how to begin learning the basic knowledge that needs to be learned, and how to overcome some of the social issues faced by young bioinformaticians. how to get started in bioinformatics bioinformatics, as already stated, comprises of three different fields: genetics, computer science and mathematics. the purpose of these three domains is important for the organization, comprehension and interpretation of the different biological information that bioinformatician handles on a daily basis. the question of studying these three areas is very difficult and few places of education provide this knowledge in one location. you've certainly learned the fundamentals of one or two of these areas as biology, computer science or mathematics graduate, you only need one or two remaining knowledge to become a bioinformatician. as you begin to learn, my tips are: 1) learn the basics, and then you will know where to go next. the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 3 of 10 2) this is about knowledge, not certification; certification will not indicate that an individual has adequate knowledge of the subject area. arab bioinformatics students are more worried about the credential than they are about the skills they need to acquire. 3) take what you need through free learning; this is your university and your home, attend those classes you need to learn, even if it is not your college. many students overlook the fact that most university lectures are not closed to free learners as long as they do not need a credential. 4) take a basic course; most of the basic courses offered in arabic countries cover weak bioinformatics skills such as primer design, sequence alignment, ncbi software and gene annotation using online tools. you may take one basic course if you don't understand the software, as long as you don't repeat courses with the same content. 5) ask as much as you can; there are many public forums for bioinformaticians that offer gatherings across all disciplines such as biology, mathematics, and computer science, where you can ask any question you want. 6) engage the scientific society as a free bioinformatician; i know it's hard for some arab students to work after graduation for 6-12 months without any kind of support. but it is about joining the research community , learning about the issues they face and seeking out how to manage it as a bioinformatician. the advantage of this would be that, if they don't pay they can't control you. you can choose to work on any type of data without any restrictions, the ability to attend any class outside the workplace, and if there is any other work opportunity you can apply. in fact, you should have a certificate of work experience and my recommendation is to look elsewhere if they don't offer you such a credential. taking in advantage that, most of the scientific institutions in arabic world can not offer payment for fresh graduates and they have limited funding, which made them in a big need for graduates who do not ask for payment. 7) read even when it is difficult to understand; bioinformatics is a science that is changing every day and few textbooks will cover all of its aspects so you need to keep reading. that you will be able to understand a few more every time you start reading new research, finally you will get a clear understanding of most of these articles. while you're reading, take care of the software that they have used and try to run it using sample data. supplementary 1 contains some simple articles and reviews you can start with. 8) self-learning is not the full answer; while self-learning through online resources such as youtube (15) and academia (16) is very helpful in expanding your knowledge, you need to deal with real problems by hand in order to sharpen your abilities, and this will not happen without joining a scientific group. 9) the way to learn more is to teach others; passing bioinformatics skills to others would open your eyes to different applications of the same tools and knowledge gaps, granting you more opportunities in the near future. operating systems and bioinformatics although most of the bioinformatics courses are interested in teaching software,i would suggest that it is not more about the tool than the environment in which you operate. many scientists in our arabic world use the microsoft windows operating system every day to manage and evaluate their data. microsoft's operating system is commercially closed and hackable (17). such an environment is not a natural space for innovation and research, especially when dealing with mega-biological data. the normal bioinformatics operating system should be opened (could be programmatically modified), highly secured, free of charge, and compatible with all bioinformatics software. of which only one operating system could have granted these privileges, linux. linux is a unix-like and often posix-compliant operating system (os) based on the design and distribution of free and open source code. the basic element of linux is the linux kernel, the very first operating system kernel introduced by linus torvalds on september 17, 1991. the free software foundation uses the name of gnu/linux to describe a complex operating system (18). most arab researchers fear linux operating system, depending on the false belief that linux is only a black and complicated command line window. on the contrary, linux can operate videos, games and handle all the types of data files you use in windows, and it has free writing programs like libreoffice (19) and a lot of wonderful programs and tools that could change your life. linux also gives users a bit of control about what occurs on their machine what does and doesn't changing. you could learn more about the advantages of linux over windows through a number of articles (20). linux has different flavor distributions such as ubuntu (21), fedora (22), opensuse (23), red hat (24) and many others. mostly there is no difference between the various linux distributions, although there is some variance between the software configuration , where it does not influence the core system performance or stability. ubuntu is one of the most common linux distributions (25), and the initial findings indicate that ubuntu do’s not require technical assistance (26), the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 4 of 10 has a defined and simple graphical user interface (gui) and has been incorporated into some arabic education systems (27). ubuntu has a great and powerful version named bio-linux (28). in 2002, dr. dawn field launched the bio-linux system under the nerc environmental bioinformatics program (29). bio-linux release allows easy access to a versatile computing environment pre-loaded with bioinformatics software from basic data analysis tools to advanced analytic framework programming packages (21). you can download and install bio-linux in an iso file format from the official website (http://environmentalomics.org/bio linux/). the only downside is that bio-linux distributions are a way behind ubuntu's latest updates, and most hardware drivers such as wifi, computer mouse, or touch pad, are not installed which involve certain installation skills. in order to overcome this issue, my recommendation is to install the latest ubuntu release and then download bio-linux packages via the linux synaptic package manager. alternatively, you can download the edition of bio-linux and then request the system to be upgraded with simple commands. with the ubuntu software center, you can quickly download and install thousands of linux software without any complications. the linux terminal is another way the software can be used (supplementary 2). although some tools require some command lines to be ready for analysis, most of them do not require installation skills. there are few linux guidelines for fresh bioinformatics students: 1) install and boot from bio-linux using flash memory for a short period of time while you are in practice (supplementary 2). 2) learn to use all the tools and software that ubuntu has provided, even if they were really basic, such as libreoffice and calculator. this would eliminate the fear of using a new operating system to allow you more comfortable with the environment. 3) bio-linux has a web page documentation, and sample data for most its bioinformatics software, try to open and use it through the desktop panel. 4) try to write your own easy manual notes for all software built on bio-linux, explain the use, input, output and how you can use this tool in your future research. in supplementary 2, i am explaining how to use a few of these tools. 5) after a few weeks try installing the latest version of ubuntu or bio-linux , update and lunch jemboss (30) using the ubuntu software manager 6) try to practice simple linux commands and use as many tools as you can. computer science and bioinformatics most arabic bioinformatics students prefer to start with programming languages such as python for biological data analysis. python has many advantages when it comes to bioinformatics and has several advantages as a computer language compared to languages such as perl, which is also a famous bioinformatics computer language (31,32). the problem, though, is that most of these learners neglect and ignore the basic rule of computer language learning, which is the need to understand the basics of computing and its core structures, such as object orientation programming (oop). on the other hand java is a very common computer language in our arabic country, and most commercial training companies offer paid courses to learn this language. my concern is that, although java has a smaller relationship with bioinformatics compared to python, few training companies offer python and fewer do so professionally in our arab world compared to java. in this regard, while you are learning computer language, you need to understand the fundamentals of computer science. these fundamentals involve algorithms that are a sequence of commands typically used to solve a problem or perform a computation, and can be expressed in a finite number of steps and a well-defined formal language (33). the best way to learn algorithms in your first step is to learn algorithms by programming languages that you have selected to learn such as java (34), python (35), and perl (36). using this method, in addition to learning the algorithm effectively, you will be able to sharpen your coding skills. after studying programming language in such a smarter way, you can easily add other programming languages to your set. among these languages is r, which is a statistical programming language and common computational tool for data analysts, and is it has become one of the most commonly utilized programming languages in bioinformatics software. this is mainly due to its performance and the richness of the libraries that could be used for data manipulation and simulation (37). r is very simple, and there are large numbers of r libraries developed for bioinformatics. it might be difficult for biological researchers to learn coding and algorithms, and my recommendations for easy learning are: 1) practice basic codes as much as you can, i would prefer to write these codes a couple times by hand and then run it under the computer language compiler. 2) don't spend much of your time on graphical user interface (gui) coding, most common bioinformatics software don't have a gui, but instead focus on learning programming skills. 3) do not forget that, for the purpose of interpreting biological data, you are studying programming language, and you are not a computer scientist. based on this, don't go deep into programming language http://environmentalomics.org/bio-%20linux/ http://environmentalomics.org/bio-%20linux/ the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 5 of 10 learning, instead of considering what it can give you to achieve your goal. 4) after learning basics, you can search for programming codes that address simple biological issues such as dna transcription and translation, reversing dna sequence, and scripts which reflect the use of simple genetic concepts without the use of external libraries. 5) do not use libraries/modules/packages for simple tasks in your first steps; you need to learn how to use computer language to apply scientific principles in algorithmic analysis. 6) organize yourself; the first lines of your scripts should indicate the use of the script and its input and expected output data. you'll use these scripts in several occasions, and it's unfair to waste your time rewriting the same code. 7) you can use the same script or tool differently; this depends on your imagination. 8) try to write clean and structured scripts where the overused programming functions can be found in wellorganized libraries. it would provide the ability to use the same collection of libraries for various uses in different projects. 9) backup your scripts every week. mathematics and bioinformatics most educational systems in our arab world are concerned with teaching students without proper application of mathematics principles where student knowledge of these basics are not linked to their use. there is an easier way to teach mathematical rules in other countries by providing real-life experiments to learners (38). most arabic students have a decent mathematical background based on their high school education, which might be enough to start learning the science of bioinformatics, but my basic concern is that, they need to get missing knowledge through straightforward courses. the main target is a sub-science of mathematics, statistics. statistics is a systematic and random analysis of variability. in many aspects of scientific research, statistical methods are valuable. they constitute the research of the right way to collect process and interpret data (40). the connection between statistical methods and bioinformatics is very critical; you might argue that most of the biological data analysis could not be conducted without a good statistical background (41). as a bioinformatician, you will continue to learn the fundamentals and implementations of this science in biology for the most part of your life. on the other hand, the basics of this science will need to be practiced in a simple way, where you can use r programming to study statistical methods. as i mentioned earlier, r is a programming language that has been written for statistical analysis in general, and studying basic statistics through r could provide an opportunity to learn a new language and to understand the basics of statistics in the main time. this doesn't mean that you don't have to solve statistical problems by hand, but it does mean that you have to understand both ways. early publishing of students and graduates although there are concerns about early scientific publishing in the arabic education system, it is very important for bioinformatics students to publish their research, scripts, and pipelines (series-connected processing systems in which the output of one component is received from another) during their early years. some of these articles would not include much, but this is aimed at improving their interest in research. it would also give them the ability to sharpen their writing skills, address the comments of reviewers and the international research society. this would add to their computing skills in early age more specificity and professionalism. you should try to write your publications for the tools, scripts, pipelines or methods you have developed with or without other colleges and you need some guidelines on how to publish in this regard: 1) first of all, you need to know that most of the software articles are one or two pages long. it must include a brief introduction, a comprehensive methodology and some discussion. 2) don't extend your article beyond two pages, the more you do, the more unstable your sentences will be. 3) discuss the benefits of your tool, how you deal with the input, where adding a flowchart algorithm would be efficient. 4) use simple english. 5) cite previously published work by comparing your tool with others. it doesn't matter if your tool doesn't add much; the most important thing is that, it's your tool. 6) start with small publications. 7) search for free journals. in this regard, biorxiv (42) is a non-profit, electronic archiving and distribution platform for the pre-printing of life science research papers. it was founded by the cold spring harbor laboratory, a scientific and academic organization. learn new programs and use published scripts hundreds of bioinformatics tools are released daily, specialized in the analysis of different data types, and written in a variety of programming languages. in order to use these tools, you need to take two steps: (1) test the configuration of the software using sample data and (2) read tutorials (if available) describing the different parameters and input data the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 6 of 10 that this tool could handle, which would enable you to understand and analyze the outputs of these tools. using sample data and reading tutorials will save you time and provide you with a simpler way to resolve the error messages. on the other hand script archives such as githhub are the most important source for software scripts with more than 10 million repositories (43). such websites offer you the ability to access open source software, script codes and pipelines in c, python, perl, r and other programming languages freely. you may be able to use such resources to know more about coding or bypass basic programming functions by using these codes as external libraries, but ethically you need to reference the published websites, articles or acknowledge the source of these scripts in your codes. it could be difficult to use these scripts and you need advice on how to handle it: 1) begin with basic codes; you can find several tutorial codes for programming language implementations in bioinformatics, which you will find useful in sharpening your programming skills. 2) in order to understand any script, you need to divide the script parts according to what they do, use the print function to show the performance of the input processing stage after each step on the screen. 3) to understand any code structural algorithm, block, remove and replace any line debug (run by compiler) and monitor performance changes. furthermore, the key to understanding the overall algorithm could be to comprehend how to evaluate script errors when ignoring certain coding lines. 4) draw a basic flowchart that demonstrates your interpretation of how this program manages input data. start learning cloud computing the management of large biological data requires sufficient computing power and storage capacity. cloud computing can provide arab research with large capabilities via open and often free websites. some of these servers are galaxy (44) and cyverse (45). from my point of view, all these clouds and others provide computing resources and storage capacity to handle biological data, but i would prefer cyverse more because it is more robust and offers a lot of software and simple gui interfaces. using such clouds can offer you the ability to manage big information and to conquer the poor and slow speed of the internet which inhibits you from accessing those data; you can do even more than that. such as: 1) cyverse gives you the ability to email your scripts to administration to handle or convert those scripts to a tool that you can use. 2) using the tools available to build your own pipeline. 3) you can transfer or upload data from a private fttp server such as ncbi (46), ensemble (47) or from your computer. 4) choose to use the storage capacity provided by these platforms to store data instead of overfilling your computer memory. 5) such platforms have their own research communities, where you can submit your questions. life as a bioinformatician in arabic countries many arab countries do not support research (48) and fresh bioinformaticians may suffer from a lack of funding. my answer to this problem is to begin your career as a freelancer. freelance employees are terms that are commonly used by a self-employed person who is not usually a long-term employer. fresh bioinformatics graduates may offer their data analysis expertise to local and international research groups in exchange for money. in this manner, they will support their continuing self-education and their personal goals. considering that, this path needs good research knowledge, free and low-cost services and communication skills. while freelance is a good way to start your life as a bioinformatician, some considerations are required: 1) don't manipulate output reports for those who want to finish their dissertations without having worked. 2) finish the work of each researcher without doing more than is necessary and not less than is essential. 3) note that freelancing is not a type of research, and you need to write any scientific papers for yourself in order to build your own scientific background. 4) once you have a successful and well-paid position, avoid freelancing and start sharing the credit for your work. bioinformatics and communication skills communication skills for bioinformatician are more than necessary and are very important for researchers, especially when working in scientific teams. communicating with other bioinformaticians, biologists and researchers is critical to understanding, resolving and addressing daily bioinformatics issues. such challenges could be software bugs, incorrect results and experimental methodologies. in most of our arabic countries, there is a weak link between different scientific groups so, for order to overcome such difficulties, arab bioinformaticians could use research communities to interact with each other and with international scientific teams. there are various advanced research, e.g. ask ubuntu (49), bioinformatics (50) and researchgate (51). such groups will help young scientists start their careers and find new bioinformatics problems that they could address. the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 7 of 10 the future for you following basic learning, one or two programming languages and the handling of different biological data through a variety of analytical techniques, the expected next phases are as follows: 1) learn c programming language; c is faster and consumes less memory than other programming languages. although learning c is difficult, most famous bioinformatics programs such as the blast (52) software package are written in c. the problem is that python, perl and r are slower than c when handling large data, and then when performing complicated processes and memory management are needed (53). you will need to write some of your codes in c in the future to make your program faster and more efficient, even if these scripts need more lines to express. like c, fortran is a very important programming language, particularly when it comes to statistical analysis, and some may argue that it is faster than c (54). 2) boost your understanding of shell scripting. shell is the programming language used by the linux system (55), improving your reading and writing skills in such a language will enhance the management of complex tasks. in some cases, you can create hybrid scripts where you compose an algorithm to manipulate data throughout different computer programming languages such as python, c, julia and r, in addition allowing you the flexibility to use what the linux system could provide. 3) don't repeat your work; try to handle different types of data and use different methods of analysis to gain more knowledge and experience. 4) machine learning (ml) is a natural outgrowth of a combination of computer science and statistics and answers the question of how to build machines that automatically learn from experience (56,57). understanding ml is very essential for your career and could aid your scientific background. python, r, java and c have specific libraries for ml, but python has the maximum attention (58). 5) begin the web programming learning process. internet development is essential in order to make massive, definitive data available to the scientific community. in fact, you could create your own online software that would make it easier for scientists with poor programming skills to use. python has its own web programming packages, such django. mental and social life of arabic bioinformatician research life is demanding, frustrating and highly competitive. where postgraduates need high marks for their courses and a variety of publications under their belts if they want to earn excellent scholarships and research positions (59). it is important to take part in activities and social structures in order to promote the academic and social growth. many current students in bioinformatics have been prepared to learn new research methodologies and programming languages and to ignore social life. sadly, this kind of behavior is very risky with several articles on psychological science relating mental illness with creativity (60). as a bioinformatician, you need to socialize with others and participate in outdoor activities to boost self-efficiency and promote team harmony. these type of activities are highly recommended in the field of mental health through a number of scientific articles (61). in fact, physical activity has been shown to be related to mental health and can play a key role in sustaining moderate to severe mental health conditions, especially anxiety (62). first of all, there is a high rate of depression and work stress, especially for those working as researchers in developing countries who need to maintain their mental health in order to thrive and thrive. conclusion bioinformatician is a person who has the ability to take advantage of three different sciences, to think innovative and to reshape complexity into simplicity. his capabilities rely on a sound scientific experience, a high level of knowledge in addition to patience, enthusiasm and productivity. our arabic world needs this kind of expertise in translating biological knowledge into practice, preserving our natural ecological resources and boosting our standard of living. as citizens, we can not change the education system, the economic climate or our society, but as researchers, we can inform others how to deal with these conditions and achieve their goals with less sacrifice as possible. the secret of learning bioinformatics relies basically on the willingness of our students and researchers to acquire new sciences. bioinformatics as a discipline is the most attractive and interesting field in which lack of resources, materials and equipment is not a concern, but requires additional knowledge and a high level of creativity that seems as easy to learn as it is difficult to obtain. my final advice is to be guided and to lead, both procedures could fill the gap in your scientific background, where opening your mind to new ideas would result in a better way, and deeper understanding and interaction with others would offer you multiple choices. acknowledgement the author would like to thank dr. morad mokhtar (molecular genetics and genome mapping lab agricultural the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 8 of 10 genetic engineering research institute, arc, egypt) and dr. abdulqader jighly (department of economic development, jobs transport and resources australia) for their valuable support in this research. supplementary files the supplementary material for this article can be found online at: http://supplementary.highlightsin.org/20193_sup.zip supplementary file 1: articles and reviews on bioinformatics could be used to initiate the learning process in this field. supplementary file 2: short tutorial for some bio-linux basics. references 1. welch l, lewitter f, schwartz r, brooksbank c, radivojac p, gaeta b, et al. bioinformatics curriculum guidelines: toward a definition of core competencies. plos comput biol. 2014;10(3):e1003496. 2. bansal ak. bioinformatics in microbial biotechnology-a mini review. microb cell fact. 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science (80). 2015;349(6245):255–60. 58. müller ac, guido s, others. introduction to machine learning with python: a guide for data scientists. ― o’reilly media, inc.; 2016. 59. ortega a. three tips to achieve academic success — while enjoying a social life. nature. 2019 60. nettle d. schizotypy and mental health amongst poets, visual artists, and mathematicians. j res pers. 2006;40(6):876–90. 61. fernee cr, gabrielsen le, andersen ajw, mesel t. therapy in the open air: introducing wilderness therapy to adolescent mental health services in scandinavia. scand psychol. 2015;2. 62. paluska sa, schwenk tl. physical activity and mental health. sport med. 2000;29(3):167–80. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202106 research article open access 1 genetics and plant improvement team, biosciences laboratory, doctoral school of sciences and technologies, joseph ki-zerbo university, 03 bp 7021 ouagadougou 03, burkina faso. 2 chadian institute of agronomic research for development (itrad), b.p. 5400, n’djaména, chad. 3 plant ecophysiology team, biosciences laboratory, doctoral school of sciences and technologies, joseph kizerbo university, 03 bp 7021 ouagadougou 03, burkina faso. 4 ziniaré university center, joseph kizerbo university, 03 bp 7021 ouagadougou 03, burkina faso. contacts of authors * to whom correspondence should be addressed: nerbewende@yahoo.fr received: january 2, 2021 accepted: march 4, 2021 published: march 18, 2021 citation: tiendrébéogo kf, sawadogo n, gapili n, ouédraogo mh, ouédraogo rf, nanema kr, ouoba a, sawadogo m . variability and relationships between characters of physic nut (jatropha curcas l.) in burkina faso . 2021 mar 18;4:bs202106 copyright: © 2021 tiendrébéogo et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. variability and relationships between characters of physic nut (jatropha curcas l.) in burkina faso kouka fidèle tiendrébéogo1, nerbéwendé sawadogo*1, naoura gapili2, mahamadi hamed ouédraogo1, razacswendé fanta ouédraogo3, kiswendsida romaric nanema1, adjima ouoba4, mahamadou sawadogo1 abstract jatropha curcas is a highly promising species for biodiesel production in burkina faso and other countries in the tropics. it is rustic, grows in warm regions and is easily cultivated. these characteristics and high-quality oil yields from the seeds have made it a priority for biodiesel programs. consequently, this plant merits genetic investigations aimed at improving yields. the present study was conducted to determine the extent of genetic variability and relationships among 30 accessions from burkina faso using 7 qualitative characters and 20 quantitative characters. for most of the traits, there were highly significant differences among the accessions. a positive and significant correlation between the 100-seed weight trait and the oil content trait was observed. the oil content trait is significantly and positively influenced by growth traits such as plant height and crown diameter. based on the petiole base pigmentation, three morphotypes were identified: green morphotype, purple morphotype and brown morphotype. the green morphotype was characterized by very high oil content and high 100-seed weight while the brown morphotype presents low oil content and low 100-seed weight. the purple morphotype registered a high oil content and medium 100-seed weight. these results are important for the continuity of breeding programs, aimed at obtaining cultivars with high grain yield and high oil content in seeds. keywords: oil content, genetic diversity, genetic correlation, morphotype, jatropha curcas, burkina faso. introduction physic nut (jaropha curcas l.) is a perennial oil plant commonly used in tropical areas as a medicinal plant, in the construction of defensive hedges against animals and in the fight against water erosion [1]. it is rustic, grows in warm regions and is easily cultivated. these characteristics and high-quality oil yields from the seeds have made this plant a priority and a highly promising species for biodiesel programs in countries in the tropics [2]. it produces seeds rich in oil which can be used pure after filtration as fuel in diesel engines with indirect injection or serve as raw material for the production of biodiesel by transesterification [3, 4]. [5] highlighted the economic interest of the use of oil of this species for the poor countries, in particular tropical africa and asia. indeed, j. curcas provides various products that contribute to poverty reduction, in particular the promotion of income-generating activities mainly for women such as sale of seeds and soap and the valuation of oil cakes as organic fertilizers. it is an opportunity for developing countries to improve farmers' incomes and even stimulate the rural economy [6]. however, in most countries of sub-saharan africa, the establishment of plantations preceded the conduct of agronomic research essential for the sustainable exploitation of the plant. as a result, plant development has been poor and yields obtained during the first years of cultivation have generally been disappointing [7]. consequently, this species merits genetic investigations aimed at improving yields [2]. so, the viability of the j. curcas-based biodiesel sector is essentially based on highly productive and oil-rich j. curcas genotypes [6]. highlights in bioscience page 1 of 11 march 2021|volume 4 https://doi.org/10.36462/h.biosci.202106 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-0448-4239 https://orcid.org/0000-0003-0136-8408 https://orcid.org/0000-0001-6415-8745 https://orcid.org/0000-0002-3037-8624 https://orcid.org/0000-0002-4241-4696 https://orcid.org/0000-0001-8913-1012 https://orcid.org/0000-0001-6456-2297 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) despite strong recommendations for improvement of j. curcas for intensive production, a very little references exist on the genetic characteristics of the plant, the level of productivity of j. curcas in relation to its genetic potential and the cultivation techniques [6]. indeed, j. curcas is still essentially a wild plant which must be the object of genetic improvement and domestication for its popularization and vaolrization [8, 9]. in burkina faso, the genetic diversity of the species remains still poorly known [10-13]. thus, within the framework of the promotion of the neglected or under-exploited local species, this study was initiated in order to contribute to a better knowledge the genetic diversity and the relationships between agro-morphological traits and seeds oil content of j. curcas. material and methods plant material the plant material consisted of 30 elite accessions selected by [11] based on the morpho-metric characteristics of the fruits as well as the oil content of the seeds. in this study, an accession is a set of seeds, from natural pollination, harvested from the same tree during prospecting. the seeds were stored at laboratory temperature without any prior chemical treatment. experimental site the agro-morphological characterization was carried out on a plantation installed in the experimental station of the institute for rural development (idr) of gampela at 1°21'0.9'' west longitude, 12°24'10.7'' north latitude and 924 m altitude. the station is characterized by very heterogeneous, deep soils, of low physicochemical fertility and a predominantly sandy-clay texture [14]. the climate of the zone is of the sudano-sahelian type characterized by the alternation of two seasons, namely a rainy season which extends from june to october and a dry season from november to may [15]. the annual rainfall recorded in the station during the experimentation varied between 728 and 984.8 mm. average air temperatures during the rainy months range between 35° c and 40° c for the maximums and between 18° c and 19° c for the minimums [15]. experimental design the experimental design used is a completely random experiment plan consisting of 30 elementary plots of 8 m x 8 m. each elementary plot contains 9 plants arranged in three lines of 8 m in length each. the spacing between the lines was 4 m. each line includes three plants with a spacing of 4 m. the aisles between the elementary plots were also 4 m. characters studied qualitative characters based on the work of [16], seven qualitative variables were chosen to characterize the phenotypic variability of the accessions studied. the table 1 presents the different characters with their modalities. table 1. modalities of qualitative traits. variables modalities brancing pattern basal intermdiate top entire petiole base pigmentation green brown purple stem colour green grey leaf colour green light green dark green latex colour cream red phyllotaxy alternate whorled growth habit shurb (< 5 m) tree (> 5m) quantitative characters based on the work of [17, 2, 18, 16, 6], 20 quantitative variables were chosen to characterize the genetic diversity of j. curcas five years after planting. these parameters were measured on three plants sampled by accession. these are first of all the parameters linked to the vegetative development of the trees, measured three months after the appearance of the first leaves. those are: • the dendrometric characters of trees such as plant height (ph) measured from the base to the apex of the main stem, crown diameter (cd) measured between both ends of the plant, stem diameter (sd) measured at the collar using a tape measure (sd = collar circumference / π) and number of main branches (nb) evaluated by counting the branches coming out from the trunk . • the characters linked to the dimensions of the leaves measured on three fresh leaves, fully developed and not parasitized, per individual: these were the width of the leaf (wl) measured between the two ends of the leaf, the length of the leaf (ll) measured from the petiole to the tip of the leaf and the length of the petiole (lp) measured from the insertion of the stem to the insertion of the leaf. the traits linked to the productivity of the accessions such as fruits weight (frw), seeds weight (sew) and pulps weight (puw) per accession were also evaluated at the ripe and dry fruit stage using an electric scale. moreover, others traits relative to the fruits were measured. they are the average length of the fruit (alf), the average diameter of the fruit (adf) evaluated using a digital caliper, the average weight of the fruit (awf) and the average weight of the pulp (awp) determined using an electric highlights in bioscience page 2 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) scale on ten fruits. the average number of seeds per fruit (ans) was determined by counting. as concerning the seeds, the characters measured were the 100-seed weight (sw100) determined by weighing a random sample of 100 seeds of each mother plant using an electric scale, the average length of the seed (als), the average width of the seed (aws) and the average thickness of the seed (ats) measured with a digital caliper on ten seeds. the seeds oil content (oil) was determined by the soxhlet extraction method using hexane as the solvent [19]. [16] proposed a descriptor with four classes based on the seeds oil content character in relation with the weight of the seed: low (0-20%), medium (21-30%), high (31-40%) and very high (> 40%). data analysis the data collected were analyzed with xlstat 2020.3.1.11 software. analysis of variance (anova) and of student newman keuls means separation test at α=5% were carried out in order to compare accessions. pearson’s r coefficient was used to measure correlations between quantitative traits while the relationships between discriminate qualitative and quantitative traits were determined through multiple correspondence analysis (mca). indeed, the quantitative traits have been previously transformed into qualitative traits according to the classes obtained in the student newman keuls means separation test. results analysis of the phenotypic variability of j. curcas using qualitative variables the results recorded in table 2 showed that all the accessions studied had green stems and green leaves, a cream-colored latex and alternate leaves. however, two characters presented variability. thus, the majority of plants (70%) had a basal branching pattern while a minority (30%) revealed an intermediate branching pattern (figure 1). the petiole base expressed several pigmentation color (figure 2): green (33.33%), brown (13.33%) and mostly purple (53.34%). analysis of the phenotypic variability of j. curcas using quantitative variables the results of the significance of the means separation test of newman keuls were performed with vegetative development parameters (table 3), fruit parameters (table 4) and seeds parameters (table 5). most of the vegetative development parameters and seeds traits significantly discriminate the accessions studied except the length of the petiole (lp), the average length of the seed (als) and seeds weight per accession (sew). for the fruit traits, only three of the seven measured characters, precisely the average length of the fruit (alf), the average weight of the fruit (awf) and the average weight of the pulp (awp) showed variability. based on the each of three characters of interest namely oil content (oil), 100-seed weight (sw100) and crown diameter (cd), three classes were obtained within accessions. indeed, according to the value of the character, three performance classes table 2. variation in the qualitative characters of the collection of j. curcas. variables modalities frequencies (%) brancing pattern basal 70 intermdiate 30 top 0 entire 0 petiole base pigmentation green 33.33 brown 13.33 purple 53.34 stem colour green 100 grey 0 leaf colour green 100 light green 0 dark green 0 latex colour cream 100 red 0 phyllotaxy alternate 100 whorled 0 growth habit shurb (< 5 m) 100 tree (> 5m) 0 (low, medium, high) were registered with the 100-seed weight trait and crown diameter, respectively. the seeds oil content character showed also three performance classes which ranged medium, high and very high according to the descriptor classes. relationships between characters correlation between characters the correlation of pearson (table 6) showed positive and significant correlations at the 5% and 1% threshold between the characters studied. thus, the character oil content was positively correlated with the characters 100-seed weight (r = 0.253), plant height (r = 0.344) and crown diameter (r = 0.260). the 100seed weight character was positively correlated with the characters linked to vegetative development such as plant height (r = 0.522), crown diameter (r = 0.443) and length of the petiole (r = 0.272). furthermore, the crown diameter character is positively correlated with all the other characters studied. these different correlations indicate that plants with significant vegetative development produce seeds of high weight and high oil content. the characters linked to the yield (frw, sew and puw) are positively correlated with the characters relating to the scale of the plant (ph, sd and cd). so, large-scale plants have also high productive potential. furthermore, the characters of the seeds (als, aws and ats) are positively influenced each other. association between characters the results of multiple correspondence analysis (mca) recorded in figure 3 showed three associations of the characters. on the plan formed by axes 1 and 2 with 45.41% of the total inertia, the f1 axis (23.14% of total inertia) opposed two groups of variables precisely group 1 and group 3. group 1 combined the characters very high seed oil content (> 40%), intermediate highlights in bioscience page 3 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) figure 1. branching pattern of j. curcas. a: basal (70%), b: intermediate (30%). figure 2. petiole base pigmentation of j. curcas a: green (33.33%), b: brown (13.33%), c: purple (53.34%) highlights in bioscience page 4 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) table 3. results of newman keuls'means separation test on vegetative development parameters. accession ph (m) sd (cm) cd (m) nb wl (cm) ll (cm) lp (cm) 1 2.450 bcde 9.076 b 2.650 abc 4.000 b 16.295 abc 14.365 abc 20.165 a 2 1.977 cde 15.711 ab 2.733 abc 6.333 ab 15.130 abc 14.127 abc 17.197 a 3 2.600 bcd 12.527 ab 3.100 abc 5.667 ab 15.040 bc 13.477 abc 17.007 a 4 2.747 b 13.896 ab 3.717 ab 6.000 ab 17.207 abc 15.497 abc 18.997 a 5 2.490 bcde 15.977 ab 3.467 abc 6.000 ab 14.410 c 13.930 abc 17.167 a 6 2.150 bcde 13.694 ab 3.350 abc 5.500 ab 15.545 abc 13.545 abc 18.430 a 7 2.033 bcde 13.694 ab 3.000 abc 5.000 ab 17.477 abc 14.720 abc 16.983 a 8 2.567 bcd 13.907 ab 3.133 abc 5.000 ab 18.850 ab 16.687 a 18.430 a 9 2.567 bcd 14.756 ab 3.150 abc 5.667 ab 18.463 ab 15.710 abc 19.820 a 10 2.383 bcde 12.909 ab 3.000 abc 5.667 ab 16.597 abc 14.797 abc 16.943 a 11 2.017 bcde 12.707 ab 2.857 abc 7.667 ab 16.333 abc 14.043 abc 16.863 a 12 3.225 a 13.854 ab 3.500 abc 9.000 a 18.415 ab 15.930 ab 19.195 a 13 2.100 bcde 12.739 ab 2.800 abc 5.000 ab 14.895 bc 12.460 c 14.945 a 14 2.233 bcde 14.862 ab 3.150 abc 6.000 ab 17.283 abc 14.997 abc 16.710 a 15 2.017 bcde 15.287 ab 2.733 abc 8.000 ab 16.763 abc 14.517 abc 15.467 a 16 2.047 bcde 15.287 ab 2.900 abc 5.333 ab 16.177 abc 13.410 abc 16.310 a 17 2.227 bcde 13.163 ab 3.167 abc 4.667 ab 16.553 abc 14.483 abc 18.073 a 18 2.275 bcde 15.287 ab 3.450 abc 8.000 ab 16.350 abc 15.015 abc 18.795 a 19 2.683 bc 13.376 ab 4.000 a 6.333 ab 18.997 a 16.630 a 19.553 a 20 2.340 bcde 13.641 ab 3.400 abc 4.333 ab 16.693 abc 14.753 abc 18.310 a 21 2.225 bcde 13.694 ab 2.900 abc 9.000 a 17.545 abc 14.910 abc 18.130 a 22 2.417 bcde 12.781 ab 2.867 abc 7.000 ab 16.910 abc 14.340 abc 17.517 a 23 1.915 de 10.987 ab 2.650 abc 4.000 b 15.530 abc 12.900 bc 13.995 a 24 1.900 de 14.411 ab 2.635 abc 7.000 ab 15.965 abc 14.130 abc 18.365 a 25 1.770 e 10.403 ab 2.100 c 5.667 ab 15.387 abc 12.683 bc 15.297 a 26 2.267 bcde 10.032 ab 2.600 abc 3.667 b 17.940 abc 15.440 abc 17.097 a 27 1.857 de 12.951 ab 2.400 bc 5.667 ab 14.953 bc 12.730 bc 14.617 a 28 2.183 bcde 16.561 ab 2.767 abc 7.000 ab 16.217 abc 13.463 abc 16.410 a 29 1.880 de 17.304 a 2.767 abc 6.000 ab 15.573 abc 13.797 abc 15.750 a 30 1.900 de 11.996 ab 2.700 abc 4.333 ab 16.420 abc 14.697 abc 16.017 a ph: plant height, sd: stem diameter, cd: crown diameter, nb: number of main branches, wl: width of the leaf, ll: length of the leaf, lp: length of the petiole. the values for each class followed by the same letters are not significantly different at the 5% level. branching of the stem, green pigmentation of the petiole base, high 100-seed weight (≥ 62.85 g) and large crown diameter (≥ 4 m) while the group 3 is formed by association between the medium oil content (21-30%), the brown pigmentation of the petiole base and the low 100-seed weight (≤ 36.9 g). group 2, correlated to axis f2 with 22.127% of total inertia, combined the high oil content (31-40%), the basal branching of the stem, the purple pigmentation of the petiole base, the medium 100-seed weight (43.30-43.85 g), the medium crown diameter (2.4-3.467 m) and the small crown diameter (≤ 2.1 m). highlights in bioscience page 5 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) table 4. results of newman keuls'means separation test on fruits parameters. accession alf (cm) adf (cm) awf (g) awp (g) ans frw (g) puw (g) 1 2.533 ab 2.110 a 2.610 ab 0.940 ab 2.550 a 158.400 a 49.900 a 2 2.265 ab 2.058 a 1.843 ab 0.790 abcd 2.500 a 207.233 a 94.867 a 3 2.425 ab 2.120 a 2.623 ab 0.920 abc 2.700 a 263.167 a 93.567 a 4 2.457 ab 2.080 a 2.460 ab 0.873 abc 2.800 a 509.200 a 189.900 a 5 2.340 ab 2.053 a 2.430 ab 0.837 abcd 2.833 a 541.667 a 211.600 a 6 2.468 ab 2.083 a 2.710 ab 1.005 a 2.950 a 816.250 a 317.450 a 7 2.383 ab 2.090 a 1.840 ab 0.670 bcd 2.200 a 136.967 a 56.733 a 8 2.478 ab 2.158 a 2.120 ab 0.757 abcd 2.467 a 163.767 a 68.467 a 9 2.535 ab 2.100 a 2.277 ab 0.837 abcd 2.667 a 463.867 a 210.133 a 10 2.425 ab 2.117 a 2.143 ab 0.720 abcd 2.500 a 249.433 a 100.367 a 11 2.402 ab 2.067 a 2.300 ab 0.930 abc 2.633 a 248.233 a 114.567 a 12 2.430 ab 2.140 a 2.500 ab 0.830 abcd 2.750 a 453.500 a 168.300 a 13 2.498 ab 2.035 a 2.175 ab 0.895 abc 2.850 a 641.400 a 256.050 a 14 2.483 ab 2.078 a 2.133 ab 0.880 abc 2.567 a 433.867 a 172.033 a 15 2.130 b 1.998 a 1.417 b 0.547 d 2.367 a 174.767 a 79.533 a 16 2.567 a 2.158 a 2.773 a 0.877 abc 2.967 a 496.000 a 190.900 a 17 2.425 ab 2.045 a 2.120 ab 0.750 abcd 2.600 a 384.400 a 161.667 a 18 2.593 a 2.145 a 2.530 ab 0.770 abcd 2.850 a 834.300 a 277.150 a 19 2.395 ab 2.093 a 2.493 ab 0.750 abcd 2.967 a 774.267 a 287.667 a 20 2.397 ab 2.078 a 2.380 ab 0.750 abcd 2.833 a 836.400 a 323.800 a 21 2.420 ab 2.073 a 2.115 ab 0.660 bcd 2.750 a 158.850 a 59.700 a 22 2.393 ab 2.112 a 1.940 ab 0.733 abcd 2.533 a 162.800 a 73.567 a 23 2.460 ab 2.088 a 2.015 ab 0.670 bcd 2.600 a 99.600 a 39.100 a 24 2.330 ab 1.940 a 1.770 ab 0.607 cd 2.230 a 86.400 a 37.350 a 25 2.205 ab 1.927 a 1.627 ab 0.608 cd 2.420 a 89.200 a 39.600 a 26 2.380 ab 2.083 a 1.797 ab 0.713 abcd 2.133 a 64,933 a 29.600 a 27 2.212 ab 1.978 a 1.537 ab 0.637 bcd 2.200 a 97.867 a 49.033 a 28 2.368 ab 2.088 a 1.830 ab 0.833 abcd 2.333 a 147.533 a 71.133 a 29 2.245 ab 1.945 a 1.800 ab 0.870 abc 2.100 a 89.300 a 40.000 a 30 2.192 ab 2.043 a 1.607 ab 0.843 abcd 2.067 a 78.100 a 43.933 a alf: average length of the fruit, adf: average diameter of the fruit, awf: average weight of the fruit, awp: average weight of the pulp, ans: average number of the seeds per fruit, frw: fruits weight per accession, puw: pulps weight per accession. the values for each class followed by the same letters are not significantly different at the 5% level. highlights in bioscience page 6 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) table 5. results of newman keuls'means separation test on seeds parameters. accession als (cm) aws (cm) ats (cm) sw100 (g) sew (g) oil (%) 1 1.830 a 1.120 ab 0.863 ab 56.100 ab 108.500 a 33.811 i 2 1.755 a 1.092 ab 0.835 ab 47.833 abc 112.367 a 31.160 lm 3 1.803 a 1.150 ab 0.868 a 63.350 a 169.600 a 35.897 g 4 1.833 a 1.113 ab 0.855 ab 62.850 a 319.300 a 34.663 h 5 1.753 a 1.068 b 0.818 ab 55.833 ab 330.067 a 39.286 c 6 1.845 a 1.173 a 0.853 ab 61.100 ab 498.800 a 31.309 l 7 1.813 a 1.092 ab 0.847 ab 55.600 ab 80.233 a 35.023 h 8 1.833 a 1.123 ab 0.865 ab 52.900 abc 95.300 a 32.874 j 9 1.890 a 1.068 b 0.835 ab 53.300 abc 253.733 a 37.931 e 10 1.827 a 1.098 ab 0.873 a 54.933 ab 149.067 a 35.474 g 11 1.753 a 1.130 ab 0.850 ab 54.450 ab 133.667 a 37.989 e 12 1.780 a 1.073 b 0.845 ab 63.700 a 285.200 a 37.931 e 13 1.753 a 1.065 b 0.813 ab 43.850 bc 385.350 a 32.360 k 14 1.868 a 1.060 b 0.835 ab 48.500 abc 261.833 a 40.383 b 15 1.688 a 1.063 b 0.813 ab 45.900 abc 95.233 a 31.331 l 16 1.840 a 1.118 ab 0.852 ab 58.333 ab 305.100 a 29.366 o 17 1.792 a 1.107 ab 0.825 ab 55.233 ab 222.733 a 31.577 l 18 1.813 a 1.120 ab 0.870 a 64.300 a 557.150 a 32.486 jk 19 1.832 a 1.152 ab 0.857 ab 59.200 ab 486.600 a 40.863 a 20 1.765 a 1.125 ab 0.832 ab 63.250 a 512.600 a 34.726 h 21 1.840 a 1.118 ab 0.853 ab 52.450 abc 99.150 a 30.800 mn 22 1.805 a 1.065 b 0.832 ab 64.000 a 89.233 a 32.463 jk 23 1.808 a 1.088 ab 0.828 ab 54.400 ab 60.500 a 32.760 jk 24 1.740 a 1.090 ab 0.868 ab 43.300 bc 49.050 a 30.474 n 25 1.663 a 1.070 b 0.800 b 50.200 abc 49.600 a 34.040 i 26 1.780 a 1.113 ab 0.847 ab 53.200 abc 35.333 a 36.909 f 27 1.707 a 1.065 b 0.817 ab 51.600 abc 48.833 a 38.640 d 28 1.782 a 1.060 b 0.832 ab 43.500 bc 76.400 a 31.617 l 29 1.672 a 1.067 b 0.837 ab 47.100 abc 49.300 a 35.549 g 30 1.687 a 1.112 ab 0.853 ab 36.900 c 34.167 a 28.137 p als: average length of the seed, aws: average width of the seed, ats: average thickness of the seed, sw100: 100-seed weight, sew: seeds weight per accession, oil: seeds oil content. the values for each class followed by the same letters are not significantly different at the 5% level. highlights in bioscience page 7 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) ta bl e 6. m at ri x of co rr el at io ns be tw ee n th e st ud ie d ch ar ac te rs . v ar ia bl es a l f a d f a w f a w p a n s a l s a w s a t s s w 10 0 o il p h s d c d n b w l l l l p f r w s e w a d f 0. 78 7* * a w f 0. 74 5* * 0. 62 7* * a w p 0. 58 8* * 0. 49 3* * 0. 70 6* * a n s 0. 59 8* * 0. 55 0* * 0. 83 6* * 0. 46 9* * a l s 0. 86 0* * 0. 78 7* * 0. 61 3* * 0. 43 3* * 0. 50 9* * a w s 0. 39 1* * 0. 46 3* * 0. 52 0* * 0. 35 9* * 0. 41 8* * 0. 43 2* * a t s 0. 42 3* * 0. 40 2* * 0. 39 2* * 0. 31 6* * 0. 04 1 0. 47 0* * 0. 52 1* * s w 10 0 0. 39 2* * 0. 35 0* * 0. 57 0* * 0. 19 1 0. 42 8* * 0. 32 4* * 0. 32 0* * 0. 22 6* o il 0. 07 6 0. 01 2 0. 15 5 0. 08 9 0. 10 0 0. 14 0 -0 .0 62 -0 .0 53 0. 25 3* p h 0. 41 8* * 0. 41 3* * 0. 49 9* * 0. 27 7* 0. 38 7* * 0. 45 9* * 0. 18 6 0. 32 6* * 0. 52 2* * 0. 34 4* * s d 0. 06 7 0. 09 7 0. 10 2 0. 19 4 0. 07 0 0. 05 9 -0 .1 75 0. 11 6 0. 05 7 -0 .0 19 0. 13 3 c d 0. 38 7* * 0. 42 8* * 0. 55 2* * 0. 37 0* * 0. 50 1* * 0. 40 2* * 0. 31 2* * 0. 30 1* * 0. 44 3* * 0. 26 0* 0. 65 1* * 0. 46 0* * n b -0 .0 10 -0 .0 18 0. 03 8 0. 01 6 0. 09 3 0. 00 1 -0 .1 05 0. 12 5 0. 02 4 0. 02 5 0. 18 9 0. 37 9* * 0. 22 5* w l 0. 14 3 0. 16 0 0. 00 2 -0 .1 71 -0 .0 27 0. 26 0* 0. 02 9 0. 26 1* 0. 04 7 0. 17 2 0. 42 9* * 0. 03 9 0. 31 7* * 0. 02 2 l l 0. 13 3 0. 20 8 0. 08 8 -0 .0 67 0. 04 7 0. 26 5* 0. 11 6 0. 35 5* * 0. 09 5 0. 21 0 0. 54 6* * 0. 15 9 0. 45 5* * 0. 08 9 0. 84 3* * l p 0. 18 3 0. 10 8 0. 26 6* 0. 06 1 0. 22 1* 0. 26 5* 0. 20 8 0. 23 4* 0. 27 2* 0. 10 8 0. 55 0* * 0. 09 6 0. 39 4* * 0. 06 9 0. 53 0* * 0. 64 1* * f r w 0. 42 6* * 0. 32 5* * 0. 52 4* * 0. 34 5* * 0. 55 6* * 0. 35 0* * 0. 25 8* 0. 13 4 0. 41 0* * 0. 14 9 0. 46 4* * 0. 30 8* * 0. 67 5* * 0. 14 1 0. 03 9 0. 16 8 0. 29 3* * s e w 0. 42 4* * 0. 31 8* * 0. 52 7* * 0. 33 2* * 0. 55 4* * 0. 34 4* * 0. 26 9* 0. 14 7 0. 42 7* * 0. 14 9 0. 47 3* * 0. 28 6* * 0. 66 7* * 0. 13 6 0. 04 5 0. 17 5 0. 30 7* * 0. 99 7* * p u w 0. 42 5* * 0. 33 3* * 0. 51 2* * 0. 36 3* * 0. 55 4* * 0. 35 6* * 0. 23 5* 0. 11 1 0. 37 5* * 0. 14 8 0. 44 3* * 0. 33 9* * 0. 68 0* * 0. 14 6 0. 02 7 0. 15 4 0. 26 6* 0. 99 2* * 0. 98 ** a l f :a ve ra ge le ng th of th e fr ui t, a d f :a ve ra ge di am et er of th e fr ui t, a w f :a ve ra ge w ei gh to f th e fr ui t, a w p :a ve ra ge w ei gh to f th e pu lp ,a n s :a ve ra ge nu m be r of th e se ed s pe r fr ui t, a l s :a ve ra ge le ng th of th e se ed ,a w s :a ve ra ge w id th of th e se ed ,a t s :a ve ra ge th ic kn es s of th e se ed ,s w 10 0: 10 0se ed w ei gh t, o il :s ee ds oi lc on te nt ,p h :p la nt he ig ht ,s d :s te m di am et er , c d :c ro w n di am et er ,n b :n um be r of m ai n br an ch es ,w l :w id th of th e le af ,l l :l en gt h of th e le af ,l p :l en gt h of th e pe ti ol e, f r w :f ru it s w ei gh tp er ac ce ss io n, s e w :s ee ds w ei gh tp er ac ce ss io n, p u w :p ul ps w ei gh tp er ac ce ss io n, * s ig ni fi ca nt ly at 5% ,* * s ig ni fi ca nt ly at 1% . highlights in bioscience page 8 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) figure 3. association of modalities of traits in the ½ plan of the multiple correspondence analysis (mca) (45.41%) . bp: branching pattern, pbp: petiole base pigmentation, oil: oil content, sw100: 100-seed weight. discussion the agro-morphological evaluation using quantitative and qualitative characters highlighted the existence of variability for some characters. similar results for quantitative traits were obtained by [20] and [11, 12]. for qualitative characters, this variability relates to branching pattern and petiole base pigmentation. [16] reported similar results on the petiole base pigmentation. however, only two types of stem branching were observed in this study compared to four types obtained by these same authors, thus indicating that the accessions studied are less diversified for this character. no color variation was noted on the stems and leaves of the studied accessions. these results are similar to those of [17], [21] and [22] but differ from those of [16]. genetic correlations indicate genetic relations between characters. knowledge of correlations among the characters is useful in designing an effective breeding program for any crops [23]. indeed, the magnitude and direction of the genetic correlation is important in the choice of breeding methods and the formulation of strategies for simultaneous selection on multiple traits [2]. the correlations noted in this study corroborate those observed by [2] and [23]. in this study, statistically significant correlation of oil content existed with crown diameter and plant height. in addition, 100-seed weight character is significantly correlated with crown diameter, petiole length and plant height. these correlations are interesting for improvement programs because according to [24], the selection of elite accessions to reproduce for oil extraction should be oriented towards those with the best crown and seed weight traits. furthermore, [25] indicate that the quantity of seeds produced on the one hand is positively correlated with crown, and on the other hand the seed weight and the oil content are positively correlated. significant and positive correlations between the characters of the seeds would favor the simultaneous improvement of these characters through selection. thus, in the study, interesting correlations were noted between the characters linked to the weight and dimensions of the seeds. the strong positive correlation between the characters of the seeds indicates that the genes which govern these characters are probably linked or have a pleiotropic effect. similar results have been reported by [2] and [23] between 100-seed weight and the dimensions of the seed. however, the study found no significant correlation between the oil content and the dimensions of the seeds studied. similar results have been also reported by [2] and [18]. furthermore, the study showed a positive and significant correlation between the 100-seed weight trait and the seed oil content trait. [26] and [23] observed a positive and significant correlation between seed weight and oil content while [27] revealed a negative correlation between these two characters. [18] indicated that these contrasting results could be explained by the high weight of the seed coat of some accessions. according to [16], qualitative characters are morphological markers that can be used to identify lines in a relatively short time. these authors reported also that the development of descriptors on perennial plants such as j. curcas can contribute to an effective use of germplasm in the improvement programs. analysis of the association of characters revealed the existence of three morphotypes differing in the color of the pigmentation of the base of the petiole, the oil content of the seeds and 100-seed weight. [16] identified also three descriptors based on the pigmentation of the petiole base. for perennial plant such as j. curcas, these morphological descriptors could make it possible to select genotypes with high productive potential in the short term. according to [23], complex plant characters such as yield are quantitatively inherited and influenced by genetic effects, as well as by genotype and environment interaction and selection may be difficult and time consuming to improve yield directly especially for perennial crops such as j. curcas. therefore, identification and use of associations between characters are appropriate. in this study, statistically significant association of 100-seed weight existed with seed oil content. therefore, seed weight can be considered as important character for early selection of seed sources. furthermore, according to [11], the characters 100-seed weight and oil content showed the highest values of broad-sense heritability (83.23% and 99.93% respectively) and high expected genetic advance (22.63% and 19.62% respectively). the high broad-sense heritability values associated with high expected genetic advance indicate that effects of the genes are of additive type for these characters and the opportunities to improve seed weight character and oil content character through the selection. conclusion this study highlighted an important agro-morphological diversity within the accessions of j. curcas from burkina faso. the variability observed within the accessions studied and the ashighlights in bioscience page 9 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) sociations between the characters obtained constitute a database for the genetic improvement programs of j. curcas in burkina faso, in particular the development of high-yielding cultivars. a positive and significant correlation between the 100-seed weight trait and the oil content trait was noted. growth traits such as plant height and crown diameter was also significantly and positively correlated with the oil content trait. three morphotypes were identified using petiole base pigmentation which are the green morphotype with very high oil content and high 100seed weight, the purple morphotype with medium oil content and medium 100-seed weight and finally the brown morphotype with low oil content and low 100-seed weight. for perennial plant such as j. curcas, these results on relationships between characters could make it possible to select genotypes with high productive potential in the short term. acknowledgments the authors gratefully acknowledge the bioscience laboratory precisely genetic and plant breeding team (egap) of the «université joseph ki-zerbo» for the financial support of this work. they are also thankful to all team members of the bioscience laboratory for their input in this manuscript. authors’ contributions this work was carried out in collaboration between both authors. the two first authors designed the experiment and analyzed the data. both of the authors interpreted the data, read the final manuscript, and agreed with all contents. references 1. pandey vc, singh k, singh sj, kumar a, singh b, singh rp. jatropha curcas: a potential biofuel plant for sustainable environmental development. renewable and sustainable energy reviews. 2012;16(5): 2870-2883. 2. freitas rg, missio rf, matos fs, resende mdv, dias las. genetic evaluation of jatropha curcas l.: an important oilseed for biodiesel production. genetic and molecular research. 2011;10(3): 1490-1498. 3. berchmans hj and hirata s. biodiesel production from crude jatropha curcas l. seed oil with a high content of free fatty acids. bioresource technology. 2008;99(6): 1716-1721. 4. hamad b . transestérification des huiles végétales par l’éthanol en conditions douces par catalyses hétérogènes acide et basique. thèse de doctorat, université claude bernard-lyon 1, france; 2009.p.197. 5. martin g et mayeux a. réflexion sur les cultures oléagineuses énergétiques ii. le pourghère (jatropha curcas l.) : un carburant possible. oléagineux. 1984;39(5): 283-286. 6. ouattara b. etude de la diversité génétique, de la variabilité agromorphologique et éco-physiologique de jatropha curcas l. au sénégal. thèse de doctorat unique, université cheich anta diop de dakar; 2013.p.120. 7. minengu jdd, mobambo p, mergeai g. influence de l’environnement et des pratiques culturales sur la productivité de jatropha curcas l. en afrique subsaharienne (synthèse bibilographique). biotechnoogy, agronomy, society and environment. 2013;18(2): 290-300. 8. fao et fida. le jatropha, une culture bioénergétique pour les pays pauvres. rapport; 2010.p.4. 9. coulibaly nd, koné d, soro d, kouadio yj. caractérisation physiologique de dix accessions de jatropha curcas dans deux zones pédoclimatiques assez contrastées de la côte d’ivoire. european sientific journal. 2015;11(36) : 61-77. 10. ouédraogo m. etude biologique et physiologique du pourghère : jatropha curcas l. ( euphorbiaceae) en vue d’une meilleure production de carburant de substitution. thèse de doctorat, université de ouagadougou; 2000.p. 284. 11. tiendrebeogo kf, sawadogo n, nanema kr, traore re, bationokando p, ouedraogo mh and sawadogo m. evaluation of morphometric characteristics of fruits and oil content of seeds of jatropha curcas l. in burkina faso. international journal of plant, animal and environmental science. 2016a;6 (2): 145-155. 12. tiendrébéogo kf, sawadogo n, nanéma kr, traoré re, bationokando p, zongo jd and sawadogo m. evaluation de la diversité génétique du pourghère (jatropha curcas l.) au burkina faso. international journal of innovation and applied studies. 2016b;16 (1): 155-165. 13. tiendrebeogo kf, sawadogo n, ouedraogo mh, kiebre z, zida wpmsf, nanema kr, batieno tbj, traore re, bationo-kando p, zongo jd, sawadogo m. genetic diversity of jatropha curcas in burkina faso revealed by microsatellite markers. in european scientific journal, esj. 2019;15(15): 229-243. 14. bureau national des sols (bunasol). etude pédologique de la station expérimentale de gampèla, échelle 1/5000 . n° 59; 1988.p. 279. 15. guinko s. végétation de la haute-volta. thèse de doctorat, université de bordeaux iii (france); 1984.p. 394. 16. sunil n, kumar v, sujatha m, rao gr, vaprasad ks. 2013. minimal descriptors for characterization and evaluation of jatropha curcas l. germplasm for utilization in crop improvement. biomass and bioenergy. 2013;48: 239-249. 17. heller j, 1996. physic nut (jatropha curcas l.) in promoting the conservation and use of underutilized and neglected crops. international plant genetic resources institute (ipigri). 1996;1: 1-66. 18. leela t, naresh b, srikanth reddy m, madhusudhan nc, cherku pd. morphological, physicochemical and micropropagation studies in jatropha curcas l. and rapd analys of the regenerants. applied energy. 2011;88(6): 2071-2079. 19. aoac. official methods of analysis .1990; 1.p.684. 20. sunil n, sujatha m, kumar v, vanaja m, basha sd and varaprasad ks. correlating the phenotypic and molecular diversity in jatropha curcas l. biomass and bioenergy. 2010;35(3): 10851096. 21. sukarin w, yamada y and sakagushis s. characteristics of physic nut, jatropha curcas l. as a biomass crop in the tropics. japan agricultural research quarterly. 1987;20 (4): 302-303. 22. tiendrébéogo kf, sawadogo n, ouédraogo rf, ouédraogo mh, sawadogo m et zongo jd. evaluation morpho-physiologique et estimation des paramètres génétiques au stade juvénile de 30 highlights in bioscience page 10 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) accessions de pourghère (jatropha curcas l.) au burkina faso. afrique science. 2020;17(2): 202-210. 23. shabanimofrad m, rafii my, wahab mpe, biabani ar, latif ma. phenotypic, genotypic and genetic divergence found in 48 newly collected malaysian accessions of jatropha curcas l. industial and crops products. 2013;42: 543-551. 24. gbemavo cj, gandji k, gnangle cp, assogbadio ae and kakai rlg. variabilite morphologiques et conservation des morphotypes de jatropha curcas linn. (euphoriaceae) au benin. journal of agriculture and environment for international development. 2015;109(1): 55-69. 25. rao gr, korwar gr, shanker ak, ramakrishna ys. genetic associations, variability and diversity in seed characters, growth, reproductive phenology and yield in jatropha curcas l. accessions. trees. 2008;22(5): 697-709. 26. kaushik n, kumar k, kumar s, kaushikb n. genetic variability and divergence studies in seed traits and oil content of jatropha (jatropha curcas l.) accessions. biomass and bioenergy. 2007;31(7): 497-502. 27. wani ta, kitchlu s, ram g. genetic variability studies for morphological and qualitative attributes among jatropha curcas l. accessions grown under subtropical conditions of north india. south african journal of botany. 2012;79: 102-105. highlights in bioscience page 11 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ abstract introduction material and methods plant material experimental site experimental design characters studied data analysis results analysis of the phenotypic variability of j. curcas using qualitative variables analysis of the phenotypic variability of j. curcas using quantitative variables relationships between characters discussion conclusion acknowledgments authors’ contributions references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20219 highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 10 research article open access 1 department of nucleic acid and protein structure, agricultural genetic engineering research institute (ageri), arc, giza, egypt 2 department of genetics, faculty of agriculture, cairo university, giza, egypt 3 department of microbiology, faculty of science, ain shams university, 11566, abbassia, cairo, egypt contacts of authors * to whom correspondence should be addressed: ghada abu el-heba citation: hassan a., abdallah a., a-bouzeid m. and abu el-heba g. (2020). blkn, a novel medicago truncatula mutant achieving black nodule phenotype. highlights in bioscience volume 3. article id 20219. dio:10.36462/ h.biosci.20219 received: july 14, 2020 accepted: august 27, 2020 published: september 10, 2020 copyright: © 2020 hassan et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. blkn, a novel medicago truncatula mutant achieving black nodule phenotype asmaa hassan 1 , naglaa abdallah 2 , mohamed a-bouzeid 3 and ghada abu el-heba 1 * abstract blkn is a medicago truncatula mutant that is achieving null function-black nodule phenotype. blkn is a tnt1-retrotransposon mutant, tnt1 is nicotiana tabacum retro-transposon which is replicated via rna copy and integrated in plant genome. interestingly, blkn exhibited double contents of phenolic compounds comparing to r108 wild type. the mutated black nodule is displaying cells abnormality in both infection and nitrogen fixation zones. transverse section of blkn nodule doesn’t display clearly characteristic shape like the control and the symbiotic cells don't totally filled with bacteroids along with high lignification at the cell wall periphery. our goal was blkn mutant; phenotype, physiological, and molecular characterizations. aflp-based pcr method was used to detect the mutated gene(s) in this mutant line. about 25 tnt1-tagged fragments ranging from ~100 to ~500 bp were isolated, sequenced and submitted to genbank. the tnt1 insertion was precisely located next to the base number 303 post atg start codon of m. truncatula l-type lectin-domain receptor kinase vii.2 gene encodes lectin_legb receptor like kinase (mtlectinrlk). mtlectinrlk contains lectin_legb domain, two transmembrane helix (tmhilex) and an extracellular receptor protein kinase (pkinase). mtlectinrlk is an ancestry related to probable l-type lectin-domain containing receptor kinase cicer arietinum, trifolium pretense, phaseolus vulgaris, vigna radiate and glycine soja. keywords: blkn mutant, medicago truncatula, lectin-domain, receptor kinase, tnt1 retrotransposon, fsts, aflp. introduction legumes are considered as one of the main plant families for thousands of years. their seeds contain large amount of protein therefore they are a vital plant protein supplier in food pyramid for human nutrition, animal feed and as a raw matter for industry [1]. additionally, they are essential in agriculture as they can grow in limited nitrogen environment with no addition of organic or inorganic fertilizers. legumes family has the ability to convert atmospheric nitrogen gas to ammonia throughout symbiotic association with soil bacteria commonly called rhizobia. soil nitrogen fixing bacteria such as mesorhizobium, sinorhizobium, bradyrhizobium, rhizobium, and azorhizobium have many common features such as; the rode shape, gram negative and non sporulated bacteria. farming such biological nitrogen fixation family reduces expansive usage of fertilizers and hence decreasing environmental related pollution. the relation between legumes and rhizobia is beneficial symbiotic relationship. https://creativecommons.org/licenses/by/4.0/ hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 10 the plant provides micro-aerobic condition and energy supplier to bacteria, and then in return it obtains organic nitrogen compounds [2,3]. when nitrogen becomes limiting for growth, legume plant roots produce flavonoid compounds in the rhizosphere which are considered as signals to the compatible bacteria. rhizobia reply to these compounds by secreting a lipochitin oligosaccharide called nod factor that activates several early steps in root hair infection progression. then plant percepts this specific node factor inducing alterations in the gene expression. additionally, perception results in physical changes as plant root hair curling to quester the bacteria. bacteroid is the form of bacteria which is considered as the function form that responsible for biological nitrogen fixation [3-6]. total reprogramming in the gene expression appears in the two organisms during the nodule development and nitrogen fixation. unfortunately, most of cultivated crucial legumes are weak model system for genetic study because of their large genome, difficult transformation, being out crossing and having polyploidy. thus medicago truncatula is considered as legume model plant which has many features to study the symbiosis mechanism between plant and bacteria for nitrogen fixation as it has diploid genome, produce numerous seeds, short life cycle and self-fertile plant [6,7]. wide ranges of mutant collections have been developed from this plant by using many genetic tools such as t-dna insertions, transposon, ionizing radiation, fast neutron bombardment and chemical mutagens [3,8]. the most effective transposon used for insertion mutagenesis in medicago truncatula is tobacco retrotransposon tnt1 [9]. tnt1 is retro-transposon defined as group of moveable dna elements which can transfer through rna intermediate using copy-andpaste technique [5], tnt1 is one of longrepeated terminal ltr retro-transposon, both ends of this subclass has internal direct repeats. additionally its length is 5.3 kb, it creates 5bp duplication in different sides during its inclusion, and it produces stable changes which effectively can move onto descendants [9]. samuel roberts noble foundation in usa is the major source for the medicago tnt1-insertion mutant collections. numerous mutant phenotypes have been separated, such as mutant has no ability to induce nodule (nod−), mutants have less effective or inefficient nitrogen fixation nodule (nod+fix+/-), mutants with very high nodule number (nod++) [3]. this study aimed to characterize blkn mutant, the medicago truncatule tnt1 insertion mutagenesis mutant line. the mutant characterization includes; phenotype, physiological and molecular characterizations. materials and methods plant material: medicago truncatula nf0478 is tnt1 mutant line was provided from noble foundation [9-11]. this line was produced in vitro through agrobacterium tumefactions transformation [3]. in this study medicago truncatula r108 was used as a wild type control plant. bacterial strain: sinorhizobium meliloti strain (sm1021) [12] was used to inoculate seedlings of blkn as well as r108 wild type. seed germination and rhizobia inoculation: both blkn mutant and r108 seeds were scraped with sand paper and the surface was sterilized with ten% (v/v) sodium hypochlorite for ten minutes. after 5 times washing using sterilize water, seeds were cultivated on basal nitrogen medium (bnm). seven day old seedlings were inoculated with sinorhizobium meliloti culture with an optical density 0.1 at wave length 600 nm to examine the nodule phenotype according to galibert 2001 [13]. genetic cross: plants homozygous for blkn allele were crossed with r108 wild type plant. mutant alleles were allowed to segregate through f1 self-fertilization. genomic dna extraction was carried out for f2 individuals and subjected to pcr amplification for analyzing the segregation pattern of blkn alleles. nodule examination with light microscopy: two weeks old nodules were directly fixed in 50ml solution containing 25ml ethanol 95%, 5ml formaldehyde, 2.5ml glacial acetic acid and 17.5ml distilled water. the fixed nodules were embedded in paraffin wax. blocks were divided with rotary microtome (euromex usa). slides were stained with safranin red followed by light green dye. finally, slides were scanned under light microscope (axio vert.a1) [14]. estimation of nitrogenase activity: root nodules were washed with sterilized water and placed in bottles sealed with rubber. 10 ml of air was withdrawn from bottle and replaced by 10 ml of acetylene using plastic syringes. the bottles were incubated for one hour at 30 ◦ c thereafter, 2 ml gas samples were withdrawn and assayed for measuring ethylene concentration using liquid chromatography [15]. determination of plant pigments: one hundred mg of fresh green leaves from each line with 10 biological replicas were frozen and grounded with liquid nitrogen rapidly. chlorophyll pigment was obtained by 3 ml of 80% acetone including 1 mm potassium hydroxide then centrifuged at 10,000 rpm for 10 min. followed by supernatant quantification with a spectrophotometer device(bio spectrophotometer, ependorf) [16]. chlorophyll and total carotenoid amounts were calculated based on [17]. phenolic compounds quantification: total phenol contents were estimated by folin ciocalteu’s technique. one gram of each mutant and control leaves were extracted in 3 ml of ethanol 95% and kept at 4°c/24 hrs in the dark, and then the samples were centrifuged at 13000rpm/5min. one ml of supernatant was transferred to 5 ml of distilled water with 0.5ml of folin ciocalteu reagent (f-c) were mixed well, after that 1.5 ml of 20% of sodium carbonate hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 10 (na2co3) was added and the total volume was adjusted to 10 ml and was incubated for 2 hrs at room temp. finally, spectrophotometer (bio spectrophotometer, ependorf) was used to read the samples at 765nm [18,19].total contents of phenolic compounds were calculated from standard curve obtained with different concentration of salicylic acid (10 to 100 mg/ml) aflp type pcr for tnt1 border characterization in blkn mutant: genomic dna of blkn seedlings in addition to control plants were isolated separately using i-genomic plant dna extraction mini kit (intron biotechnology). dna pool of blkn isolated samples and dna of control sample were separately double digested by ecori mfei and asei ndei respectively according to enzyme producer protocol biolabs®, eco and ase adaptors were ligated to the digested dna using takara t4 dna ligase separately. amplification of tnt1 borders was carried out by nested pcr to amplify all tnt1 -flanking sequence tags (fsts) in blkn mutant line. all pcr reactions were prepared using takara la polymerase in biorad . cloning of amplified fragments: qiaquick pcr purification kit was used to purify the amplified tagged fragments before cloning into pgem-teasy vector (promega®). after cloning, colony pcr method was done using sp6 and t7 common oligonucleotide primers. later fragments with different sizes were purified and sequenced. this method was used as confirmation test to success transformation. data analysis: tnt1 fsts were analyzed against medicago truncatule genome using national center for biotechnology information data base ncbi (http://blast.ncbi.nlm.nih.gov and http://www.medicagohapmap.org/home/view). statistical analysis: data was exposed to analysis using spss20.0 (one-way anova) and the value of the least significant difference (lsd) was calculated at significance level of 5%. results phenotype characterization blkn seedling phenotype: for phenotype characterization, blkn mutant was tested against r108 for roots and shoots lengths, 10 biological replicas were used for each line. the lengths were measured seven days post germination on ½ ms medium, measurements were recorded every seven days’ time interval. blkn average root lengths were significally longer than r108 at (p=0.01). while the average shoot lengths in blkn was similar to that of r108 (p>0.05) (figure 1a and 1b). on the other hand, mature pods of blkn had different shape; it displayed compressed pod and shorter spines than r108 but the two lines showed anti-clock wise spines direction (figure 1c and 1d) and the same number of seeds which is about from 5 to 8. the average number of produced pods has no significant difference between blkn and r108 at (p=0.2) (figure 1e). figure 1. phenotypic characters of m.truncatula wild type and blkn. (1a) comparison between average root lengths of blkn and r108 is indicating a significance difference between the two types of plants at (p = 0.01). (1b) the mean value of blkn shoot length was close to mean value of r108 shoot length at (p>0.05). (1c) mature pods of r108 is showing long spine with anti-clock wise spines direction. (1d) mature compacted pods of blkn with short spines and anti-clock wise direction. (1e) the average number of the produced pods in blkn and r108 with no significant difference at (p=0.2). http://blast.ncbi.nlm.nih.gov/ http://www.medicagohapmap.org/home/view hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 10 blkn nodule phenotype: blkn nodules were examined three weeks post inoculation with sinorhizobium meliloti strain (sm1021) on bnm. blkn nodules showed null functionblack color phenotype with longer size than r108 nodule (figure 2a). according to statically analysis, nodules number of blkn mutant is significally more than r108 at (p=0.03) as shown in (figure 2b). figure 2. (2a) one month-old blkn seedling showing black color nodule in a cluster arrangement. (2b) the average nodules number of blkn mutant was significally more than r108 wild type at p=0.03. transfer sections of blkn nodule were compared to those of r108 through light microscopy examination. nodules of r108 prompted by rhizobia displayed the characteristic layers and the cells in each layer have their definite structure and function (1, meristem zone; 2, bacterial infection zone; 3, nitrogen-fixing zone; 4, senescent zone) (figure 3a). figure 3. nodule examination by light microscope. (3a) transfer section of wild type nodule, (3b) transfer section of blkn nodule. (1) bacterial infection zone; (2) nitrogen-fixing zone; (3) senescent zone. blkn transfer section is completely different from this of r108 in both number and size of cells in bacterial infection zone and nitrogen fixation zone. while the phenotype of blkn nodule that was induced with the same symbiotic bacteria strain showed an unusual nodule progressive process. the zones in blkn nodule didn’t have clearly characteristic shape like the control. cells of infection zone were longer in size than those in the same layer of control nodule. additionally, there was abnormal number of cells in nitrogen fixation zone although almost of these cells were free from bacteroid and had accumulated phenolic compounds. physiological characterization determination of blkn pigmentation: plant pigment is any kind of colored material produced by the plant. plant pigments can absorb visible radiation between violet (380 nm) and ruby-red (760 nm). plant pigments responsible for the color to the leaves, the flowers, and the fruits and are essential in regulating photosynthesis and development. plant has many different pigments found in several organic compounds. chlorophyll, the green pigment that can absorb light energy as much as possible and carotenoids, the pigments that can absorb blue and indigo light are the two most important pigmentation in the plant. the two pigments were estimated in blkn and r108 as well. both type of chlorophyll (chlorophyll a and chlorophyll b) don’t show any significant difference between their content in blkn and r108 (p=0.7) for chlorophyll a and p=0.06 for chlorophyll b). while blkn produces more than double amounts of carotenoids than r108 with a significant differenc (p=0.02) as shown in (figure 4a). figure 4. (a) measurement of pigment contents in r108 and blkn showing no significant difference in both types of chlorophylls (a and b) between r108 and blkn at (p=0.7) for chlorophyll a and at (p=0.06) for chlorophyll . while blkn produces more than douple amounts of carotenoids than r108 with a significanc differenc at (p=0.02). (b) blkn roots contain more than double content of phenolic compounds than r108 (p=0.00). hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 10 phenolic compounds quantification in blkn: plant phenolics are secondary native metabolites which perform a very wide range of physiological characters in plants. higher plants produce several thousand of diverse phenolic compounds. plant phenolics are fundamental defense compounds during different biotic and abiotic environmental stresses like; high and low temperatures, high light, ultraviolet radiation, different oxidants, nutrient deficiency, herbivores and pathogen infection. due to the black nodule colour and the dark colour of blkn root, total phenols were estimated in root of blkn and r108 one day pre rhizobium inoculation and 72 hrs post rhizobium inoculation. the total phenolic compound estimation was performed colorimetrically using spectrophotometer. there was no significant differences between blkn and r108 pre rhizobia inoculation while blkn seedlings have the ability to accumulate large amount of phenolic compounds than r108 and the difference was significantly at p=0.00 as shown in (figure 4b). molecular characterization tnt1-tagged loci identification and characterization in blkn mutant: to characterize tnt1-flanking sequence tags in blkn mutant, oligonucleotide primers (ltr3, ltr4, ltr5, ltr6) for tnt1 transposon against asei, aseii and ecoi, ecoii for ase and eco adaptor respectively [20]were used. all tnt1 tagged fragments were separated and visualized on 1.5% agarose running at 80 v, data presented in (figure 5a, b, c, and d). tnt1-tagged fragments with different sizes were exposed to cloning in pgem-teasy and sequencing process. about 25 fsts from blkn mutant line were isolated and subjected to data analysis against genbank http://www.ncbi.nlm.nih.gov/ database. some of them were blasted with coding regions of medicago genome, others were blasted with non-coding regions and the rest were unknown sequences. fsts corresponding to blkn mutant were submitted to genbank http://www.ncbi.nlm.nih.gov/. genbank accession numbers, e-value and reference genes are shown in (table 1). figure 5. blkn-tnt1 border amplification. (5a) mfei and ecori -double digestion the pcrii result using ltr4and ecoii oligonucleotide primers. (5b) ecori and mfei-double digestion pcrii result using ltr6 and ecoii oligonucleotide primers. (5c) ndei and ase i -double digestion test pcrii result using ltr4 and aseii oligonucleotide primers. (5d) ndei and asei double digestion pcrii test using ltr6 and aseii oligonucleotide primers. lanes from 1-16 are, ag ,at, aa, ac, cg, ct, ca, cc, gt, gg, gc, ga, tt, tg, tc, and ta separately at the end of oligonucleotide primers. aseii and ecoii. m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m a b m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m a b c d hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 10 table1: blkn tnt1-tagged sequences. sequence name length accession number organism reference e-value blkn2 560 mn529997 medicago truncatula mwmb01000023.1 0 blkn3 291 mn529998 medicago truncatula mwmb01000001.1 8.00e-152 blkn4 289 mn529999 medicago truncatula mwmb01000001.1 1.00e-125 blkn5 288 mn530000 medicago truncatula mwmb01000001.1 4.00e-150 blkn6 291 mn530001 medicago truncatula mwmb01000001.1 8.00e-152 blkn8 284 mn530002 medicago truncatula mwmb01000001.1 6.00e-148 blkn9 171 mn530003 medicago truncatula xm_024776685.1 3.00e-79 probable l-type lectindomain containing receptor kinase vii.2 blkn10 250 mn530004 medicago truncatula mwmb01000001.1 4.00e-129 blkn11 71 mn530005 medicago truncatula mwmb01000031.1 1.00e-23 blkn13 250 mn530006 medicago truncatula cu179894.1 3.00e-106 blkn15 251 mn530007 medicago truncatula mwmb01000011.1 1.00e-129 blkn16 292 mn530008 medicago truncatula ac130200.30 2.00e-152 blkn17 252 mn530009 medicago truncatula mwmb01000011.1 3.00e-130 blkn18 294 mn530010 medicago truncatula ac130200.30 6.00e-103 blkn19 156 mn530011 medicago truncatula mwmb01000006.1 5.00e-77 blkn20 255 mn530012 medicago truncatula mwmb01000011.1 7.00e-132 medicago truncatula l-type lectin-domain receptor kinase locus is interrupted by tnt1 insertion in all f2 black nodule individuals from our data analysis, three medicago truncatula tnt1-insertion loci in blkn were subjected to additional investigation; blkn_2 (tnt1 insertion located in f-box/lrrrepeat protein), blkn_4 (tnt1 insertion located in unknown protein) and blkn_9 (tnt1 insertion located in l-type lectindomain receptor kinase). two oligonucleotide primers blkn2–r (5-gcttggaagagtctaaggttaa-3) and blkn4–r (5-ccaagttgacttgtttcatc-3) were used separately with ltr4 to test blkn_2 and blkn_4 insertion loci respectively. pcr was performed on genomic dna extracted from f2 population entities resulting from blkn mutant and r108 wild type back cross. only entities displaying black nodule phenotype with the other mutation phenotype characters were tested (about 1/4 population of f2 progeny). pcr result indicated that tnt1 insertion wasn’t confirmed in all of the entities means that the insertion on those loci aren't charged for this mutation phenotype. oligonucleotide primer pair blkn9-f (5-ccctcaaaaaat accaacaaaacc-3) and blkn9-r (5-caacttttccaa ttcatcacca-3) were designed to be used in compatible with the two ltrs oligonucleotides of tnt1 to test the incidence of tnt1 within the predicted mutated locus, l-type lectin-domain containing receptor kinase (blkn_9), in all blkn mutant individuals. pcr result confirmed the incidence of the two tnt1 side borders in all f2 entities displaying the mutation phenotype at the proper locus while showed a negative result in r108 entities. when using the two specific oligonucleotides for blkn_9 locus amplification, only r108 gave the proper band at 358bp (data not shown) while the amplification at this position failed in all blkn mutant individuals (1/4 population of f2 progeny). the rest of segregation individuals which are showing wild type phenotype (3/4 population of f2 progeny) gave a ratio of 60/30 positive to negative pcr result for tnt1 border amplification. this result is obeying mendel law, indicating that (blkn_9) insertion could be accountable for null function black-nodule, phenol accumulation and other mutation phenotype. the mutation at this locus was confirmed also at rna level, rt-pcr performed on all blkn mutant individuals using the two specific primers of the proper locus was giving negative result in all entities and positive amplification in r108 (data not shown). tnt1 insertion was precisely located next to the base number 303 post atg start codon of medicago truncatula l-type lectindomain receptor kinase gene which is 2079 bp in length with one exon encode lectin_legb receptor like kinase 692 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000023 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nucleotide/xm_024776685.1?report=genbank&log$=nuclalign&blast_rank=1&rid=svnduw9d015 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000031 https://www.ncbi.nlm.nih.gov/nucleotide/cu179894.1?report=genbank&log$=nuclalign&blast_rank=2&rid=s83xbg0y014 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000011 https://www.ncbi.nlm.nih.gov/nucleotide/ac130200.30?report=genbank&log$=nuclalign&blast_rank=1&rid=rex0cu3v014 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000011 https://www.ncbi.nlm.nih.gov/nucleotide/ac130200.30?report=genbank&log$=nuclalign&blast_rank=1&rid=reyeg4pb01r https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000006 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000011 hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 10 amino acids in length. mtlectinrlk consists of three domains; two transmembrane helix (tmhilex) the first is from 4bp to 26bp and the other from 288 to 310 predicted by tmhm (http://www.cbs.dtu.dk/services/tmhmm/). the second domain is lectin_legb domain at the position from 22bp to 285bp, and third one is the extracellular receptor protein kinase (pkinase) at position from 357bp to 641bp as shown in (figure 6). figure 6. mt-lectinrlk is 692 aa in length contains two transmembrane helixes (tmhelix) from 4 to 26 and from 288 to 310, lectin_legb domain at position from 22 to position 285, and extracellular receptor protein kinase (pkinase) at position from 357 to position 641. phylogenetic tree was constructed using embl-ebi (www.uniprot.org/align) as shown in (figure 7). mtlectinrlk is an ancestry related to probable l-type lectin-domain containing receptor kinase vii.2 cicer arietinum, putative l-type lectin-domain containing receptor kinase-like protein trifolium pretense, protein kinase domain-containing protein lupinus angustifolius, protein kinase domain-containing protein phaseolus vulgaris, probable l-type lectin-domain containing receptor kinase vii.2 isoform x1 vigna radiate, putative l-type lectindomain containing receptor kinase vii.2 glycine soja, putative l-type lectin-domain containing receptor kinase vii.2 mucuna pruriens, lectin-domain containing receptor kinase a4.2 cajanus cajan. figure 7. the predicted medicago truncatula mtlectinrlk is ancestry related to probable l-type lectin-domain containing receptor kinase vii.2. phylogenetic tree was constructed using embl-ebi (www.uniprot.org/align). discussion unique medicago truncatula mutant line nf0478 is a tnt1-retrotransposon mutant that is verified as black nodule phenotype mutant (blkn). we utilized reverse genetic tool to isolate and characterize the medicago truncatula “knockout” mutations in a gene that is supposed to be involving in nodulation process and other physiological processes. previously, about 2801 tnt1 flanking sequence tags were isolated from 156 medicago truncatula symbiotic mutant lines. the insertions were in nodulating genes like; nodule inception (nin), nodulation signaling pathway (nsp1, nsp2), doesn't make infections (dmi1, dmi2 and dmi3) and a number of super-nodulation genes sunn and skl [3]. blkn the black nodule mutant is showing defective in infection zone and nitrogen fixation zone alongside cell wall periphery lignification in symbiotic organ. characterization of blkn mutation depends mainly on phenotype monitoring and various biotechnology tools for investigation. linking between mutation phenotype with the changes occurred in the native physiological process due to this mutation along with the molecular characterization gave us clear evidence about the defected protein responsible for the emergence of this mutation. more than ~25 tnt1 insertion sites was isolated, sequenced and analyzed using genbank (http:// www.ncbi.nlm.nih.gov/) database during our study. some insertions were located within non-coding regions while the others were located in non-matching sequences. our results enable us to confirm that the medicago truncatula l-type lectin-domain receptor kinase locus is interrupted by the tnt1 insertion in all blkn mutant line individuals. tnt1 insertion locus was tested with pcr at both dna and rna levels and confirmed genetically through the segregation ratio of tnt1 borders in f2 population. our results indicating that the tnt1 insertion is precisely located next to the base number 303 post atg start codon of medicago truncatula l-type lectin-domain receptor kinase gene in blkn mutant line. this medicago truncatula gene encodes lec_legb-rlk which is 692 bp amino acids in length was scanned by http://pfam.xfam.org for functional domains. our data showed that medicago truncatula lec_legb-rlk has two transmembrane helix domains (tmhilex) the first is from 4bp to 26bp and the other from 288 to 310 predicted by tmhm (http://www. cbs.dtu.dk/services/tmhmm/). the second is lectin_legb domain (start from 22 to 285). each leguminous lectin subunit contains sole carbohydrate-binding site with beta pleated sheets consist of two conserved amino acids. the lectins are binding galactose, mannose or glucose and this interaction requires a firmly bound manganese and calcium ions. the third conserved domain is the protein kinase pk domain (start from 357 to 641) and it acts as switch on/off for majority of cellular activity in plant cell by phosphorylation and any defect in this domain can cause dysfunction abnormality. native biotic environment of plants root contains both symbionts and pathogens. the plants are responding to http://www.cbs.dtu.dk/services/tmhmm/ http://www.uniprot.org/align http://pfam.xfam.org/ hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 10 various microbes either by establishing a symbiotic interaction with friendly symbionts bacteria and mycorrhiza [21], or by inducing plant innate immunity to defend against pathogens attack [22]. medicago truncatula is an ultimate opportunity to study such crosstalk between symbiosis and defense in the environment. innate immunity in plants is triggered through recognition of microbe-associated molecular patterns (mamps) by plants pattern recognition receptors (prrs) [23,24]. this recognition process elicits defense program cascades enable the plant to respond to the pathogen attack in a precisely and effective manner. such recognition is switching on all the downstream protection including: production of reactive oxygen species (ros), accumulation of salicylic acid signal, inducing nonexpressor of pr gene (npr1) protein that convert into its monomeric form then translocate to the nucleus to activate pr–related genes, increasing ca2+ influx, and finally activation of mitogenactivated protein kinase (mapk) cascade [22]. this fast response is leading to the resistance against wide range of pathogens attack [25]. plant receptor-like kinases (rlks) include a superfamily of trans-spanning proteins involved in pathogen detection and defense signaling transduction [26] which is closely related to ros production [27]. lectin receptor kinases (lec-rks) is one class of the plants rlks that plays essential role through plant development and during the various stress responses. it contains an extracellular lectin domain and divided into three subclasses according to the diversity in this motif; gtype (s-locus), c-type (calcium dependent) and l-type (legume) [28]. g-type lect-rks are found in flowering plants and known as s-domain rlks they are involving in self-incompatibility [29] .c-type lec-rks are calciumdependent lectin that are found regularly in the mammalian proteins and involving in pathogen recognition and trigger the various immune responses [30]. arabidopsis has a single ctype lec-rk motif but its function is not been established so far [28], while arabidopsis contains about 45 l-type lec-rks [28,31]. lec-rks were proposed to play essential role in both abiotic stress tolerance [32-34] and biotic stress signals transduction [28]. this study is reporting the knock out mutation of medicago truncatula lec_legb-rk gene (blkn), this mutation is driving null function black color root nodule phenotype. the mutated nodule is displaying abnormality in both infection zone and nitrogen fixation zone. transverse section of the mutated nodule was displaying an empty symbiotic zone with higher lignification at the cell wall periphery compared with the wild type nodule phenotype. the released bacteriods in blkn infection zone were less than those in r108. in addition, the total phenolic compound accumulation was dramatically and significantly excessive in blkn root 72 hours post inoculation with sinorhizobium comparing to r108. this ultimate modification wasn’t observed pre sinorhizobium inoculation. the first published mutation in lectin-rk was reported [35] in arabidopsis lecrk-i.9. this mutation is exhibited an increasing susceptibility to pseudomonas syringae, phytophthora brassicae, and phytophthora capsici, while the over expression of this gene enhanced the resistance of the plant against these three pathogens [35]. although most of plant lectin receptor kinases function is not yet so clear, the role of these kinases in innate immunity is developing. some other lrk were reported to be involving in plant protection against various pathogen attacks. pi-d2 is g-lrk isolated from rice can provides defense against rice blast derived by magnaporthegrisea, the parasitic fungal pathogen [37]. nblrk1 is lecrk in nicotiana benthamiana was suggested to recognize the inf1 elicitor of phytophthora infestans and mediates plant defense [38]. while the expression of g-lrk is up-regulated in tobacco by lipopolysaccharides signal [39]. according to our data, lec_legb-rk switching off due to the tnt1 insertion in blkn mutant line is resulting in defective nodule, so what is the proposed function of medicago truncatula lec_legb-rk? our results in addition to the previous data enabled us to suggest that mtlec_legbrk is like a switch key. mtlec_legb-rk can recognize the symbionts microorganisms and directed the plant cells toward being involved in symbiotic interaction, in addition to recognize the invading microorganisms and trigger the signal defense pathway. thus, the mutation of medicago truncatula lec_legb-rk altered the plant cells unable to distinguish between the symbionts and invaders. we are suggesting that in case of blkn mutant, although the rhizobia-derived n-acetylglucosamine signal were normally perceived via lysm proteins [40] otherwise the mutant plant cells treated them as attackers instead of treating them as symbionts. consequently, the mutant cells produced greater amount of phenolic compound in roots and nodules compared to those of wild type r108. normally, such phenolic compounds are produced in plant cells due to several biotic and abiotic stress conditions such as: salt stress, drought, extreme temperature, nutrient deficiency, uv radiation, heavy metals and herbicides and other pathogen attacks. all these kind of plant stresses can induce unbalance between production of reactive oxygen species (ros) and its scavengers. plants (ros) are extremely reactive, toxic, and can cause severe damage to various cell components; dna, carbohydrates, proteins, and lipids leading to oxidative stress. plants have their own enzymatic and non-enzymatic mechanisms for ros detoxification. peroxidase (pox) is one of main enzymatic system, it oxidize phenolic compound by using them as an electron donor [41,42]. so, what is the fate of high contents of phenolic compound in blkn mutant plant? according to [43], phenolics are oxidized by pox at h2o2 expense hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 9 of 10 throughout lignin formation. thus, the strong lignin deposits around the cells wall periphery of both infection zone and nitrogen fixation zone confirmed our suggestion that the root cells in blkn mutant failed to distinguish between symbionts and attackers through producing a high amount of phenolic compound which is consider as ros scavenging compound. in turn these phenolic compounds were oxidized into lignin via pox within the nitrogen fixation organ. this suggestion may also explain the low frequent bacteroids in infection zone of blkn mutant than r108 infection zone. this result is obeying with the previous published data by roopashree [44]. they proposed that in native condition the legume lectin gene has two reverse functions depending on the perceived microorganisms and also the gene may be involved in the attachment of nitrogen-fixing bacteria to the legumes and in reverse function as a 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sudan. contacts of authors * to whom correspondence should be addressed: yousifassad12@gmail.com received: september 24, 2020 accepted: february 7, 2021 published: march 30, 2021 citation: adam eiy , assad yohm, eltohami mme, bashir nhh, abdelrahman sh, nour bym . natural anophelin mosquito repellents and phytochemical analysis of ethanol and hexane leaf extracts from four plant species. 2021 mar 30;4:bs202107 copyright: © 2021 adam et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. natural anophelin mosquito repellents and phytochemical analysis of ethanol and hexane leaf extracts from four plant species emad i. y. adam1 >< , yousif o.h.m. assad*1 >< , marwa m. e. eltohami1 >< , nabil h.h. bashir1 >< , samira h. abdelrahman2 >< , bakri y. m. nour2 ><  abstract currently mosquitoes control is the cornerstone to minimize the rising number of mosquito borne diseases. there is an urgent need looking for alternatives to the current reliance on synthetic insecticides for the vectors control. application of active toxic agents from plant extracts as an alternative control strategy was available from ancient times. these are non-toxic, easily available at affordable prices, biodegradable and show broadspectrum target-specific activities against different species of vectors. natural products with repellency properties are urgently needed. an insect repellent works by masking human scent, and a number of natural and synthetic mosquito repellents were studied. the main aim of this study was to identify the phytochemicals and compare their potentials as mosquito repellents from the ethanol (et. oh) and hexane (hex) leaf extracts (les) of ocimum basilicum, coleus forskohlii, eucalyptus camaldulensis and cymbopogon flexuosus under laboratory conditions at 50 and 100% concentrations. phytochemical analysis showed that alkaloids, saponins, flavonoids, terpenes, tannins and terpenoids were present/or absent in the 4 plant-les. at 50% concentration of et. oh extracts, c. forskohlii exhibited higher repellency potential on anopheles gambiae with protection time of 137.3 min, while o. basilicum, e. camaldulensis and cy. flexuosus registered protection time of 30.6, 15.3 and 19 min, respectively. at 100% concentration of ethanolextracts, c. forskohlii caused the highest protection time against the vector with protection time of 182 min. o. basilicum, e. camaldulensis and cy. flexuosus were equally less potent against the insect, with protection time 42.6, 32.6 and 28 min, respectively. regarding hex-extract, at 50% concentration of c. forskohlii, the highest repellency potential , with protection time of 174 was registered, while o. basilicum, e. camaldulensis and cy. flexuosus registered protection times of 44, 18 and 28.6 min, respectively. however, the 100% concentration of et. oh-extracts of, c. forskohlii and o. basilicum exhibited the highest protection time, with protection times of 228 and 116min, respectively. e. camaldulensis and cy. flexuosus were less potent, with protection times 28.6 and 54.6 min, respectively. the eos of c. forskohlii and o. basilicum l eos proved to have potentials as repellency agents against anopheles. gc-ms analysis of the extracts identified the compounds of both solvents extracts and the terpens were the major compounds. according to the results, further studies are required, especially on the extracts of basil and coleus since they proved to be effective. keywords: mosquito repellent, anopheles, essential oils, sweet basil, wild thyme, kafure, lemon grass introduction soonwera and phasomkusolsil [1] stated that mosquitoes are pestiferous insects to public health; transmit several deadly dangerous diseases, e.g. dengue (df), filariasis, malaria, yellow fever (yf) and japanese encephalitis (je). several researchers reported that every year at least 500 million people in the world suffer from one of the tropical diseases that include df, malaria and filariasis [2, 3]. highlights in bioscience page 1 of 10 march 2021|volume 4 https://doi.org/10.36462/h.biosci.202107 https://creativecommons.org/licenses/by/4.0/ strongmass2020@gmail.com yousifassad12@gmail.com https://orcid.org/0000-0003-2928-3051 marwatoxic@gmail.com bashirnabilhh@gmail.com samhamid2002@yhoo.co.uk https://orcid.org/0000-0001-7714-9266 bakrinour@gmail.com https://orcid.org/0000-0002-2066-4176 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species salih et al., [4] reported mosquitoes are among the most disturbing blood -sucking insects afflicting human beings. the mosquito female's behavior, involving feeding on human blood, is responsible for the transmission of a number of diseases. one to two million deaths is reported annually, due to malaria worldwide. pest/vector management is facing economic and ecological challenges worldwide, due to human and environmental hazards caused by majority of the synthetic pesticides. identification of novel effective insecticidal compounds is essential to combat the increasing resistance rates. botanical pesticides have long been touted as attractive alternatives to synthetic pesticides for pest/vector management, because botanicals reputedly pose little threat to the environment or to human health. the body of scientific literature documenting bioactivity of plant derivatives to arthropod pests/vectors continues to expand, yet only a handful of botanicals are currently used in agriculture, public health, industrialized world, and there are few prospects for commercial development of new botanical products. the medical vectors control is considered the most important corner-stone to prevent the diseases; however, the control is either by the synthetic pesticides or the repellents that prevent the biting of the mosquitoes. personal repellents are substances applied to skin, clothing, or other surfaces to repel or discourage insects and other arthropods, e.g. ticks, from feeding on humans. repellents help people to avoid bites from mosquitoes, and other biting arthropods that may transmit diseases, and allow people to engage freely in outdoor activities. insect repellents may also be used to exclude insects from an area [5]. with increasing public concern regarding pesticide safety and problems of insecticide resistance, new safer active ingredients are becoming necessary to replace the existing compounds. furthermore, the use of repellents in an integrated pest/vector management (ipm/ivm) program has been ignored to a large extent [5]. salih et al., [4] reported that the personal protection from mosquito bites is currently the most important measures to control mosquito transmitted diseases. the mosquito repellent products commonly available on the market contain n, n-diethyl3-toluamide (deet) as the active ingredient. the synthetic repellents are not safe for human, especially children, because they may cause skin irritation, hot sensation rashes or allergy [6]. recently, as results of the public health concern on the safety of many synthetic products, many research scientists renewed the interest on the use of natural products from plant origin for mosquito's management [1]. in addition, plant essential oils (eos), in general, have been recognized as an important natural resource of insect repellents. the plant eos base repellents according to soonwera and phasomkusolsil [1] are effective for mosquito control, and are environment friendly. vector borne disease such as malaria is a major global health problem; half of the world's populations, mostly the poorest countries living in the tropical world's are at risk of malaria. the disease caused by plasmodium parasites transmitted by infected female anopheles mosquitoes bit is serious problem in africa, where one in every five (20%) childhood deaths is due to malaria. most malaria cases and deaths are in sub-saharan africa most malaria cases and deaths are in sub-saharan africa [7]. therefore, herbal insect repellents gained importance recently; detailed knowledge concerning them is still lacking. as a result, it is high time to launch extensive search to explore ecofriendly biological/botanical materials for controlling pests. curtis [8] choochote et al., [9] reported that eos have been investigated and described as potentially natural sources of insect repellents. the effective repellency against female anopheles results varied with different species of the plant and the concentration/dose. according to trongtokit et al., [10] study on an. stephensi in brazil using cymbopogon nardus citronellal 40% eos was applied topically, reported 100% protection for 7-8 hr. the present study investigated the potential of sweet basil (ocimum basilicum l), wild thyme (coleus forskohlii), kafure (eucalyptus camaldulensis) and, lemon grass (cymbopogon flexuosus), les as mosquitoes repellents. curtis [8] stated in many parts of the world, plant derived natural products have traditionally been used as repellents against insects bites. particularly essential oils have been investigated and described as potentially natural sources of insect repellents it is time to search for effective alternatives to synthetic repellents as reported by choochote et al., [9]. many research studies were conducted on various medicinal plants and the effective repellency against female anopheles mosquitoes, the result is varying according to the plant and the concentration. the present study investigates to figure out the effectiveness of ocimum basilicum l., coleus forskohlii, eucalyptus camaldulensis and, cymbopogon flexuosus steud, (leafs) extract as mosquitoes repellent. materials and methods collection of plant material and extraction fresh leaves of the four above-mentioned plants were collected from gezira state, sudan. the collected leaves were washed, shade-dried and powdered. the powder was extracted in ethanol (et. oh) and hexane (hex) by soaking in et. oh, and clevenger apparatus was used for preparation of hexextract; 50 g of each plant leaves powder (lp) was extracted separately in conical flasks by et. oh. analytical grade et. oh was added until the lp was fully submerged, thoroughly stirred to ensure proper mixing, and subjected to shaking at regular intervals to allow percolation for 4 days. on the 5th day, the extracts were filtered using whatman no.1 filter paper into another conical flask and the et. oh was evaporated. the resultant viscous substance was dried and stored in amber-colored vials in a refrigerator at 4oc pending formulation. for hex-extracts the eo was isolated by steam-distillation, using a clevenger apparatus for 6 hr; moisture in the oil was removed by anhydrous sodium sulfate and stored at 5oc for further use. highlights in bioscience page 2 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species formulation of plant leaves extracts the plant leaves extracts to be tested were formulated in pure fragrance-free vaseline jelly. the formulations tested were made into two concentrations of 50% and 100%. to make 50% of the formulation, 5g of the pure vaseline jelly was weighed and transferred to a clean 100 ml beaker, placed in a water bath at 80°c, stirred with a rod up to the melting point. five g of each of the 8 extracts was then added to the melted jelly, followed by continuous stirring to be completely mixed with the jelly. the resultant 8 formulations of eight plant extracts were stored at 4°c for the bioassay. to make 100% of the formulation, 1 g of pure vaseline jelly was melted as above; 10 g of each of the 8 extracts were added and treated as above. the two concentrations (50 and 100%) were used in bioassay. anopheline mosquito the anopheles gambiae (3-7 days old) was obtained from blue nile national institute for communicable disease (bnnicd) insectary, university of gezira, colony maintained at 12: 12 ld photoperiod, 70±10% r.h. and 24±2oc. the adult females of anopheles used for the bioassay before any blood meal. females were starved for 24 hr and provided with only 10% sugar solution in water. tests were conducted in triplicate 25 individual of starved adult mosquitoes rearing cage was used and topical treated arm with plant extract exposed to the mosquitoes in the cage to observe the mosquitoes bite. bioassay test the reared mosquitoes were taken from the insectary of blue nile institute for communicable disease (bnicd) (24±2cº and 80% rh) and the experiment was conducted in the insectary lab of bnicd, the arm cage method was used. three gram of formulated plant leaves extract was applied onto the forearm of a human subject. the treated arm was exposed until the first bite to 25 female mosquitoes, in a (252x3.14x60 cm3) cage, and any mosquitoes landing and biting were counted. after any bite the time was recorded at which the first bites occurred the bioassay terminate and repeated for the other 50 mosquitoes to make triplicate at successive time intervals. for the negative and positive control, the arm with vaseline jelly and with deet was used respectively. numbers of mosquitoes landing and biting in a minute were recorded. the application of plant extract was used following frances methods [11]. two concentrations (50 and 100%) for each plant, ethanol and hexane extract and mixture of ethanol and hexane for each plant to account the number of mosquitoes land and the complete protection time (cpt) in the treated area (20-15cm2), with 3g formulated plant extract. phytochemical analysis the phytochemical analysis was carried out at chemistry lab, faculty of agricultural sciences, university of gezira by using the method of balbaa [12] and harborne [13] to investigate the presence of the saponins, tannins, sterols, triterpens, flavonoids, terpenoids and alkaloids. gas chromatographymass spectrometry the qualitative and quantitative analysis of the sample was carried out by using gas chromatography-mass spectrometer (gc/ms-qp2010-ultra) from japans simadzu company, with the serial number 020525101565sa and capillary column (rtx-5ms30m[2e3?]0.25mn[2e3?]0.25μm).the sample was injected by using split mode, helium as a carrier gas passed with flow rate 1.61 ml/min, the temperature program was started from 60c with rate 10oc/min to 300oc as a final temperature degree with 5minutes hold time, the injection port temperature was 300oc as the ion source temperature was 200oc and the interface temperature was 250oc. the sample was analyzed by using scan mode in the range of m/z 40-500 charges to ratio and the total run time was 29 minutes. identification of the components for the sample was achieved by comparing their retention index and mass fragmentation patents with those available in the library, the national institute of the standards and technology (nist). data analysis for each plant species, the number of mosquitoes landed on the arm was counted at different time intervals and the percentage repellency (pr) per each plants leaf extract/solvent/concentration was determined using the following equation (1) according to teklani and perera [14] as follows: pr = n iu − n it 25 (is ) 100 (1) where: • pr=percentage repellency. • niu=number of individual in the untreated group. • nit=number of individual in the treated group. • is=number of insects used. at least triplicates were carried out for all the plants leaves extracts for selected extracts concentration with significant mosquito repellent activity. results phytochemical analysis phytochemical analysis of ethanolic and hexane extracts of the four plants leaves were carried out to identify the presence of alkaloid, tannin, saponin, terpenoids, flavonoids and terpens. in this study four plant species (basil, wild thyme, kafure and lemon grass) belonging to different families were collected. most of these plants were reported to treat a variety of diseases in traditional medicine system. ethanol extracts the preliminary phytochemical screening had shown the presence of alkaloid, tannin, flavonoids, saponin and terpens in the leaves extracts the summary of the results are presented in table 1. the alkaloid, tannins, saponin and terpens were detected in the basil leaves extract; alkaloid, tannins, and terpens in wild thyme leaves extract; alkaloid, tannins, flavonoids and saponins in kafure leaf extract and alkaloid,tannins, and saponins in lemongrass, highlights in bioscience page 3 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 1. the phytochemical constituents (pc)of ethanol (e) and hexane (h) leaf extracts from the studied four plants. pc oc col eu cy e h e h e h e h alkaloid + + + + + + + tannins + + + + + + + flavonoids + + saponins + + + + + terpens + + + oc=ocimum, col=coleus, eu=eucalyptus, cy= cymbopogon. (+)= presence, (-) = absence hexane extract the preliminary phytochemical screening had shown the presence of alkaloid, tannin, flavonoids, saponin and terpens in the hexane leaves extracts the summary of the results are presented in table 1. in basil leaves extract alkaloid, tannins was only detected. while in wild thyme leaves extract terpens only was detected. in kafure leaves extract alkaloid, tannins, flavonoids and saponins were detected and terpens is absent. in lemongrass extract alkaloid, tannins, and saponins were detected. bioassay the results of bioassay were collected as a complete protection time (cpt), and the number of mosquitoes landing for the concentrations (50 and 100%), ethanol, hexane and hexane plus ethanol extract. ethanol extracts presented in tables 2 and 3 shows that wild thyme 50% and 100% were recorded the highest protection time 137.3 and 182 min respectively compare to the other plant extracts, with percent repellency 92 and 89% respectively. for the hexane extracts presented in tables 4 and 5 prove that thyme 50% and 100% were recorded the highest protection time 124 and 228 min respectively compare to the other plant extracts, with percent repellency 90 and 92% respectively. regarding the combination of the two extracts presented in tables 6 and 7 also the wild thyme 50% and 100% were recorded the highest protection time 182 and 215 min respectively compare to the other plant extracts except basil in 50% recorded higher protection time (86 min), with percent repellency 84 and 88% respectively. by the way the selected four leaf plant extracts offers protection against an. arabiensis adult female mosquitoes but varied in terms of repellency up to 228 minutes of exposure periods recorded by wild thyme. chemical constituents detected in kafure leaf extracts table 8 shows the chemical constituent of kafure leaves ethanol and hexane extracts; their concentrations, peak area, retention time and chemical group of each compound. a total of 13 compounds detected in ethanol and hexane extract which represent 63.63% and 68.54% for ethanol and hexane extract, respectively. eucalyptol was the main compounds (33.11% and 26.93%), respectively. table 9 shows the chemical constituent of kafure leaf's ethanol extract; their concentrations, peak area, retention time and chemical group of each compound. a total of 20 compounds detected in ethanol extract and represent 36.37% of the total of the extract. globulol 9.94% and squalene 8.10% were the main compounds. table 10 shows the chemical constituent detected in kafure leaf's hexane extract; their concentrations, peak area, retention time and chemical group of each compound. a total of 25 compounds detected in hexane extract, as follows, where globulol was the main compound (18.06%). discussion the synthetic chemicals used to control insects leads to several documented environment and human health hazards. an alternatives method suggested using natural products that possess good efficacious results and environmental friendly. among those alternative methods the use of essential oils from plants belonging to several plant species subjected intensive studies which provide successful results against field crop pests and public health pests and disease vectors control, including mosquitoes (anopheles, culex and aedes). these eos were extensively tested by several scientists for their repellent properties as valuable natural resources and to elucidate their chemical and physical properties, in addition to their modes and sites of action, formulation, toxicological profiles, side-effects, etc... today medical vector management has to face environmental and economic costs. table 2. spatial repellent activity of 50% and 100% evaluated plants leaves ethanol extract against anopheles mosquito based on the number of landed mosquitoes (nlm) and protection time per minute (pt). treatment 50% 100% nlm pt nlm pt 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66 wild thyme 1 2 1 1.3 113 122 177 137.3 2 3 1 2 210 154 182 182 sweet basil 2 3 1 2 25 36 31 30.6 4 2 6 4 42 31 45 42.6 kafure 4 2 1 2.3 15 10 21 15.3 2 3 4 3 25 32 41 32.6 lemongrass 2 3 5 3.3 15 17 25 19 4 2 3 3 31 25 28 28 table 3. percent repellency (pr) of 50% and 100% of evaluated plants leaves ethanol extract against anopheles mosquito based on the number of landed mosquitoes (nlm). treatment 50% 100% nlm pr nlm pr 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 68% 8 9 5 7.33 68% wild thyme 1 2 1 1.3 92% 2 3 1 2 89% sweet basil 2 3 1 2 89% 4 2 6 4 81% kafure 4 2 1 2.3 88% 2 3 4 3 85% lemongrass 2 3 5 3.3 84% 4 2 3 3 85% highlights in bioscience page 4 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 4. spatial repellent activity of 50% and 100% evaluated plants leaves hexane extract against anopheles mosquito based on the number of landed mosquitoes (nlm) and protection time per minute (pt). treatment 50% 100% nlm pt nlm pt 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66 wild thyme 1 2 2 1.6 191 142 189 174 1 2 1 1.3 230 208 246 228 sweet basil 3 2 5 3.3 46 36 50 44 2 1 3 2 90 118 140 116 kafure 4 6 8 6 18 21 15 18 3 1 2 2 36 21 28 28.3 lemongrass 3 4 6 4.3 22 36 28 28.6 2 3 5 3.3 59 41 63 54.3 table 5. percent repellency according to the number of landed mosquitoes (nlm) of 50% and 100% percent of evaluated plants leaves hexane extract against anopheles mosquito. treatment 50% 100% nlm pr nlm pr 1st 2nd 3rd mean 1st 2nd 3rd mean untreatedcontrol 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 68% 8 9 5 7.33 68% wildthyme 1 2 2 1.6 90% 1 2 1 1.3 92% sweetbasil 3 2 5 3.3 84% 2 1 3 2 89% kafure 4 6 8 6 73% 3 1 2 2 89% lemongrass 3 4 6 4.3 80% 2 3 5 3.3 84% table 6. spatial repellent activity and protection time per minute (pt)of 50 and 100% of evaluated plants leaves hexane and ethanol extract mixture against anopheles mosquito. treatment 50% 100% nlm pt nlm pt 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66 wild thyme 2 3 5 3.3 164 194 190 182.6 4 2 1 2.3 211 233 201 215 sweet basil 4 1 3 2.6 48 51 31 43.3 2 6 4 4 66 94 101 87 kafure 4 6 7 5.6 17 11 19 15.6 6 5 8 6.3 20 15 23 19.3 lemongrass 6 3 4 4.3 15 24 19 19.3 1 2 4 3.3 27 20 31 26 table 7. percent repellency (pr) according to the number of landed mosquitoes (nlm) of 50% and 100% of evaluated plant leaves hexane and ethanol extracts mixture against anopheles mosquito. treatment 50% 100% nlm pr nlm pr 1st 2nd 3rd mean 1st 2nd 3rd mean deet 8 9 5 7.33 68% 8 9 5 7.33 68% wild thyme 2 3 5 3.3 84% 4 2 1 2.3 88% sweet basil 4 1 3 2.6 86% 2 6 4 4 81% kafure 4 6 7 5.6 74% 6 5 8 6.3 72% lemongrass 6 3 4 4.3 80% 1 2 4 3.3 84% the synthetic repellents deet and pyrethroids containing formulations commonly sold in markets and insecticides sales vender. the side effects of these two chemical groups was reported by several research scientists, accordingly plant-based repellents not only minimize the use of synthetic chemicals and economic cost and save huge amount of money spent on synthetic compounds, also protects the environment, health of users. the plants screened for phytochemical analysis seemed to have the potential to act as a source of useful chemicals and also to improve and help in the health status of the people exposed to mosquito's bits (table 1). this study justifies the use of plant species in medicinal field to protects human from mosquito's bits and hence minimize the malaria spreading. there are different plants which have mosquito repellent activity and this activity affected with species the environment and other factors. and the chemical constituent of these show different results on the mosquito repellent. the different plant extract contains different chemical constituent shows different results on the mosquito repellent activities (tables 2-7). this study investigated the mosquito spatial repellent effihighlights in bioscience page 5 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species cacy of the basil, wild thyme, kafure and lemongrass, (ethanol, hexane and mixture of both ethanol extract and hexane extract). the present study results provided information and observations on the repellency activity of tested four plant species les against anopheles mosquitoes. mosquitoes usually make surreptitious landings on exposed skin to feed this why most people wearing protective clothing stands as a good measure of preventing them from vector contact. application of these plants les prevented landing attempts and bites by mosquitoes; this verifies already documented evidence that some plants le is a potent mosquito repellent. sweet basil o. basilicum eos are materials that are traditionally used in sudan the mosquitoes by burning leafs and flowers. however, coker et al., [15] stated that certain characteristics, e.g. volatility, limited its effectiveness. active ingredients, e.g. terpenes (2◦ metabolites) and oil components are responsible for the repellency activity. for a plants material to be valuable as a mosquito repellent it must effectively prevent insect bites on the treated area for several hours, and it must work in different environmental conductions, should be environmentalfriendly when applied to human skin, it must acceptable and having a pleasant odor, it should also be harmless to clothing, it should have a relatively low cost and effective against other biting insects such as flies. basil leaves extract provide different spatial repellency against mosquitoes with different concentration, in ethanol, hexane and mixture of ethanol and hexane extract (tables 2, 4 and 6). ethanol extract provided 30.6 and 42.6 min 32 and 45% for 50% and 100% ethanol extract respectively and the hexane extract was provided 44 and 116 min and evaluated with 46% and 122% for 50% and 100% respectively when compared with deet, while the mixture of ethanol and hexane extract shows that basil was provides the protection time in both (50% and 100%) was 43 min and 87 min respectively and this repellency act as 45% and 91.9% when compared with standard mosquito repellent deet (94.6 min protection time) respectively (tables 3, 5 and 7). basil hexane leaves extract provide the highest repellency against mosquitoes (116 min), baba et al., [16] conclude that hexane basil leaves extract provided 183 min and 303 min cpt from concentration 50% and 100% respectively (tables 4 and 6). in this study; the presence of ethanol extract in the mixture was reduced the repellency of mixture from the 116 to 87 min in 100% concentration; so the tannin and alkaloids may responsible for the repellent activity in hexane extract according to the phytochemical analysis (table 1). the phytochemical analysis was preceded an alkaloids, tannins, saponins and terpens were found in the ethanol extract, while alkaloid and tannins only detected in hexane extract.. wild thyme ethanol extract, the wild thyme was provides the highest protection time in both (50 and 100) %137.3 min and 182 min respectively when compared with other plant ethanol extract, and repellency at 154% and 192%, respectively when compared with standard mosquito repellent-deet (94.6 min protection time). active movement away from treated arm within minute of application was observed in wild thyme, and this strong repellent activity may caused by terpens group which appear in ethanol and hexane extract (according to phytochemical analysis) as main compounds and thymol (monoterpenoid) was highest concentration (54.96% and 57.42%) in ethanol and hexane extract respectively according to gc-ms analysis, and this was confirm by the study done by pandey et al., [17] they prove the activity of thymol that repel an. stephensi adults at the dose of 25.0 mg/mat after 1 h duration, while same degree of repellency was obtained by the oil at the dose of 55.0 mg/mat, which indicating thymol has double activity than the essential oil obtained from seeds of trachyspermum ammi (linn.). in present study beside the alkaloids and tannins present in ethanol extract only (phytochemical analysis), the hexane extract of the wild thyme is provides the highest protection time in both (50 and 100) % 174 min and 228 min respectively when compared with other plant leaves extracts and this highest cpt and repellency as 183% and 241% respectively, when compared with standard mosquito repellentdeet (94.6 min cpt). regarding the mixture of ethanol and hexane extract of each of the tested plants, wild thyme is provides the highest protection time in both (50 and 100%) 182.3 min and 215 min with spatial repellency at 193% and 227% respectively, compared to deet. the main compounds detected in ethanol and hexane extract was thymol which shows considerable anti-feedant activities against insects in many studies conducted by gonzalo et al., [18] and won et al., [19] shows that the wild thyme essential oil was rich source monoterpenes, including thymol, p-cymene, carvacrol, linalool, and o-terpinene, were assessed to determine their repellent activities to the mosquitoes. o-terpinene had a potent spatial repellent activity with a protection rate of essential oil topical treatment while the carvacrol and thymol showed an equivalent level of repellency. the wild thyme was the best one compared with the rest plants it was provided highest cpt; in both ethanol and hexane extract. however, the hexane 100% was provided the protection until 228 min, this period even longer than deet (2.4 times). according to phytochemical analysis the spatial repellency of wild thyme could be rise from terpens mainly because the hexane extract shows the terpens only. although provided the highest cpt and the ethanol extract which contain terpens beside the alkaloids and tannins show low cpt. our study show that thymol and eucalyptol (monoterpenoid) was main compounds in either ethanol and hexane extract and its concentration was corresponding with spatial repellency period, the repellency of plant extract could be due to the eucalyptol and this was proved by james et al., [20] they found that mosquito feeding and ovipositional repellency due to the presence of major monoterpenoid in the chemical volatile oil (i.e., 1,8-cineole (eucalyptol) identified in plant extract of hemizonia fitchii (asteraceae). phytochemicals are the principal active components that are highlights in bioscience page 6 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 8. chemical constituent detected appear in the gc-mass chromatogram in both ethanol and hexane extract of kafure leaf’s and area percentage of peaks. id name area% ethanol area% hexane 1. alpha-pinene 3.38 6.77 2. d-limonene 0.19 0.55 3. eucalyptol 33.11 26.93 4. gamma.-terpinene 0.30 0.47 5. pinocarvone 1.06 1.94 6. 2-oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl-, acetate 0.25 0.37 7. bicyclo(3.1.1)heptane-2,3-diol, 2,6,6-trimethyl0.18 0.24 8. copaene 0.19 0.42 9. alloaromadendrene 0.39 0.26 10. caryophyllene 0.76 0.92 11. aromandendrene 7.02 7.07 12. alpha.-guaiene 1.60 2.37 13. 2-naphthalenemethanol, decahydro-.alpha.,.alpha.,4a-trimethyl-8-methylene-, [2r-(2.alpha.,4a.alpha.,8a.beta.)] 15.20 20.23 table 9. chemical constituent detected appear in the gc-mass chromatogram in ethanol extract of kafure leaves and area percentage of peaks. id name peak area(%) 1. alpha-phellandrene 0.79 2. p-cymene 1.78 3. 3-methyl-4-cyclohexene-1,2-dicarboxylic anhydride 0.20 4. benzene, 1-methyl-4-(1-methylethenyl)0.67 5. isoledene 0.11 6. 1-tridecene 0.12 7. bicyclo[2.2.2]octa-2,5-diene, 1,2,3,6-tetramethyl0.15 8. cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, [1s-(1.alpha.,2.beta.,4.beta.)]0.04 9. naphthalene, decahydro-4a-methyl-1-methylene-7-(1-methylethenyl)-, [4ar-(4a.alpha.,7.alpha.,8a.beta.)]0.18 10. 1h-cyclopropa[a]naphthalene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,7,7a-tetramethyl-, [1ar-(1a.alpha.,7.alpha.,7a.alpha.,7b.alpha.)0.21 11. (s)-(-)-4-isopropenyl-1-cyclohexene-1-carboxylic acid 1.98 12. azulene, 1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1r-(1.alpha.,3a.beta.,4.alpha.,7.beta.)]1.42 13. guaia-1(10),11-diene 1.37 14. 7-epi-cis-sesquisabinene hydrate 0.79 15. globulol 9.94 16. phytol, acetate 3.03 17. 3,7,11,15-tetramethyl-2-hexadecen-1-ol 0.55 18. 9-eicosyne 1.04 19. n-hexadecanoic acid 3.90 20. squalene 8.10 believed to exhibit the medicinal activity of the plants and possibly the repellent activity of the oils. according to banthorpe [21] and heinrich et al., [22] terpenoids could be responsible for the flavor of fruits, the fragrance of the flowers and the quality of agricultural products. therefore coker et al., [15] prove that the presence of terpens is speculated to be associated with fragrance material and spatial repellent activity of oils. kafure kafure leaves extract provided different cpt by (50% and 100%) concentration, in ethanol; hexane and ethanol and hexane extract (mixture) (tables 2, 4 and 6). ethanol extract provide 15 min and 32 min by 50% and 100% concentration respectively. however the hexane extract provide 18 min and 28 min complete protection by 50% and 100% concentration respectively when the mixture provided 15 min and 19 min complete protection by 50% and 100% concentration respectively. the result shows that hexane extract was slightly stronger repellent than the ethanol and the mixture; the phytochemical analysis shows the presence of the: saponins, tannins, and alkaloids and absence of triterpens in both ethanol and hexane extract; while the gc-ms detected some of monoterpenes like euclyptol, (cyclic) 33.11% and 26.93% and 2-naphthalenemethanol, decahydro-alpha, alpha, 4a-trimethyl-8-methylene-, [2r-(2alpha, 4aalpha, 8abeta )]15.20% and 20.23%, in ethanol and hexane extract respectively, and globulol was 9.94% in ethanol extract and hexane extract was contained (-)-globulol 18.06% so this variety may responhighlights in bioscience page 7 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 10. chemical constituents detected appear in the gc-mass chromatogram in hexane extract of kafure leaves and area percentage of peaks. id name peak area(%) 1. camphene 0.14 2. bicyclo[3.1.0]hex-2-ene, 4-methylene-1-(1-methylethyl)0.07 3. beta-pinene 0.22 4. beta-myrcene 0.06 5. o-cymene 3.58 6. doconexent 0.20 7. butanoic acid, 3-methyl-, 3-methylbutyl ester 0.28 8. alpha-campholenal 0.14 9. terpinen-4-ol 1.58 10. l-.alpha.-terpineol 1.67 11. ethanone, 1-(6,6-dimethylbicyclo[3.1.0]hex-2-en-2-yl)0.19 12. 2-cyclohexen-1-one, 3-methyl-6-(1-methylethyl)0.08 13. citral 0.15 14. 1h-cyclopropa[a]naphthalene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,7,7a-tetramethyl-, [1ar-(1a.alpha.,7.alpha.,7a.alpha.,7b.alpha.) 0.20 15. 12-oxatetracyclo[4.3.1.1(2,5).1(4,10)]dodecane, 11-isopropylidene0.17 16. naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2-(1-methylethenyl)-, [2r-(2.alpha.,4a.alpha.,8a.beta.)]1.38 17. beta-humulene 0.31 18. 2h-3,9a-methano-1-benzoxepin, octahydro-2,2,5a,9-tetramethyl-, [3r-(3.alpha.,5a.alpha.,9.alpha.,9a.alpha.)]0.22 19. gamma-muurolene 0.21 20. aromadendrene, dehydro0.20 21. 2-oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl0.26 22. 1h-cycloprop[e]azulen-4-ol, decahydro-1,1,4,7-tetramethyl-, [1ar-(1a.alpha.,4.beta.,4a.beta.,7.alpha.,7a.beta.,7b.alpha.)]1.32 23. (-)-globulol 18.06 24. caryophyllene 0.47 25. 1h-benzocycloheptene, 2,4a,5,6,7,8,9,9a-octahydro-3,5,5-trimethyl-9-methylene-, (4as-cis)0.27 sible of different repellency and the 32 min was highest cpt achieved by kafure ethanol extract. in the study conducted by fathelrahman [23] showed ethanol extract 10% concentration provide 17% repellency for 60min. the result was corresponding with this study that increasing extract concentration of kafure was increase the repellency in both extract and the kafure extract, although was weak in spatial repellency. according to the result of gc-ms analysis of the chemical compound of kafure leaves extract of the current study (tables 8, 9 and 10); the results were corresponding with the gc-ms analysis of essential oil yield and composition in eucalyptus species in montenegro coastline by slavenko, et al; [24] show the presence of α-pinene, β-pinene, β-myrcene, α-phellandrene, α-terpinene, p-cymene, eucalyptol γ-terpinene, terpinene-4-ol, α-terpineol, aromadendrene, alloaromadendrene, also was found in this study. according to the gc-ms analysis of the present study prove the presence of o-cymene 3.58%, terpinen-4-ol 1.58%, l-.alpha.terpineol1.67%, naphthalene, 1, 2, 3, 4, 4a, 5, 6, 8a-octahydro4a, 8-dimethyl-2-(1-methylethenyl), l-alpha -terpineol 1.67% [2r-(2.alpha.,4a.alpha.,8a.beta.)]-1.38%, 1h-cycloprop[e] azulen-4-ol,decahydro-1,1,4,7-tetramethyl,[1a r (1a alpha, 4beta, 4a beta, 7alpha, 7a beta, 7b alpha)]-1.32%, (-)-globulol 18.06%, 2-naphthalenemethanol, decahydro -alpha, alpha, 4a-trimethyl -8-methylene-,[2r-(2alpha,4a alpha, 8a beta)]20.23%, was found in the hexane leaves extract and not found in ethanol extract, and alpha-pinene was 6.77% in hexane extract and 3.38% in ethanol extract. lemongrass application of cymbopogon oil prevented landing attempts and bites by mosquitoes; and this verifies already documented evidence by baldacchino et al., [25] and prabhakar et al., [26] that cy. citrates extract is a potent mosquito repellent. however, the interval of protection is a matter of concern; in this study, lemon grass leaf extracts provided protection from mosquito for 3 hours after which landing attempts and bites were experienced. this observation corroborates previous records of plants essential oils by bhupen et al., [27]. according to bhupen et al., [27] has demonstrated the essential oils from a variety of plant species been used as insect repellents; such as cymbopogon. in the present study lemongrass leaves extract provided different cpt with different concentration (50% and 100%) in ethanol, hexane and ethanol and hexane extract (mixture). ethanol extract provided 19 min and 28 min by 50% and 100% concentration respectively (tables 2 and 4). however the hexane extract provide 28 min and 54 min complete protection by 50% and 100% concentration respectively (table 6). while the mixture provided 19 min and 26 min complete protection by 50% and 100% concentration respectively. highlights in bioscience page 8 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species the 54 min was maximum cpt provided by lemon grass (hexane extract) with spatial repellency is equal 57% compared with deet, and also was low when compared with wild thyme and basil extract. in phytochemical studies both ethanol and hexane extract was showed the alkaloid, tannins and saponins while the terpens and flavonoids were absent and those groups were important as insect repellent. although monoterpenes was detected by the gc-ms as main compounds as 2,6-octadienal, 3,7-dimethyl, (z)-in ethanol extract and 2,6-octadienal, 3,7-dimethyl-, (z)and 2,6-octadienal, 3,7-dimethyl-, (e)in hexane extract. in the study conducted by vinutha et al., (28) on essential oil yield from aerial part of cymbopogon flexuosus; myrcenol, linalool, trans-chrysanthemal, 3,6,6-trimethyl -cyclohex-2-enol, citronellal, (-)-isopinocampheol, 1-pentanol, 5-cyclopropylidene-3undecyne, 3-carvomenthenone, (z)-linalool oxide, (furanoid), neral, geranial, β-vatirenene, citronellol, dextro-carvone, cycloisolongifolene, trans-(-)-carveol, cis-carveol, nerol, oxiranmethanol, 3-methyl-3(4-mathyl-3-pentenyl), bicyclopentylone, geranic acid, geranyl acetate, most of those compounds were detected in lemongrass extract in this study. according to the result, undiluted hexane leaves extract ( 100% ) provide maximum cpt according to the follows: 228 min wild thyme, 116 min basil, 54 min lemongrass and 28 min kafure, thus the hexane extract was seem more stronger in repellency than the ethanolic extracts, and the coleus is the best and effective as mosquitoes repellent than other plants, followed by basil and lemon grass and kafure. the results of the current work indicated that the spatial repellent potential of the leaves extract oils is concentration dependent. however it is observed that all the thyme leaves extract oils indicated protection of at least 2.5 h at 50% concentration and 2.5-5 h protection at 100%. accordingly the use of plants leaves extract essential oil in prevent mosquito bite is expected to reduce the cost and environmental effects of mosquito repellents. according to annamaria et al., [29] showed volatile compounds detected in bixa orellana samples the major volatile compound present in seed extracts proved to be source of repellent activity (mainly of sesquiterpenes, monoterpenes, arenes and as α-humulene). these volatile compound found in most of our chemical analysis to the four plants which reflects the repellent activity of the test extract especially thyme. in conclusion the preliminary findings of this laboratory evaluation bioassay of the repellent potential of the essential oils of ocimum basilicum l., coleus forskohlii, eucalyptus camaldulensis and, cymbopogon flexuosus steud, leaves extracts have confirmed their traditional use as a broad mosquito repellent agent. further investigations leading to identification of the lead compounds exhibiting the repellent activity is recommended. this would enhance a robust development of plant based oil for protection against mosquito bite. basil provided considerable repellency against mosquitoes. wild thyme was most effective which provided 105 min cpt evaluated with 205% in compare to the control (deet, lavender). the examined plants will be useful if the further studies have been conducted and it will replace the commercial mosquitoes repellent (deet, lavender). terpenoids were found in most of the extracts, but in the c. forskohli with the highest concentration. the presence of the (+)-4-carene, benzene, 1methyl-3-(1-methylethyl)-, 2-naphthalenemethanol, decahydrowere related with hexextracts of c. forskohli only, and it has strong repellency activity. acknowledgements we appreciate the assistance from blue nile institute for communicable diseases for providing the mosquitoes for the studies. we thank ministry of higher education and scientific researchcommission of scientific research and innovation in supporting the research fund and thanks to all colleagues helping in a way or another to let this work done. references 1. soonwera m, phasomkusolsil s. mosquito repellent from thai essential oils against dengue fever mosquito (aedes aegypti (l.)) and filarial mosquito vector (culex quinquefasciatus(say)). african journal of microbiology research. 2014; vol. 8(17), pp. 18191824. 2. madhumathy ap, aivazi aa, vijayan va. larvicidal efficacy of capsicum annum against anopheles stephensi and culex quinquefasciatus. j vector borne dis. 2007; 44: 223-226. 3. kumar sv, mani p, john btmm, ravi kg. mosquito larvicidal, oviposition deterrent and repellent properties of acalypha indica l. extracts against aedes aegypti, anopheles stephensi and culex quinquefasciatus. international journal medicine bioscience. 2012; no 1: 33-41 4. salih aoa, salih mm, ali mm. bioassay of herbal mosquito repellent formulated from the essential oil of ocimum basilicum. american journal of applied chemistry. 2018; vol. 6, no. 4, pp. 132-141. 5. peterson c, coats j. insect repellentspast, present and future, review the history of insect repellents from the 1920s down to recent work on plant-derived repellents. pesticide outlook .2001; 154-158pp. 6. das ng, baruah i, talukdar pk, das sc. evaluation of botanicals as repellents against mosquitoes. journal of vector borne disease. 2003;40: 49-53. 7. pronczuk j, bruné mn, gore f. children's environmental health in developing countries. in encyclopedia of environmental health. jerome o. nriagu, ed. 2011, pp. 601–610. 8. curtis cf. personal protection methods against vectors of disease. rev med vet entomol, 1992; 80: 543-553. 9. choochote w, chaithong u, kamsuk k, jitpakdi a, tippawangkosal p, tuetun b, champakaew d, pitasawat b. repellent activity of selected essential oils against, aedes aegypti. fitoterapia. fitoterapia. 2007; 78:359-364. 10. trongtokit y, curtis cf, rongsriyam y. efficacy of repellent products against caged and free flying anopheles stephensi mosquitoes. southeast asian j trop. med public health, 2005; 36:14231431. highlights in bioscience page 9 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species 11. frances sp, klein ta, hildebrandt dw, burge r, noigamol c, eikarat n, sripongsai b, wirtz ra. laboratory and field evaluation of deet, cic-4 and ai3-3722o against anopheles dirus (diptera: culicidae) in thailand. j med entomol. 1996; 33:511515. 12. balbaa ay, zaki ay, shamy am. qualitative and quantitative study of the flavonoid content of the different organs of sophora japonica at different stages of development. planta med. 1974; 25: 325–330. 13. harborne aj. phytochemical methods: a guide to modern techniques of plant analysis. 3rd ed. springer netherlands; 1998. 14. teklani pwnn, perera bgk. mosquito repellent and larvicidal activities of acalypha indica leaf extracts. int. j pharm. pharmacol. 2017; 1(2): 107. 15. coker hb, chukwuanim cm, ifudu nd, aina ba. the malaria scourge, concepts in disease management. nig. j. pharm. 2000; 32: 19-47. 16. baba g, lawal ao, shariff hb. mosquito repellent activity and phytochemical characterization of essential oils from striga hermonthica, hyptis spicigera and ocimum basilicum leaf extracts. british journal of pharmacology and toxicology. 2012 apr 25; 3(2) :43-8.. 17. pandey sk, shikha u, tripathi ak. insecticidal and repellent activities of thymol from the essential oil of trachyspermum ammi (linn) sprague seeds against anopheles stephensi. parasitol res. 2009; 105(2):507-512. 18. gonzalo ortiz de elguea-culebras; raúl sánchez-vioque; maría isabel berruga; david herraiz-peñalver and omar santana-méridas. antifeedant effects of common terpenes from mediterranean aromatic plants on leptinotarsa decemlineata. journal of soil science and plant nutrition. 2017; 17 (2), 475-485. 19. won-sik c, byeoung-soo p, sae-kwang k, sung-eun l. repellent activities of essential oils and monoterpenes against culex pipiens pallens, journal of the american m osquito control association. 2002;vol.18, no.(4):348-351. 20. james ak, mark vd, manuel fb. 1,8-cineole (eucalyptol), a mosquito feeding and ovipositional repellent from volatile oil of hemizonia fitchii (asteraceae). journal of chemical ecology. 1987(13), pp:2131–2141. 21. banthorpe dv. classification of terpenoids and general procedures for their characterization. in: charlwood, b.v. and d.v. banthorpe, (eds.), methods in plant biochemistry. terpenoids. academic, london, 1991; 7: 1-41. 22. heinrich m, pieroni a, bremner p. "medicinal plants and phytomedicines" in: the cultural history of plants. taylor and francis, new york, routledge, 2005;pp: 205-238. 23. fathelrahman ie. effect of organic extracts of cafure leaves (eucalyptus camaldulensis dehn.) on mosquitoes (anopheles arabiensis patton.)gezira journal of agricultural science, 2011; vol., 9 no2. 24. slavenko g, dejan o, maria c, emilija j, dušan b, kristina b, neda md. variation of essential oil composition of eucalyptus camaldulensis (myrtaceae) from the montengero coastline, biblid: 1450-7188 (2010) 41, 151-158. 25. baldacchino f, tramut c, salem a, lienard e, deletre e, franc m, martin t, duvallet g, jay-robert p. the repellency of lemongrass oil against stable flies tested using video tracking, parasite, 2013; 20:21. 26. prabhakar k, kinnera k, priya kk, peele ka. investigation of the repellence activity of bio-out, a natural mosquito repellent. international journal of life sciences, biotechnology and pharma research. 2013; 2:3. 27. bhupen k, somi b, sharma ak. plant essential oils as mosquito repellent: a review. international journal of research and development in pharmacy and life sciences, 2013; 3(1): 741-747. 28. vinutha m, suchetha m. and k. j. thara saraswathi. effect of different a biotic stress on essential oil yield from aerial part of cymbopogon flexuosus (nees ex steud) wats. int. j. adv. res. 2016; 4(12), 1490-1504. 29. annamaria g, pietro de m, giuseppe g, luca mc, sara p. secondary metabolite profile, antioxidant capacity, and mosquito repellent activity of bixa orellana from brazilian amazon region, journal of chemistry volume 2013; /article id 409826/ 10 pages https://doi.org/10.1155/2013/409826. highlights in bioscience page 10 of 10 march 2021|volume 4 https://doi.org/10.1155/2013/409826 http://bioscience.highlightsin.org/ abstract introduction materials and methods collection of plant material and extraction formulation of plant leaves extracts anopheline mosquito bioassay test phytochemical analysis gas chromatography–mass spectrometry data analysis results phytochemical analysis ethanol extracts hexane extract bioassay chemical constituents detected in kafure leaf extracts discussion sweet basil wild thyme kafure lemongrass acknowledgements references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202205 review open access 1 department of animal hygiene and zoonoses, faculty of veterinary medicine, university of sadat city, egypt. 2 faculty of veterinary medicine, cairo university, egypt. 3 department of biophysics, faculty of science, cairo university, egypt. 4 biotechnology program, faculty of science, cairo university, egypt. 5 department zoology and chemistry, faculty of science, cairo university, egypt. 6 department of bacteriology, mycology and immunology, faculty of veterinary medicine, university of sadat city, egypt. * to whom correspondence should be addressed: vet_noura@yahoo.com editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): santosh k maurya, department of biochemistry, central university of punjab, bathinda, punjab, india. amira m. elsherbini, department of oral biology, faculty of dentistry, mansoura university, mansoura 35116, egypt. received: september 7, 2022 accepted: december 15, 2022 published: december 29, 2022 citation: eissa n, badrkhan sm, mohamed ma, shaban jy, shahban rs, dawoud m. xenotransplantation: past, present, and future directions. 2022 dec 29;5:bs202205 copyright: © 2022 eissa et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. xenotransplantation: past, present, and future directions nourhan eissa*1 >< , salma m. badrkhan2 >< , maha a. mohamed3 >< , joumana y. shaban4 >< , rahma s. shahban5 >< , mai dawoud6 >< abstract xenotransplantation, in its broadest sense, is the transplantation, implantation, or infusion of cells, tissues, or organs from one species to another. while there is a high demand for human tissues, cells, and organs for use in clinical transplantation, they are often in short supply. recent scientific and biotechnological advancements, coupled with the scarcity of human allografts, have led to renewed interest in developing exploratory treatment strategies that use xenotransplantation products in human recipients. however, despite its potential benefits, the use of xenotransplantation is still limited due to various considerations, as discussed in this review of the past, present, and future directions of xenotransplantation. one of the key ethical concerns surrounding xenotransplantation is the potential impact on the animals from which the cells, tissues, or organs are obtained. as with genetic modification to fix genetic defects or prevent disease, the ideal outcome for these animals is that they will be better off as a result of the change. however, unless there are major changes in the way science is taught to incorporate ethics into recognized scientific theory and practice, these concerns will not be adequately addressed. keywords: donor animals, ethical issues, immunological barriers, religious considerations, xenotransplantation introduction despite the fact that there are over 135,000 transplants carried out annually throughout the world, this still only accounts for less than 10% of the true global needs for failing organs (such as kidneys, skin, testicles, hearts, livers, lungs, bones, small bowels, and pancreas, etc.) due to a lack of donors. this is true even though living donor transplants have been performed since the 1960s [1]. this fact has prompted medical professionals and researchers worldwide to develop a "bridge the gap" technique called xenotransplantation (cross-species transplantation, implantation or even infusion of live cells, tissues or even organs, especially from pigs and nonhuman primates to humans) in order to provide an immediate and limitless supply of transplantable organs that could aid in the treatment of many disorders [1; 2]. while an in-depth discussion of the history of numerous successful clinical attempts at xenotransplantation is impractical for the current review paper, it is important to highlight the key contributions that helped the field get to where it is nowa shining example of the power of science and medicine working together for the greater good. the cultural backdrop of xenotransplantation, religious beliefs, ethical considerations, desirable qualities of donor animals, challenges that the xenotransplantation procedure faces, and the influence of xenotransplantation on zoonotic risk are all briefly reviewed in this study. another important consideration is the potential for the spread of diseases from animals to humans. because the cells, tissues, or organs used in xenotransplantation come from another species, there is a risk that they may carry diseases that are not present in humans. this could potentially lead to the spread of new diseases or the exacerbation of existing ones. to minimize this risk, it is important to carefully screen the cells, tissues, or organs before they are used in xenotransplantation, and to implement strict protocols to prevent the spread of disease. despite these challenges, researchers are continuing to explore the potential of xenotransplantation as a way to overcome the shortage of human allografts. highlights in bioscience page 1 of 15 december 2022|volume 5 https://doi.org/10.36462/h.biosci.202205 https://creativecommons.org/licenses/by/4.0/ vet_noura@yahoo.com https://orcid.org/0000-0002-3622-6023 shamms951@gmail.com https://orcid.org/0000-0002-0407-1151 mhmohamed2013@gmail.com jomanayousef12@gmail.com https://orcid.org/0000-0002-5038-0270 rrsaid315@gmail.com https://orcid.org/0000-0002-2082-7692 mai_dawoud30@yahoo.com http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions in the future, it is likely that advances in science and technology will make it possible to overcome many of the challenges currently facing xenotransplantation , paving the way for its wide– spread use in clinical transplantation. chimeras in folklore historically, folklore had long contained accounts of chimaeras (i.e. monstrous creatures composed of parts of multipleăspecies) before the technique of xenotransplantation was even considered. people were sporadically shown in prehistoric cave paintings, but the sole example of a human is a man with a bird’s head in the lascaux cave in france (about 15,000 bc), which is where the stories of vampires and werewolves (half man, half beast) originated. the great sphinx of giza (about 2500 bc) features a lion body with a human head in contrast to the gods of ancient egypt (anubis), who were commonly depicted with a human body and an animal (jackal) head. additionally, a sanskrit document from the 12th century bc has the first account of xenotransplantation in indian mythology, which describes ganesha, a huge infant with an elephant-like head (a son of two indian gods, shiva and parvati). in addition, xenotransplantation was depicted in greek mythology through the likes of the minotaur (a man with a bull head), esfinge (a winged lion with a woman head), and centaurs (horses with a man’s head and trunk), as well as in homer’s odyssey, which featured chimaeras that were half-swine, half-man (about 750 bc) [3; 4; 5; 6]. history of clinical experiences with xenotransplantation the idea of human xenotransplantation attempts actually got started in the 17th century with the first attempt to transfuse sheep blood into people in 1667 (figure ??)[7]. in reality, scientists and doctors are unable to create true human-animal chimaeras followed by an opacified human cornea was replaced with a transparent porcine cornea [8] and a kidney xenotransplantation from a rabbit occurred in 1905 [9; 10], the clear pig cornea was then used to replace an opaque human cornea, and in the early 1970s, successful corneal xenotransplantations from fish and gibbons were performed [10; 11]. additionally, a clinical study of kidney xenotransplantation from a chimpanzee to humans was conducted between 1963 and 1964 [12]. this was followed by the first attempts at heart xenotransplantation from chimpanzee and baboon donors in 1964 [13] and 1984 [14], respectively. using baboon donors, the first successful liver xenotransplantation procedure was carried out in 1992 [15]. clinical xenotransplantation experiments have not been conducted in the united states or the majority of european nations since the 1990s because of certain xenozoonoses, immunological concerns, surgical effectiveness, and other regulatory concerns [16]. but according to reported reports, between 2013 and 2017 china and russia used xenotransplantation to cure diabetes patients using transplanted neonatal pig islets [17]. figure 1. historical recorded trials concerning xenotransplantation in different organs. blood xenotransfusion if we delve beyond myth and folklore, we find jean baptiste denis started the therapeutic practise of transfusing animal blood into humans [18; 19]. results were conflicting and not surprising. consequently, xenotransfusion was outlawed in france for a while. a strong case could be made for using pigs as a source of blood cells and blood products (if they are maintained in ideal "clean" conditions and are periodically checked to ensure no infectious agent is being passed) given the current threat of infectious pathogens being transferred and the need for future human blood transfusions [19]. in actuality, this method has been reevaluated by a number of studies [20]. blood vessel anastomosis more scientific developments had to wait until the 20th century, when french experimental surgeon alexis carrel devised surgical methods for anastomosing blood arteries, enabling the first successful organ transplant to occur. carrel worked first in france and subsequently in north america [21]. skin xenotransplantation various animal species and humans began using skin grafts in the 19th century when either pedicle or free skin grafts were used as the skin transplants. the donor, which may be a sheep, a rabbit, a dog, a cat, a rat, a chicken, or a pigeon, had to stay immobile while connected to the patient for a period of days so that the recipient could reportedly vascularize the graft. the perfect transplant would have looked like it was taken from a frog since they occasionally had "skinned alive" skin. when used to cover skin ulcers, it’s likely that some of these grafts were "successful" in the sense that they provided protection, at least for a few days, as the ulcer healed below them. but it’s likely that none of the grafts turned out to be long-lasting [22; 23]. corneal xenotransplantation corneal xenotransplantation, the process of transplanting corneas from one species to another, has a long and fascinating history. highlights in bioscience page 2 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions the first recorded corneal xenotransplantation was actually performed from a human to a dog in 1838 by dr. samuel d. gross [24]. however, it wasn’t until 1905 that the first corneal allograft, a transplant from a person to a pig, was successfully completed [10; 25]. since then, the field of corneal xenotransplantation has made significant progress, with various animal species, including pigs, rabbits, and monkeys, being used as potential donors [26]. despite these advances, the use of animal-derived corneal grafts in humans remains controversial, with concerns surrounding the potential transmission of diseases, the ethical implications of using animals as organ donors, and the potential immunological reactions of the recipient [27]. despite these challenges, research into corneal xenotransplantation continues, with the hope of eventually finding a reliable and safe alternative to human corneal transplantation, which is currently limited by the shortage of donor tissue [28]. cell xenotransplantation serge voronoff, a russian immigrant who settled in paris, had the concept of transplanting cells that produced a hormone that the recipient lacked. given the small number of human pancreases that become available each year, there is tremendous interest in utilising pig islets for this. but for older guys who had lost their "zest for life," voronoff’s main goal was to slow down ageing. he implanted chimpanzee or baboon testicles into a sizable number of male human patients [29; 30]. his method involved cutting the animal testicle into slices and inserting the pieces into the testicles of the recipients. on both sides of the atlantic, the treatment gained popularity, and several hundred of these surgeries were carried out. it is improbable that any of them had any positive effects besides psychological ones, yet there have been tales of extraordinary "rejuvenation" in men who have undergone surgery and reported having considerably more energy. because donor testicle slices may have necrosed and created infectious or inflammatory problems occasionally, the surgeries must have had significant complications. furthermore, the first kidney allotransplant was carried on 1933 [31] john brinkley maintained the concept of transplanting goat glandular tissue to produce hormones that the recipient would benefit from in the united states [32]. nevertheless, the development of several clinics, particularly in europe, where patients get injections of animal tissue or serum to treat a variety of disorders has ensured that the concept of cell xenotransplantation has endured to the current day. controversy has been created by the results [33]. xenotransplantation of the kidney by the 1960s, keith reemtsma of tulane university in louisiana had proposed that transplanting human recipients with nonhuman monkey kidneys might successfully treat renal insufficiency. at that time, french and american surgeons had spent a lot of effort on the concept of kidney transplantation, but there were not enough deceased person kidneys accessible, and chronic dialysis had not yet been invented. so long as organs from nonhuman animals couldn’t be procured, reemtsma thought the patient had no alternative but to pass away. he decided to get the organs from chimpanzees because of their close evolutionary relationship to humans. he carried out 13 of these transplants, each of which included giving the patient both kidneys from a chimpanzee (which generally weighs considerably less than an adult human) [12]. during autopsy, the chimpanzee kidneys showed no abnormalities or signs of acute or enduring rejection. the notion of employing non-human primates as kidney donors was pioneered by several surgeons, most notably by tom starzl who used baboons as donors in colorado [34], and his findings were comparable to those of reemtsma. others had insignificant contacts in the us and france [35]. xenotransplantation of the heart when james hardy visited reemtsma in 1963 and conducted the first human lung allotransplant, he was struck by the recipients of chimpanzee kidney transplants who were all in good condition. hardy decided to buy some chimpanzees as possible "donors" in 1964 in order to execute the first clinical heart transplant in the event that he was unable to find a deceased human donor. he had a less-than-ideal patient who would not be allowed for heart transplantation today due to his patient’s significant atheromatous vascular disease, for which he had both of his legs amputated, and the fact that he was semicomatose at the time the surgery was carried out. however, the patient’s rapid decline prompted hardy to perform a chimpanzee heart transplant [21]. because the chimpanzee heart was too tiny to maintain the circulation, it failed within a short period of time. contrary to the attempted lung allotransplantation, the heart xenotransplantation received a negative response from the public and medical community, which deterred hardy and his colleagues from trying again. the heart allotransplantation procedure was later developed by barnard and his collaborators in 1967 [21]. later, they carried out two heart xenotransplantations [36]. lung xenotransplantation only the maryland team has lately engaged in active lung xenotransplantation research. platelet sequestration and activation during gtko was discovered by [37]. the hcd46 pig lung perfusion by human blood was mostly caused by gpib, gpiib/iiia, and von willebrand factor. gtko is reduced by transgenic expression of the human leukocyte antigen (hla-e). the hcd46 pigs with xenograft pulmonary injury. ex vivo human blood perfusion models of the lungs of genetically altered pigs with drugs that suppress complement activation, coagulation, and inflammation dramatically improved lung xenograft survival in vivo [38]. liver xenotransplantation tom starzl, one of the most important pioneers in the area of kidney and liver allotransplantation, tried a few liver transplantations on young patients and nonhuman primates in colorado in highlights in bioscience page 3 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions the 1960s without long-term success [39; 40; 41]. in the 1990s, he and his pittsburgh team performed two liver transplants from baboons in adult patients, with one patient enduring 70 days of survival after tacrolimus was added to the immunosuppressive arsenal [15]. the results, however, were not convincing enough to warrant continuing this exploratory clinical trial. the pig [42] and other nonprimate mammals have been used in a few efforts, but they haven’t been very effective. most early attempts at therapeutic organ xenotransplantation obtained their organs from nonhuman primate species [35]. the first islet xenotransplantation an estimated 2 to 3 million persons in the united states alone have type 1 diabetes. since pig insulin varies from human insulin by just one amino acid and has been used successfully to treat diabetic patients for decades before recombinant human insulin became available, it is reasonable to anticipate that normoglycemia will result from a successful pig islet transplant. the first effort at pig islet transplantation in diabetic patients was undertaken in 1993 by a swedish team under the direction of carl groth [43]. features of the perfect donor animal include when we analyse the ideal qualities of animals suitable as organ donors for humans, a large list forms. the animal’s anatomy and physiology must first be compatible with humans for the desired organ to work well in them. the risk of an infection from one species (i.e., an animal) to another should also be eliminated. even human viral infections would not be able to pass through an excellent animal donor organ. this animal species should also be inexpensive to feed and produce because to its short gestation periods and frequent births each litter to achieve economies of scale. additionally, no immunologic obstacles to transplanting into humans should be present in such an animal. finally, there shouldn’t be much ethical debate about using this animal in this way. there is no animal species that satisfies all of the aforementioned requirements. apes and monkeys are nonhuman primates that resemble humans the most anatomically and physiologically. they might also be resistant to some human diseases. in reality, because of their hepatitis b and hiv resistance, baboon liver xenografts have been used in research [15]. but the xenotransplant community appears to have given up on the idea of utilising nonhuman primates as xenograft donors, mainly due to the hazards of infection for human patients and those who come in contact with them. some monkey viruses, like herpes 8, can kill people in a couple of days [44]. it is thought that raising pathogen-free herds in sufficient numbers to satisfy therapeutic demand would be prohibitively costly. last but not least, using nonhuman primates as human organ donors has serious ethical problems [45; 46]. due to its large litter sizes (up to 10 littermates), short gestation periods (4 months), anatomical and physiological similarities to humans, widespread use for human consumption (an estimated 90 million pigs are consumed annually in the usa), and lengthy history of providing medictable 1. the benefits and drawbacks of using pigs vs baboons as a source of organs and cells for people, as described by [18]. comparison pig baboon organ size in adults sufficient insufficient maintenance costs significantly inferior elevated human anatomy similarities moderately related very related human-like physiological similarities moderately related very related accessibility adequate inadequate relation with the immune system to humans distant related very related data of tissue typing significant (in selected herds) inadequate age of sexual maturity 4-8 months 3-5 years breeding potential good quality poor quality pregnancy period 114 ± 2 days 173-193 days offsprings per time 5-12 1-2 development fast (adult human size within 6 months) sluggish (9 years to reach maximum size) blood type compatibility with humans probably insignificant vital knowledge of genetic engineering significant none risk of transfer of infection (xenozoonosis) low high availability of specific pathogen-free animals yes yes public opinion more in favor mixed inals (skin, insulin, cardiac prostheses, and clotting factors) for humans, the pig has emerged as the most likely candidate for consideration as an organ donor. undoubtedly, considerable hurdles may arise due to significant discrepancies in the coagulation cascade and other aspects of porcine physiology [47; 48]. even though they are becoming more recognised, immunologic obstacles still need to be overcome. in addition, several diabetes treatments, such as immunosuppressive regimes and pancreatic islet transplantation procedures, were initially developed using the dog model. primate models with induced diabetes are being used more frequently as a result of recent developments toward the use of monoclonal antibody treatments for immunosuppression in human islet transplantation. researchers in several domains are thinking about using naturally occurring illness models in client-owned pets in addition to induced-disease models in large animals. this article will discuss how naturally existing canine diabetes can be used as a translational model for creating islet transplants for diabetic patients who are humans [49]. other pharmaceuticals of animal origin in table 2 we provide a list of various xenotransplantation products and their origins, generic names, product names, and therapeutic class. the table includes products from a variety of animal sources, including horses, pigs, mice, cows, and others. one of the key observations from the table is the wide range of therapeutic applications for xenotransplantation products. these products are used to treat a wide range of conditions, including respiratory problems, anticoagulants, antivenoms, and vaccines. this highlights the potential benefits of xenotransplantation as a way to overcome shortages of human allografts and provide treatments for a variety of medical conditions. another interesting aspect of the table is the diversity of animal sources used in xenotransplantation. the table includes products from horses, pigs, mice, and cows, among others. this highlights in bioscience page 4 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions suggests that a wide range of animals can be used as sources for xenotransplantation products, depending on the specific needs of the recipient and the availability of appropriate cells, tissues, or organs. overall, the table provides a useful overview of the past, present, and future directions of xenotransplantation. it highlights the potential benefits of using xenotransplantation products in clinical transplantation, as well as the ethical considerations and technical challenges that need to be addressed in order for it to be widely used. in addition to the observations mentioned above, the table also highlights the potential challenges of xenotransplantation. for example, one of the main challenges is ensuring that the cells, tissues, or organs used in xenotransplantation are compatible with the recipient’s immune system. if the transplant is rejected, it may be necessary to use immunosuppressive drugs to prevent rejection, which can have negative side effects for the recipient. another challenge is the potential for the spread of diseases from animals to humans. because the cells, tissues, or organs used in xenotransplantation come from another species, there is a risk that they may carry diseases that are not present in humans. this could potentially lead to the spread of new diseases or the exacerbation of existing ones. to minimize this risk, it is important to carefully screen the cells, tissues, or organs before they are used in xenotransplantation, and to implement strict protocols to prevent the spread of disease. despite these challenges, the potential benefits of xenotransplantation are considerable. in the future, it is likely that advances in science and technology will make it possible to overcome many of the challenges currently facing xenotransplantation, paving the way for its widespread use in clinical transplantation. this could help to alleviate the shortage of human allografts and provide new treatment options for a variety of medical conditions. issues with several xenotransplantation cases complications include immunological incompatibility, cell death, abnormal cell differentiation and proliferation, virus transmission from animals to humans, and ethical concerns hinder the clinical application of xenogeneic stem cell transplantation [50]. immune rejection immune rejection is unquestionably the problem with xenogeneic stem cell transplantation that worries people the most. immunological rejection is avoided using the following methods: only a few of the variables that need to be taken into account include the use of cellular desensitisation technology, immunosuppressive medications, suitable stem cell type selection, gene editing technology, encapsulated cell technology, the use of immunosuppressive drugs, and the regulation of cytokine levels. these procedures have increased the success rate of transplantations. selecting stem cells with low immunogenicity, immunosuppressive, and immunomodulatory traits may help to alleviate this problem [51]. injected immunocompetent mice with stem cells obtained from human umbilical cord stroma. the results showed that this kind of human stem cell has immunosuppressive and immunomodulatory properties [51]. later research showed that xenogeneic stem cells, in particular xenogeneic mscs, have low immunogenicity along with immunosuppressive and immune-modulatory capabilities [52]. porcine mscs have been used in xenotransplantation investigations because to their low immunogenicity attributes and immunomodulatory qualities [53]. pig umbilical cord mscs and swine escderived neural progenitors were implanted in non-immunocompr– omised rats [54]. their investigation revealed similar cell immunosuppressive effects [53]. the potential of these cells to suppress the immune system and have minimal immunogenicity was proven by the transplantation of rabbit umbilical cord mscs with hyaluronic acid/tricalcium phosphate scaffolds in rats [55]. by co-implanting rat mscs and pig neuroblasts in immunocompetent rat striata, [52] demonstrated the immunosuppressive characteristics of these cells. according to study by [56], rat adscs can protect themselves from human xenoantibodies and complement-mediated lysis. gal, or galactose-1, 3-galactose, is related with low expression and this capacity is cd59 dependent [56]. hyperacute rejection antibodies that are spontaneously generated against blood type antigens are similar to xenoreactive natural antibodies (xna). the epitope that these antibodies primarily target is the nonreducing trisaccharide group galactosyl a-(1, 3)-galactosyl b-1,4n-acetyl glucosaminyl, also known as the gal epitope15. man does not have this epitope because he lacks the enzyme that makes it. higher primates thus recognise the gal epitope as "non-self" and produce an immune response to it. numerous microbes16 contain the gal epitope, and humans are exposed to the antigen through their guts, where they develop anti-gal antibodies. the key mechanisms by which xna exerts its effects include natural killer (nk) cells, complement activation, and endothelium phenotypic alterations. the goal of research to date has been to lessen the effects of xna [60; 61]. acute humoral xenograft rejection (ahxr) the following challenge is delayed xenograft rejection, which is frequently seen. the primary histological features of ahxr are endothelial swelling or disruption, vascular thrombosis with blood extravasation, and interstitial oedema [62]. within 24 hours of transplantation, this generally develops, gets worse over the next few days, and finally kills the graft. the first response, which is mostly but not solely specific for the gal epitope, is mediated by igm, and is thereafter followed by an increase in igg levels [63]. by themselves, these xenograft natural antibodies induce a procoagulant state that develops into disseminated intravascular coagulation. even the best practises for limiting complement activation, lowering t-cell and b-cell driven immune responses, and diminishing xenograft natural antibodies highlights in bioscience page 5 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. different pharmaceutical products derived from non-human mammalian cells as represented by [57; 58; 59]. origin generic name product name therapeutic class equine (horse) conjugated oestrogen premarin gonadal hormone, oestrogen antithymocyte immuglobulin (atg) atgam immunosuppressant snake antivenom red back spider antivenom antivenom tiger snake antivenom green pit viper antivenin sea snake antivenin cobra antivenin taipan antivenom king cobra antivenin polyvalent snake antivenin medroxyprogesterone acetate premia gonadal hormone stonefish antivenom stonefish antivenom antivenom porcine (pig): coagulation factors ii, ix, x, v & vii prothrombinex-vf haemostatic agent heparin sodium heparinised saline anticoagulant amylase, lipase, pancrelipase, protease panzytrat digestive supplement poractant alfa curosurf respiratory agent danaparoid orgaran haemostatic agent human rotavirus live attenuated vaccine rotarix vaccine dalteparin fragmin anticoagulant rotavirus vaccine live oral pentavalent rotateq vaccine pancrelipase pancreatin creon digestive supplements & cholelitholytics enoxaparin clexane anticoagulant, antithrombotics zoster virus vaccine live zostavax vaccine vancomycin hydrochloride vancomycin hcl antibiotic, miscellaneous murine (mouse) trastuzumab herceptin antineoplastic agent cetuximab erbitux antineoplastic agent infliximab remicade monoclonal antibody antihemophilic factor viii (human) hemofil m antihemophlic agent bevacizumab avastin antineoplastic agent rituximab mabthera antineoplastic agent; monoclonal antibody golimumab simponi antirheumatic agent abciximab reopro anticoagulant palivizumab synagis immunomodifier somatropin saizen pituitary hormone basiliximab simulect immunomodifier bovine (cow) epinephrine adrenaline neurotransmitter sealerprotein solution+ thrombin solution tisseel vhs/d solution haemostatic agent collagen zyderm collagen implants dermatological preparations calfactant infasurf treatment of premature infant lungs hepatitis a vaccine vivaxim vaccine allantoin allantoin cosmetics, treatment of wounds & ulcers polygeline haemaccel plasma volume expander varicellazoster vaccine, live varivax vaccine calporo calporo herbal daily supplements insulin hypurininjection insulin preparations bovine colostrums travelan anti-diarrhoeal survanta beractant treatment of premature infant lungs cartilag cartilag herbal analgesics & anti-inflammatories continued on next page highlights in bioscience page 6 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. – continued from previous page. origin generic name product name therapeutic class bovine-manufacture acitretin novatretin antipsoriatic measles, mumps & rubella vaccine priorix vaccine itraconazole itrazol antifungal, azole derivative inox mebeverine hcl mebetin antispasmodics amoxycillin synamox antibiotic, penicillin loperamide colodium antidiareal modim mycophenolate mofetil cellcept immunosuppressant agent essential phospholipids livovid cholelitholytics rabies vaccine merieux vaccine rabipur hepatitisavaccine avaxim vaccine havrix hydrocortisone hydrocortisonorion corticosteroid clindamycin hcl tidact antibiotic, lincosamide recombinant antihaemophilic factor recombinate haemostaticagents nilotinib tasigna antineoplastic agent, thyroxine kinase inhibitor clofazimine fazim antibiotics, leprostatic ampicillin sod+ sulbactam sod unasyn antibiotic, penicillin rabies human diploid cell vaccine verorab vaccine hepatitis b vaccine engerix-b vaccine omeprazole omeprazole gastric acid secretion inhibitor, proton pump inhibitor calcitriol osteocap vitamin d analog diphtheria, tetanus & acellular pertussis vaccine adacel vaccine cyclosporin sandimmun immunosuppressant, calcineurin inhibitor pneumococcal vaccine prevenar vaccine doxycycline xidox antibiotics, tetracyclines derivatives celecoxib celebrex nsaid, cyclooxygenase-2 inhibitor phenytoin sodium dilantin anti-epilepsy dutasteride avodart 5-alpha-reductase inhibitor oseltamivir phosphate fluhalt antiviral, influenza, neuraminidase inhibitor diphtheria toxoid adt booster vaccine boostrix pancreatin creon pancreatic enzyme replacement danazol nazo androgen oxycodone hcl oxynorm opioids analgesic pregabalin lyrica anticonvulsant didanosine aurobindo antiretrovirals haemophilus b influenzae vaccine hiberix vaccine heparin sodium injection heparinol anticoagulant isotretinoin acnotin anti acne, antineoplastic agent recombinant antihaemophilic factor recombinate haemostatic agents influenza virus vaccine fluarix vaccine tacrolimus prograf immunosuppressant agent fluconazole fluconazole antifungals rivastigmine rivadem acethylcholinesterase inhibitor gem fibrozil gem fibrozil dyslipidaemic agents yellow fever vaccine 17d vaccine vaccine continued on next page highlights in bioscience page 7 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. – continued from previous page. origin generic name product name therapeutic class egg/chicken measles, mumps and rubella virus vaccine l m-m-r ii vaccine influenza virus vaccine agrippal vaccine measles, mumps and rubella virus vaccine priorix vaccine measles, mumps, rubella and varicella vaccine priorix-tetra & proquad vaccine rabies vaccine rabipur vaccine coxiella burnetii vaccine q-vax & q-vax skin test vaccine influenza virus vaccine vaxigrip vaccine risperidone rixadone antipsychotic agent verteporfin visudyne ophthalmic medication quadrivalent influenza vaccine afluria quad vaccine propofol propofol sandoz propofol-lipuro 1%/2% provive 1% & provive mct-lct 1% anaesthetics yellow fever vaccine stamaril vaccine olive oil and soya oil clin oleic 20% parenteral vitamins, minerals and nutrition sebelipase alfa kanuma endocrine and metabolic agent influenza virus vaccine fluarix vaccine quadrivalent influenza vaccine fluad quad vaccine quadrivalent influenza vaccine fluquadri vaccine influenza virus vaccine fluad vaccine trivalent influenza vaccine fluzone highdose vaccine clevidipine cleviprex antihypertensive agent influenza virus vaccine influvac vaccine propofol diprivan anaesthetics propofol fresofol 1% injection & fresofol 1% mct/lct anaesthetics soya oil intralipid parenteral vitamins, minerals and nutrition chinese hamster ovary (cho) cells aflibercept eylea ophthalmic medication follitropinalfa gonal-f pituitary hormone erythropoeitin alfa binocrit hematopoietic agent laronidase aldurazyme enzyme replacement therapy abatacept orencia immuno-modifier interferon beta-1a avonex immunomodifier rebif omalizumab xolair other respiratory agent etanercept enbrel tumour necrosis factor inhibitor panitumumab vectibix antineoplastic agents eptacog alfa novosevenrt haemostatic agent octocogalfa advate haemostatic agent kogenatefs lenograstim granocyte supportive therapy follitropinbeta puregon pituitary hormone nonacogalfa benefix haemostatic agent lutropin alfa luveris 75 iu pituitary hormone imiglucerase cerezyme enzyme replacement therapy dornasealfa pulmozyme respiratory agent continued on next page highlights in bioscience page 8 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. – continued from previous page. origin generic name product name therapeutic class dornasealfa pulmozyme respiratory agent alemtuzumab mabcampath antineoplastic agent trastuzumab herceptin antineoplastic agent choriogonadotropin alfa ovidrel pituitary hormone tenecteplase metalyse fibrinolytic agent darbepoietin aranesp haemopoietic agent recombinate antihaemophilic factor recombinate haemostatic agent agalsidasebeta fabrazyme enzyme replacement therapy epoietin alfa eprex haemopoieticagent rituximab mabthera antineoplasticagent methoxy polyethylene glycol-epoetinbeta micera hematopoietic agent denosumab prolia monoclonal antibody xgeva moroctocogalfa xyntha haemostaticagent epoetin lambda novicrit haemopoieticagent bevacizumab avastin antineoplastic epoietin beta neorecormon haemopoieticagent corifollitropin alfa elonva pituitary hormones sheep box jellyfish antivenom box jellyfish antivenom antivenom digoxin binding antibody digoxin-specific antibody fragment digifab antidote fish, shark and shell fish house dust mite extract acarizax antiallergy preparation chondroitin chondroitin complementary osteoarthritis inactivated influenza vaccine fluad vaccine glucosamine glucosamine complementary osteoarthritis phleum pratense. grazax antiallergy preparation insulin human insulin (rys) & protaphane mixtard 30/70. mixtard 50/50 insulin preparation rabbit funnel web spider antivenom (rabbit) funnel web spider antivenom antivenom highlights in bioscience page 9 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions sometimes fall short of addressing these issues. diffuse intravascular coagulation and thrombotic microangiopathy, which are related to postpone xenotransplant rejection, are caused by unknown processes. ahxr is the least well-known of the early xenograft rejection phases [64]. cell proliferation, aberrant differentiation, and death similar to the problems with cell replacement treatment, cell death and abnormal cell differentiation and proliferation directly led to the failure of xenogeneic stem cell transplantation and even injured the recipients. researchers have shown that the microenvironment of the cell culture affects cell differentiation and death. several researchers have attempted to change the microenvironment of the cells to prevent cell death and abnormal differentiation. here, we will discuss two common methods for changing the microenvironments of cell cultures to resemble the in vivo natural growth niche. one tactic is to change the traditional two-dimensional (2d) culture into a three-dimensional (3d) culture. umbilical cord msc single-cell derived spheres were produced by [65] using cell chips, a device to restrict cells to specific spatial locations. they combined a 3d culture with a 2d arrayed pattern of single or multiple cells on one patch of the cell chip in order to improve msc survival and migratory ability and to promote angiogenesis in xenotransplantation [65]. the other technique requires changing the scaffold. materials used as scaffolds in tissue engineering xenogeneic stem cell transplantation may promote cell survival and differentiation. [66] employed a hyaluronic acid-based scaffold that has been covalently modified by poly-l-lysine as a delivery vehicle to deliver hbmscs to rats with injured spinal cords. rats receiving hbmscs/hyaluronic acid-poly-l-lysine showed improved in vivo survival of transplanted hbmscs, according to [66]. in contrast, when sheep mscs were injected into immunocompromised rats, a ceramic hyaluronic acid/tricalcium phosphate carrier led to ectopic osteogenesis, adipogenesis, and hematopoietic-support activities [67]. the necessity of selecting an adequate substrate for tissue creation while taking into account the anticipated direction of cell differentiation was established by these findings [67]. ipscs and escs may be tumorigenic due to their capacity for cellular growth in cell transplantation and other treatments. this problem was addressed by [68] by implementing optimised directed differentiation protocols to generate the desired precursor cell types and by using cellular enrichment techniques to eliminate unnecessary cells in order to choose only the cells with a restricted proliferation potential for transplantation. religious restrictions in table 3 we provide information about the restrictions on xenotransplantation products in different countries based on the religions practiced in those countries. it is important to note that these restrictions are based on the beliefs and practices of individual religions and do not necessarily reflect the views or laws of the countries in which they are practiced. one of the main observations from the table is that many religions place restrictions on the use of certain animal products. for example, islam prohibits the use of porcine products and requires that all animal products be slaughtered in a specific way. similarly, judaism prohibits the use of porcine and shellfish products and has strict rules about the types of land animals, birds, and fish that can be consumed. hinduism and sikhism also place restrictions on the use of animal products, with many hindus abstaining from all animal products and sikhs prohibiting the use of halal sources. another important aspect of the table is the diversity of religions represented. the table includes information about islam, judaism, seventh day adventism, hinduism, sikhism, and jehovah’s witnesses, among others. this highlights the fact that religious beliefs and practices can vary widely and may influence the use of xenotransplantation products in different parts of the world. overall, the table provides useful information about the potential restrictions on xenotransplantation products based on the religions practiced in different countries. it is important to consider these restrictions when developing and implementing xenotransplantation treatments in order to respect the beliefs and practices of different religious communities. ethical concerns ethics around xenogeneic stem cell transplantation are becoming more widely accepted. some people believe that xenotransplantation consistently transgresses the lines between species and lowers the dignity of humans. animal welfare organisations also opposed xenotransplantation on the grounds that nonhuman creatures shouldn’t be seen of as re-designable systems [70]. in reality, a wide range of animal products are now used by humans. for instance, bioactive bones from decellularized bovine femoral bone and freeze-dried bone marrow stem cell paracrine factors are widely used in large-sized bone lesions. these successes are gradually changing people’s opinions and paving the way for xenogeneic stem cell transplantation. however, any applications must consider regional variations in culture, legislation, beliefs, and other factors [71]. risk of zoonotic infections potential benefits of xenotransplantation over allotransplantation (transplantation between members of the same species) include an almost limitless supply of grafts, animal species resistance to certain human infections (baboons, for example, are immune to the hepatitis b virus (hbv) and the human immunodeficiency virus (hiv)), and the ability to lower the risk of xenograftassociated infections by using specific pathogen-free animals with lifelong controversies, and the ability to reduce the risk of xenograft [72; 73]. however, if the risk to public health arises from introducing novel zoonotic infectious diseases into the human population that aren’t typically present there, the prospect of spreading germs from animals to people via xenotransplantation cannot be completely precluded [74]. the characteristics of highlights in bioscience page 10 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 3. religious restrictions as published by others [59; 69]. religion countries where widely practiced restrictions islam indonesia, india, pakistan, bangladesh, egypt, turkey, iran, nigeria, ethiopia, afghanistan, sudan, iraq, malaysia, tanzania, somalia, cote divoire, congo, philippines, sierra leone, thailand, eritrea, lebanon porcine products prohibited all animal products not killed in the prescribed ritualistic way (halal) prohibited products containing alcohol prohibited judaism usa, israel, france, canada, uk, russia, argentina, ukraine, brazil and south africa all porcine and shellfish products prohibited . other rules about animal products that can be ingested: land animals must be mammals which chew their cud and have cloven hooves birds of prey are prohibited . fish must have scales and fins. meat and milk (or any other dairy product) cannot be combined; shrimp and other non-fish seafood are forbidden. observers follow a stringent set of regulations and only eat kosher food. seventh day adventist australia, usa, south america, some african countries some abstain from meat, but eggs are permissible. hinduism india, nepal, bangladesh, indonesia, pakistan, sri lanka, philippines, fiji, uk, mauritius, bhutan, south africa, burma, singapore for the vast majority of vegetarians, all animal products, including eggs, are forbidden. bovine and porcine goods continue to be prohibited for persons who are not vegetarians. sikh india, pakistan, malaysia, singapore, fiji, new zealand, usa and uk for some who are vegetarian all animal products including egg prohibited for those who are not vegetarian, restrictions still include bovine and porcine products all animal products from halal sources prohibited products containing alcohol prohibited. jehovahs witnesses australia, usa, mexico, brazil and many other countries (240 in total) the use of fractions derived from the primary components of blood is not absolutely prohibited buddhism tibet, bhutan, india, nepal, sri lanka, burma, thailand, laos, cambodia, malaysia, vietnam, china, bangladesh, korea, japan, singapore, parts of russia for some vegetarian buddhists all animal products prohibited however, no fixed rules. highlights in bioscience page 11 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions the particular organism, the amount of the organism transferred, the presence of the necessary equipment (such as receptors and nutrients in the host), and the immunological proficiency of the host all affect the likelihood of contracting a zoonotic infection. even the wide range of potential clinical signs cannot be predicted for previously undetected animal-derived illnesses in human hosts [75]. recipients and their contacts should be routinely screened for zoonotic infectious agents, either by direct methods (which depend on detecting the presence of the agent itself or its products) or even by indirect methods (which depend on detecting the production of antibodies against specific microbes and antigens) [76]. this is to prevent a potential new zoonosis from spreading among humans as a result of xenotransplantation. conclusion biotechnology has the power to drastically modify human existence, as we indicated at the beginning of our discussion and as the rise of xenotransplantation amply indicates. furthermore, according to gaskell’s research, moral objections to biotechnology are allegedly more significant to society than even safety objections. even non-problems, like "violating god’s will" or "going against nature," are elevated to the status of the most severe ethical concerns as a result of society’s lack of scientific and ethical understanding, which makes it challenging to come up with reasonable answers. such an error might restrict the use of biotechnology to save lives and alleviate suffering, as our discussion has shown. this in turn emphasises the critical social illiteracy of science that we mentioned at the beginning of our argument, as well as the urgent need for expanded education of the general public, the scientific community, and society at large on ethics. although it is relatively easy to see this issue, solving it is much more difficult. we have both taught and pushed for the inclusion of ethical considerations in science education as a necessary precursor to rational solutions to ethical difficulties arising out of scientific findings. we have also seen that such education produces better scientists who have a sense of social responsibility. additionally, we have argued that it is critical to discuss and elucidate ethical issues while instructing students in science, particularly biological science. this has proven to be considerably more difficult. obstacles to it include the fact that such an approach is historically uncommon and that the majority of scientists believe research is "value-free in general, and ethics-free in particular," as demonstrated in our debate. the majority of people who teach science do not have formal training in the ethical issues that arise from science or even how to start addressing such obstacles, which creates additional challenges. determining when and how to begin integrating an ethical component into scientific instruction might be difficult as a result of these difficulties. the casual attitude of the research community toward the ethical issues associated to animal usage in research, which further distances the scientific community from the general people, is evident as social concern over the treatment of animals grows dramatically. if, as leaders in the scientific community have repeatedly remarked, scientific growth is entirely dependent upon the use of animals, then it is the responsibility of the scientific community to address social ethical concerns associated to animal exploitation. the development of animal ethics as we have described it is predicated on the notion of an animal, and society seems to concur with this. furthermore, we brought this up throughout our discussion. the ethical viability of genetic modification, which is readily acknowledged to impact both large and small changes in telos, therefore inevitably arises. it is obvious that this is not the place for a comprehensive examination of this annoying problem. as long as the telos changes do not negatively affect the animals’ quality of lifethat is, as long as the animals produced through genetic modification are not worse off than their unmodified forebears and, ideally, better offwe have argued that there is no morally wrong with carrying out such genetic modifications. in other words, it’s important to make sure that animal genetic engineering doesn’t do any harm. as with genetic alteration to fix genetic defects or prevent disease, the ideal outcome for animals is that they will be better off as a result of the change. such problems will not be resolved unless major changes in scientists’ thinking, which can only be made by significantly modifying the way science is taught, are made. at that point, ethics can be incorporated into recognised scientific theory and practise. reference 1. white s, hirth r, mah’illo b, dom’inguez-gil b, delmonico f, noel l, et al. the global diffusion of organ transplantation: trends, drivers and policy implications. bulletin of the world health organization. 2014;92:826-35. 2. ekser b, ezzelarab m, hara h, van der windt d, wijkstrom m, bottino r, et al. clinical xenotransplantation: the next medical revolution? the lancet. 2012;379(9816):672-83. 3. o’flaherty w. hindu myths: a sourcebook translated from the sanskrit. hammondsworth: penguin; 1975. 4. kahan b. ganesha: the primeval hindu xenograft. in: transplantation proceedings. vol. 3. elsevier; 1989. p. 1-8. 5. bhandari m, tewari a. is transplantation only 100 years old? british journal of urology. 1997;79(4):495-8. 6. deschamps jy, roux fa, saï p, gouin e. history of xenotransplantation. xenotransplantation. 2005;12(2):91-109. 7. denis j. ’excerpt from a letter from mr. denis, professor of philosophy & mathematics, am* concerning blood transfusion,”;6(1667):69-72. 8. kissam r. ceratoplastice in man. nyj med. 1844 mar;2:281-2. 9. zirm e. eine erfolgreiche totale keratoplastik (a successful total keratoplasty). journal of refractive surgery. 1989 jul;5(4):258-61. highlights in bioscience page 12 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions 10. hara h, cooper d. xenotransplantation: the future of corneal transplantation? cornea. 2011 apr;30(4):371. 11. haq m. fish cornea for grafting. british medical journal. 1972 june;2(5815):712. 12. reemtsma k, mccracken b, schlegel j, pearl m, pearce c, dewitt c, et al. renal heterotransplantation in man. annals of surgery. 1964 sep;160(3):384. 13. hardy j, chavez c, kurrus f, neely w, eraslan s, turner m, et al. heart transplantation in man: developmental studies and report of a case. jama. 1964 jun;188(13):1132-40. 14. bailey l, nehlsen-cannarella s, concepcion w, jolley w. baboon-to-human cardiac xenotransplantation in a neonate. jama. 1985 dec;254(23):3321-9. 15. starzl t, fung j, tzakis a, todo s, demetris a, marino i, et al. baboon-to-human liver transplantation. the lancet. 1993 jan;341(8837):65-71. 16. yoon c, choi h, kim m. corneal xenotransplantation: where are we standing? progress in retinal and eye research. 2021 jan;80:100876. 17. wang y, lei t, wei l, du s, girani l, deng s. xenotransplantation in china: present status. xenotransplantation. 2019 jan;26(1):e12490. 18. cooper d, hara h, yazer m. genetically engineered pigs as a source for clinical red blood cell transfusion. clinics in laboratory medicine. 2010 jun;30(2):365-80. 19. roux f, saï p, deschamps j. xenotransfusions, past and present. xenotransplantation. 2007 may;14(3):208-16. 20. cooper d, hara h, yazer m. genetically engineered pigs as a source for clinical red blood cell transfusion. clinics in laboratory medicine. 2010 jun;30(2):365-80. 21. murthy r, bajona p, bhama j, cooper d. heart xenotransplantation: historical background, experimental progress, and clinical prospects. the annals of thoracic surgery. 2016 apr;101(4):1605-13. 22. gibson t. zoografting: a curious chapter in the history of plastic surgery. british journal of plastic surgery. 1955 jan;8:234-42. 23. cooper d. xenografting: the early, early years. xeno. 1997;5:21-2. 24. knapp ec. the first corneal transplant. cornea. 1989;8(2):107-8. 25. hara h, cooper d. the immunology of corneal xenotransplantation: a review of the literature. xenotransplantation. 2010 sep;17(5):338-49. 26. khan aj, dohlman at, chew dw. corneal xenotransplantation: a review. current opinion in ophthalmology. 2010;21(6):582-6. 27. spalton la. corneal xenotransplantation: an ethical perspective. progress in retinal and eye research. 2013;33:101-11. 28. khan aj, dohlman at, chew dw. corneal xenotransplantation: challenges and opportunities. american journal of transplantation. 2014;14(2):367-76. 29. hamilton d. the monkey gland affair. chatto windus; 1986. 30. poncelet a, denis d, gianello p. cellular xenotransplantation. current opinion in organ transplantation. 2009 apr;14(2):168-74. 31. matevossian e, kern h, hüser n, doll d, snopok y, nährig j, et al. surgeon yurii voronoy (1895-1961) a pioneer in the history of clinical transplantation: in memoriam at the 75th anniversary of the first human kidney transplantation. transplantation international. 2009 dec;22(12):1132-9. 32. lee r. the bizarre careers of john r. brinkley. university press of kentucky; 2002. 33. sgroi a, bühler l, morel p, sykes m, noel l. international human xenotransplantation inventory. transplantation. 2010 sep;90(6):597-603. 34. starzl t, marchioro t, peters g, kirkpatrick c, wilson w, porter k, et al. renal heterotransplantation from baboon to man: experience with 6 cases. transplantation. 1964 nov;2:752. 35. taniguchi s, cooper d. clinical xenotransplantation: past, present and future. annals of the royal college of surgeons of england. 1997 jan;79(1):13-9. 36. barnard c, wolpowitz a, losman j. heterotopic cardiac transplantation with a xenograft for assistance of the left heart in cardiogenic shock after cardiopulmonary bypass. south african medical journal. 1977 dec;52(26):1035-8. 37. burdorf l, riner a, rybak e, ii s, de meyer s, shah a, et al. platelet sequestration and activation during galtko. hcd46 pig lung perfusion by human blood is primarily mediated by gpib, gpiib/iiia, and von willebrand factor. xenotransplantation. 2016 may;23(3):222-36. 38. laird c, burdorf l, french b, kubicki n, cheng x, braileanu g, et al. transgenic expression of human leukocyte antigen-e attenuates galko.hcd46 porcine lung xenograft injury. xenotransplantation. 2017;24(2):e12294. highlights in bioscience page 13 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions 39. starzl t, marchioro t, faris t, mccardle r, iwaski y. avenues of future research in homotransplantation of the liver with particular reference to hepatic supportive procedures, antilymphocyte serum, and tissue typing. the american journal of surgery. 1966 sep;112(3):391-400. 40. starzl t, ishikawa m, putnam c, porter k, picache r, husberg b, et al. progress in and deterrents to orthotopic liver transplantation, with special reference to survival, resistance to hyperacute rejection, and biliary duct reconstruction. transplantation proceedings. 1974 dec;4(1):129. nih public access. 41. giles g, boehmig h, amemiya h, halgrimson c, starzl t. clinical heterotransplantation of the liver. transplantation proceedings. 1970 dec;2(4):506. nih public access. 42. makowka l, wu g, cramer d, hoffman a, podesta l. immunohistopathologic lesions associated with the rejection of a pig-to-human liver xenograft. transplantation proceedings. 1994;26(3):1074-5. 43. groth c, tibell a, tollemar j, bolinder j, östman j, möller e, et al. transplantation of porcine fetal pancreas to diabetic patients. the lancet. 1994 nov;344(8934):1402-4. 44. allan j. xenotransplantation at a crossroads: prevention versus progress. nature medicine. 1996 jan;2(1):18-21. 45. vanderpool h. critical ethical issues in clinical trials with xenotransplants. the lancet. 1998 may;351(9112):134750. 46. hughes j. xenografting: ethical issues. journal of medical ethics. 1998 feb;24(1):18-24. 47. delriviere l, havaux x, gibbs p, gianello p. basic anatomical and physiological differences between species should be considered when choosing combinations for use in models of hepatic xenotransplantation: an investigation of the guinea pig-to-rat combination. transplantation. 1998 jul;66(1):112-5. 48. robson s, am esch j, bach f. factors in xenograft rejection. annals of the new york academy of sciences. 1999 jun;875(1):261-76. 49. adin c, gilor c. focus: comparative medicine: the diabetic dog as a translational model for human islet transplantation. the yale journal of biology and medicine. 2017 sep;90(3):509. 50. platt j, cascalho m, piedrahita j. xenotransplantation: progress along paths uncertain from models to application. ilar journal. 2018;59(3):286-308. 51. ding d, chou h, chang y, hung w, liu h, chu t. characterization of hla-g and related immunosuppressive effects in human umbilical cord stroma-derived stem cells. cell transplantation. 2016;25(2):217-28. 52. lévêque x, mathieux e, nerrièredaguin v, thinard r, kermarrec l, durand t, et al. local control of the host immune response performed with mesenchymal stem cells: perspectives for functional intracerebral xenotransplantation. journal of cellular and molecular medicine. 2015;19(1):124-34. 53. yang j, liao c, pang c, huang l, chen y, shiue y, et al. transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model. cytotherapy. 2013;15(2):201-8. 54. medicetty s, bledsoe a, fahrenholtz c, troyer d, weiss m. transplantation of pig stem cells into rat brain: proliferation during the first 8 weeks. experimental neurology. 2004;190(1):32-41. 55. li k, wang y, sun q, li m, chen j, liu l. rabbit umbilical cord mesenchymal stem cells: a new option for tissue engineering. the journal of gene medicine. 2021;23(1):e3282. 56. jia y, zhao y, wang l, xiang y, chen s, ming c, et al. rat adipose-derived stem cells express low level of ś-gal and are dependent on cd59 for protection from human xenoantibody and complement-mediated lysis. american journal of translational research. 2016;8(5):2059. 57. bozoglanian v, butteri m. the diverse and promising world of animal derived medications. pharos alpha omega alpha honor med soc. 2015 jan:16-22. 58. anjum c, chia y, chan m. presence of neu5gc in animalderived products. friend or foe. stem cells regen med. 2020;4(1):1-7. 59. the state of queensland (queensland health); 2020. https://www.health.qld.gov.au/__data/assets/ pdf_file/0024/147507/qh-gdl-954.pdf. 60. galili u, mandrell r, hamadeh r, shohet s, griffiss j. interaction between human natural anti-alpha-galactosyl immunoglobulin g and bacteria of the human flora. infection and immunity. 1988;56(7):1730-7. 61. artrip j, kwiatkowski p, michler r, wang s, tugulea s, ankersmit j, et al. target cell susceptibility to lysis by human natural killer cells is augmented by ś (1, 3)-galactosyltransferase and reduced by ś (1, 2)fucosyltransferase. journal of biological chemistry. 1999;274(16):10717-22. highlights in bioscience page 14 of 15 december 2022|volume 5 https://www.health.qld.gov.au/__data/assets/pdf_file/0024/147507/qh-gdl-954.pdf https://www.health.qld.gov.au/__data/assets/pdf_file/0024/147507/qh-gdl-954.pdf http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions 62. shimizu a, meehan s, kozlowski t, sablinski t, ierino f, cooper d, et al. acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pigto-nonhuman primate renal grafts. laboratory investigation. 2000;80(6):815-30. 63. holmes b, richards a, mclaughlin m, cruz g, copeman l, bycroft s. antibody responses in early graft rejection in pig-to-primate renal xenotransplantation. in: transplantation proceedings. vol. 33; 2001. p. 717-8. 64. robson s, cooper d, d’apice a. disordered regulation of coagulation and platelet activation in xenotransplantation. xenotransplantation. 2000;7(3):166-76. 65. qiao y, xu z, yu y, hou s, geng j, xiao t, et al. single cell derived spheres of umbilical cord mesenchymal stem cells enhance cell stemness properties, survival ability and therapeutic potential on liver failure. biomaterials. 2020;227:119573. 66. raynald ly, yu h, huang h, guo m, hua r, jiang f, et al. the hetero-transplantation of human bone marrow stromal cells carried by hydrogel unexpectedly demonstrates a significant role in the functional recovery in the injured spinal cord of rats. brain res. 2016;1634:21-33. 67. mccarty r, gronthos s, zannettino a, foster b, xian c. characterisation and developmental potential of ovine bone marrow derived mesenchymal stem cells. journal of cellular physiology. 2009;219(2):324-33. 68. zygogianni o, kouroupi g, taoufik e, matsas r. engraftable induced pluripotent stem cell-derived neural precursors for brain repair. in: stem cells and tissue repair. humana, new york, ny; 2020. p. 23-39. 69. jenkins e, yip m, melman l, frisella m, matthews b. informed consent: cultural and religious issues associated with the use of allogeneic and xenogeneic mesh products. journal of the american college of surgeons. 2010;210(4):402-10. 70. fung r, kerridge i. gene editing advance re-ignites debate on the merits and risks of animal to human transplantation. intern med j. 2016;46:1017-22. 71. karalashvili l, kakabadze a, uhryn m, vyshnevska h, ediberidze k, kakabadze z. bone grafts for reconstruction of bone defects. georgian medical news. 2018;282:44-9. 72. michaels m, simmons r. xenotransplant-associated zoonoses: strategies for prevention. transplantation. 1994;57(1):1-7. 73. fishman j. infection and xenotransplantation: developing strategies to minimize risk. annals of the new york academy of sciences. 1998;862(1):52-66. 74. onions d, cooper d, alexander t, brown c, claassen e, foweraker j, et al. an approach to the control of disease transmission in pig-to-human xenotransplantation. xenotransplantation. 2000;7(2):143-55. 75. fishman j, scobie l, takeuchi y. xenotransplantationassociated infectious risk: a who consultation. xenotransplantation. 2012;19(2):72-81. 76. food, administration d. guidance for industry (final guidance): source animal, product, preclinical, and clinical issues concerning the use of xenotransplantation products in humans; 2003. available from: http://www.fda.gov/ cber/gdlns/clinxeno.htm. highlights in bioscience page 15 of 15 december 2022|volume 5 http://www.fda.gov/cber/gdlns/clinxeno.htm http://www.fda.gov/cber/gdlns/clinxeno.htm http://bioscience.highlightsin.org/ abstract introduction chimeras in folklore history of clinical experiences with xenotransplantation blood xenotransfusion blood vessel anastomosis skin xenotransplantation corneal xenotransplantation cell xenotransplantation xenotransplantation of the kidney xenotransplantation of the heart lung xenotransplantation liver xenotransplantation the first islet xenotransplantation features of the perfect donor animal include other pharmaceuticals of animal origin issues with several xenotransplantation cases immune rejection hyperacute rejection acute humoral xenograft rejection (ahxr) cell proliferation, aberrant differentiation, and death religious restrictions ethical concerns risk of zoonotic infections conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202301 research article open access 1 vegetable research department, horticulture research institute, agricultural research center, giza, egypt. 2 institute of environmental studies, arish university, north sinai, egypt. 3 plant production department, faculty of environmental agricultural sciences. arish university, north sinai, egypt. 4 department of family and community health nursing, faculty of nursing, suez canal university, ismailia, egypt. 5 department of food technology, faculty of agriculture, suez canal university, ismailia, egypt, p.o. box 41522. * to whom correspondence should be addressed: mobark_mohamed99@yahoo.com editor: aladdin hamwieh, international center for agricultural research in the dry areas (icarda), giza, egypt. reviewer(s): khaled h. radwan, agricultural genetic engineering research institute (ageri), agricultural research center (arc), giza, egypt. tawffiq istanbuli, international center for agricultural research in the dry areas (icarda), beirut, lebanon received: october 20, 2022 accepted: january 3, 2023 published: january 26, 2023 citation: abuo el-kasem saa, naiel mhf, mubarak mh, megahed fia, el-deeb gss. assessment of pesticide residues in vegetables selected from different egyptian governorates. 2023 jan 26;6:bs202301 copyright: © 2023 abuo el-kasem et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. assessment of pesticide residues in vegetables selected from different egyptian governorates sameh a. a. abuo el-kasem 1 >< , mohamed h. f. naiel 2 >< , mohamed h. mubarak *,3 >< , fatma i. a. megahed 4 >< , gehad s. s. el-deeb 5 ><  abstract this study aimed to assess the levels of contamination by pesticide residues in several types of vegetables collected from different regions in egypt. a total of 100 samples of vegetables (pepper, tomato, cucumber, and strawberry) were collected from markets in five cities (al-obour, al-salheia el-gadida, giza, zagazig, and fayed) and analyzed for the presence of 42 different pesticide residues. the quick, easy, cheap, effective, rugged, and safe (quechers) method was used to extract the target pesticides, which were then quantified using gas chromatography-mass spectrometry (gc-ms/ms) and liquid chromatography-mass spectrometry (lc-ms/ms) techniques. the results showed that 72% of the vegetable samples contained detectable levels of pesticide residues, with 21% exceeding the european union maximum residue levels (eu-mrls) and 51% containing residues below the mrls. the detected residues were primarily insecticides (56.4%) and fungicides (43.6%), with tomato and strawberry samples showing the highest frequency of both types of pesticides. tomato also had the highest absolute intake from consumption (2.89 g/kg bw/day), followed by strawberries, peppers, and cucumbers (0.47, 0.159, and 0.096 g/kg bw/day, respectively). a hazard index (hi) was used to assess the dietary risk posed by the pesticide residues, with tomato having the highest contribution value. these findings highlight the need for integrated pest management (ipm) programs to reduce the excessive use of pesticides, particularly in relation to raw food commodities. action is required to minimize the unacceptable risks identified in this study. keywords: food safety, pesticide residues, risk assessment, estimated daily intake, monitoring introduction fruit and vegetables have been a cornerstone of healthy dietary recommendations. they have potential health-promoting effects beyond providing basic nutrition needs in humans, including their role in reducing inflammation and their potential preventive effects on various chronic disease states such as cardiovascular disease and cancer leading to premature mortality decreasing years loss of individuals’ life and years-to-come lived with disability/morbidity. consumers are now choosing fruits and vegetables not only for their content of vitamins, minerals and fiber, but also for their concentration of dietary bioactive with its anti-inflammatory effects [1]. in agriculture, pesticides are considered a quick, and easy solution for controlling weeds and insect pests, improving production and productivity of agriculture commodity to feed the ever growing population, controlling vector borne disease like malaria and reducing the resultant mortality and morbidity. surprisingly; the global consumption of pesticides is about two million tons per year and out of which 45% is used by europe alone, 25% is consumed in the usa, and 25% in the rest of the world. despite their benefits, pesticides can be hazardous to humans and environment and non-intended organisms ranging from beneficial soil microorganisms to insects, plants, fish and birds. environmental contamination or prevailing use of pesticides can expose the general population to pesticide residues leading to serious and prolonged toxicity. it was estimated that a minimum of 300,000 people die from pesticide poisoning each year, with 99% of them from lowand middle-income countries in 2009 [2]. highlights in bioscience page 1 of 13 january 2023|volume 6 https://doi.org/10.36462/h.biosci.202301 https://creativecommons.org/licenses/by/4.0/ samehaoelkaseem7@gmail.com https://orcid.org/0000-0001-8275-0945 mohamednail123@yahoo.com https://orcid.org/0000-0003-1850-1308 mobark_mohamed99@yahoo.com https://orcid.org/0000-0002-2981-381x dr.fm_2013@yahoo.com https://orcid.org/0000-0003-3255-1926 gehadeldeeb@yahoo.co.uk https://orcid.org/0000-0002-9132-8508 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates pesticides in food are monitored by the environmental protection agency (epa) and the food and drug administration (fda). one of the top priorities in securing and preserving community and public health, is food safety. food safety is particularly important to ensure the healthiness of food, especially to fruits and vegetables as they are consumed in substantial and considerable quantities customarily without any processing. they are susceptible to pests at any point in the production chain, from the field through storage ahead till food consumption. their pesticide residues are yet present in the vegetable-treated products, which may constitute a potential hazards for consumers. some of these hazards were identified in fruits and vegetables were because of incorrect application of pesticides either by the producers’ application or the insufficient monitoring of the contaminated soil and/or water [3]. major causes of the environment pollution were from prejudicial human activities and improper application, spillage, and decomposition. however and despite all precautions, a very minute amount of pesticide-residues can remain in the treated crop. the maximum residue level (mrl), the maximum quantity of residue that is legally permitted on a food material, ensures that both imported and exported goods are moderately safe to consume. pesticide misuse, false positives from naturally occurring compounds, variances in national mrls standards, a lack of registered pesticides, and improper pesticide application can all cause mrls to be exceeded [4]. the sources of the mrls, however, can affect the frequency of infractions. however, the creation of mrls is based on information from supervised trials mean residues (stmr), acceptable daily intake (adi), acute reference dose (arfd), as well as data from good agricultural practice (gap) that has been registered nationally. the percentages of violation will be very different, for instance, because the maximum permissible residue of profenofos on tomatoes is 10 mg/kg in the codex and 0.01 mg/kg in european standards. in order to determine the actual risk of exposure, it is crucial to compare the findings with a more reliable toxicological endpoint, such as adi or arfd. the egyptian ought to take into account creating mrls based on regional best practices for agriculture and locally administered paths. the major tool for ensuring that the pesticides were applied in assembly with good agricultural practices is the monitoring program. these programs when applied in conformity with good agricultural practices, treated goods should not have exceeded levels of pesticide residue [5]. the health risks of pesticides are regularly evaluated through monitoring programs for eu nations. according to the yearly dg sanco report, 47% of the fruits, vegetables, and grains eaten in europe in 2004 had pesticide residues [6]. pesticides that can be used in certain foods and feed commodities have maximum residual limitations, or tolerances, determined by the environmental protection agency (epa). these restrictions are put in place to safeguard people from hazardous pesticide levels in their food [7]. a variety of pesticides, including organochlorine, organophosphorus, carbamate, insecticides, fungicides, and herbicides, are used by farmers all over the world to prevent the devastating crop loss that can result from pests and diseases as well as to boost agricultural productivity to ensure a sufficient supply of food for the expanding population [8]. food contamination creates severe health issues worldwide, ranging from minor ailments to fatal ones [9]. therefore; it is well established that contaminated food poses a risk to the general public health. however; producing, processing, moving, and handling food can all lead to food contamination [10; 11; 9; 12]. there are two types of pollution: short-term pollution at high concentrations of chemicals (induced by inadvertent release or contamination from the source) and long-term pollution of low concentrations of chemicals (produced by the progressive diffusion of pollutants in food) [13]. different chemical classes or families typically produce dissimilar symptoms. the amount of pollutants in food ingested in relation to the daily amount of food can be used to estimating the level of pollutants in the human body [14]. numerous studies have demonstrated that persistent organic pollutants, such as organochlorine insecticides, have a variety of negative impacts, including aberrant immune system development, birth abnormalities, and foetal death [15; 16]. because of this, pesticides are regarded as one of the world’s top environmental and health risks [17]. many nations and international organizations, including the european union, the world health organization, and the united nations environment program, have acclaimed that both organic and inorganic pollutants pose a serious risk to health, particularly the health of children [18]. they have gradually released a number of suggestions or guidelines intended to limit or outlaw the use of these pollutants or pollutant products. for instance, the amount of lead in the environment has decreased as a result of several countries banning the addition of tetra-methyl lead to gasoline [19]. for consumers protection from exposure to unacceptable levels of pesticide residues in food and feed, the european commission has set maximum residue levels (mrls), defined as the highest possible level of a pesticide residue that is legally authorized in food and feed. based on the results obtained from environmental sample analysis, it has been proven that the modified quechers method coupled to gas chromatography gc-ms/ms with electron capture detector (gc-ecd) which are analytical procedures for routine analysis and simultaneous determination of selected electronegative pesticides in fruits and vegetables with high water content. these procedures are suitable not only for fruits and vegetables with high water content, but also for samples containing large amounts of pigments and dyes [3]. for risk assessment analyses that could be linked to accidental intakes of contaminants at very high levels and could have severe unfavorable effects on the human body, quantitative data on the concentration of contaminants in food are an essential tool [20] . though pesticides assist increase crop productivity, the amount and variety of food consumed, and likewise the development of some diseases [21]. pesticides can be categohighlights in bioscience page 2 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates rized according to their unique biological activity or their target species, such as fungicides, herbicides, insecticides, and acaricides, in addition to functional groups [22; 23]. the maximum daily consumption that a person is permitted to consume during their lifetime without posing a significant risk to that person has been determined by numerous health and environmental protection agencies as "acceptable daily intake" (adi) levels. environmental pollution is, without any doubts, a serious global concern. many nations have made positive efforts to limit the use of pesticides. procedures and approaches are utilized to evaluate the detrimental impacts brought on by pollutants as a result of pollutant risk assessment. to evaluate past, present, and future exposure to any environmental pollutants, a risk assessment can be carried out. the majority of the time, analyses of material hazards are based on scientific research on the activities, exposure, quantity, and toxicity of chemicals. the amount of pollutants present in the environment, food, and/or products; the number of people exposed to the pollutants; and the damages of pollutants all affect the risks [24]. many organochlorine pesticides have been outlawed or have had their usage severely restricted in europe and north america, yet they are still sold and used in africa. residential pesticide use in egypt is high. adolescents in egypt are exposed to pesticides through non-occupational and para-occupational pathways. in addition to the hormonal and physiological changes associated with puberty, there are also significant developmental changes in the brain, primarily the prefrontal cortex. the impact of environmental exposures can vary across developmental periods and consequences of prolonged exposure can last into adulthood [25]. the purpose of this study was to assess the levels of a group of pesticide residues in the commonly consumed vegetables in egypt, and to evaluate their health risk according to estimated quantity of exposure. material and methods samples a total of 100 vegetable samples (pepper, tomato, cucumber, and strawberry) were collected from local markets of five egyptian cities: al-obour (qalyubia governorate), al-salheia elgadida (sharqia governorate), giza (giza governorate), zagazig sharqia (governorate), and fayed (ismailia governorate)). each representative vegetable sample was made up of 10 identical commodity subsamples that were simultaneously obtained from each market using random sampling. vegetable samples were packed in proper bags and stored at 4oc until analysis. sample preparation and analysis about 2 kg of each vegetable sample (pepper, tomato, cucumber, and strawberry) was thoroughly washed with tap water, chopped and blended using a waring laboratory blender. each sample was chopped and ground in accordance with the generally suggested procedure described by the codex alimentarius commission in 1993 for no more than two days prior to analysis. according to anastassiades et al., [26] the quechers method was used to extract pesticides from the vegetable samples. a 50 ml polypropylene (pp) tube containing 10 gm of each sample was weighed, 10 ml of acetonitrile was added, and the tube was forcefully shaken for one minute. phase separation was achieved by centrifuging the liquid at 4000 rpm for 5 min after adding buffering citrate salts (ph 5 to 5.5), containing 4g of magnesium sulphate, and 1g of sodium chloride. for analysis, an aliquot of the organic phase was directly loaded into lc-ms/ms. dispersive solid phase extraction (dspe). extracts from the samples were evaporated and then redissolved along with injection standard for gc-ms/ms analysis after cleaning with primary secondary amine sorbent (psa). aldrin was used as an internal standard for quantification, and it was added to the gc-ms/ms system right before injection. gc ms/ms and lc-ms/ms were used for the identification and confirmation of pesticide residues in the samples. calculation of adi and hi comparing the established acceptable daily intake (adi) with the estimated acceptable daily intake (edi), which is based on the concentration of pesticide residues and food consumption, gives the risk assessment. the edi (mg/kg bw/day) for each pesticide residue that was violated was computed by multiplying the mean pesticide residue concentration (mg/kg) x food consumption and then dividing by the typical adult’s body weight (60 kg) of each commodity. based on gems/foods from the who’s global environment monitoring system [27], acceptable daily intake was determined. edi = concentration o f pesticideresidue × food consumed body weight (1) the daily consumption rate of vegetables was derived conclusively for this study from the reports of who/ fao [28], who/ global environment monitoring system-food contamination, monitoring and evaluation program average consumption cluster diets [27], and gad alla et al., [29]. if data from food balance sheets are unavailable for a commodity, the consumption level for a comparable food is used (who 1997). because there isn’t a strawberry consumption rate available, the consumption level of a comparable food is used. the scientific names and daily intake rate (g/day) for the used vegetable samples are given in table 1 . the eu pesticides database served as the source for both the maximum residue limits (mrls) and the established acceptable daily intake (adi) values. using the health risk index, the health risk for consumers from consuming pesticide-contaminated samples was described (hi). it is calculated by dividing the estimated daily intake (edi) by the corresponding values of the acceptable daily intake (adi in mg/kg) specified by who/fao as stated in the equation 2: hi = edi adi 100 (2) highlights in bioscience page 3 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 1. scientific names and consumption rate of studied commodities in g/day based on gems/food total diet food balance sheet. consumption rate (g/day) based on who/global environment monitoring systemfood contamination, monitoring and assessment program average consumption cluster c diets[27]. common name scientific name family name crop type consumption (g/day)* cucumber cucumis sativas l. cucurbitaceae vegetable 5.9 pepper capsicum annum l. solanaceae vegetable 13 strawberry fragaria ananassa l. rosaceae vegetable 20 tomato solanum lycopersicum solanaceae vegetable 118 according to the european food safety authority (efsa) [30; 31] el-sawy et al. and [32]. when the hi is less than 100%, the food concerned is considered acceptable. if it is above 100%, the food concerned is considered a risk to the consumer [33]. results and discussion use of excessive pesticides contaminates soil, water and finally enters the food chain and contaminates the food produced. the international agency for research on cancer has found sufficient evidence of carcinogenic potential in most of the pesticides beyond the threshold limit. the united nations environment program estimates accidental pesticide poisoning causing 20, 000 deaths and 1 million cases of illness per year worldwide [34]. forty-two commonly used pesticides in agriculture were identified in this study. the broad scope analyzed includes numerous groups of pesticides such as organophosphorous, organo– chlorine, pyrethroids and other groups of pesticides widely used or outlawed in egypt. according to a directive issues by egypt’s agriculture pesticides committee (codex+eu), pesticide residue levels should be compared to codex alimentarious when it is available and to eu-mrls when codex mrls are not accessible. in this investigation, only the codex alimentarious mrls and the agriculture pesticides committee decree were used to compare the monitoring data. table 2 listed the number of samples evaluated, the range of pesticides found, the average in mg/kg, the number of chemicals violated in the samples analyzed, and the status of each pesticide/commodity combination in the registration system established by the agricultural pesticide committee (apc). a number of 32 out a total of 42 pesticides were detected in strawberry fruits. fluopyram had the highest pesticide concentration in the samples, whereas iprodion had the lowest pesticide residue (table 2). data revealed that 13 (52%) of the strawberry fruit samples had no detectable pesticide residues. whereas, a total of 12 samples (48%) contained pesticide residues, of which 10% were contaminated samples and contained residues at levels below the mrls, and 8% had residues over the allowed limits (table 2). however, according to the apc regulation, the breach was found in 8% of cases when comparing the results to (codex + eu restrictions). bifenazate, methamidophos, fluopyram, metalax, captan, propargite, and pyrimethanill are seven pesticide residues that recorded greater amounts than their regulated eu mrl values (table 2 and figure 1). the discovered quantities of pyrimethanill contamination (loq to 0.076, average 0.048 mg/kg) were not too far from the mrl (0.05 mg/kg). otherwise, the residues of both fluopyram and methamidophos exhibited a serious issue because their concentrations in strawberries exceeded their mrl values by 4 and 9 fold, respectively. this indicates that it is necessary to regulate their use. figure 1. the percentage of detected pesticide residues in samples based on pesticide type. regarding the outcomes of the tomato samples, which were the second crop of the vegetables under study, roughly 17 pesticides were detected in the tomato samples (25), as shown in table 3. among the pesticides found, only 8 (bifenazate, captan, fluopyram, chlorfenapyr, chlorpyrifos, dimethoate, lambdacyhalothrin, and thiofanat-methyle) had concentrations greater than the mrls reported for tomato (table 2 and figure 2). however, data in table 3 revealed that 28% (7) of tomato fruit samples had no detectable pesticide residues (25). while pesticide residues were discovered in 18 samples (72%) of which 48% (12 samples) were contaminated tests recorded residual amounts comparable to the worldwide mrls for them in tomato and 24% had residues over the allowed levels, pesticide residues were also discovered in other samples. fenarimol fungicide, followed by malathion insecticide, had the highest pesticide mean in samples, whereas fenpropathrin and metalaxyl had the lowest pesticide residue levels (table 4). however, pesticide residues were discovered in 21 samples (84%), of which 36% (9 samples) had residues over the allowed limits and 48% (12 samples) of contaminated samples had residues at levels higher than the mrls (table 4). according to the decision of the agriculture pesticides committee in egypt, a violation was found in one sample when results were compared to codex and eu limitations, and in the other eight samples when findings were compared to solely eu limits. data in table 5 regarding cucumber fruits showed that the majority of the investigated pesticides (12 out of 42) were detected in cucumber samples. only 2 insecticides (abamectin & acetamiprid) and 2 fungicides (captan & penconazole) out of the detected 12 pesticides had concentrations greater than the mrls indicated for cucumber, as shown in table 5. highlights in bioscience page 4 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates figure 2. frequency percentages of most detected pesticide residues in vegetable samples. in total, 16% of the cucumber samples had no detectable pesticide residues, while the remaining 84% contained detectable residues which 68% of contaminated samples contain resides at levels lower than the mrl’s and 16 % (4 samples) had residues above the permissible limits as shown in figure 3. the mrls are often set well below the thresholds deemed safe for humans. it is important to understand that mrls are not safety limits; food residues might have levels beyond mrls while still being safe to eat [35]. according to ifoam [36], mrls are not a guarantee of "zero health risk" in this situation; rather, they are only indicators of whether or not good agricultural practices (gap) are being violated, not a sign of a health danger. risk exposure should therefore be assessed using toxicological endpoints like acceptable daily intake (adi). when it comes to earlier egyptian investigations, the pattern of pesticides in tomato indicated that the detection levels of dimethoate, pirimiphos-methyl, and profenofos were 0.461, 0.114, and 0.28, respectively [37]. figure 3. the contamination and the violation percentages per each commodity of vegetables. otherwise, greater residual levels of profenofos and malathion were found in tomato samples taken from various areas in egypt, according to dogheim et al., [38]. additionally, greater profenofos residue levels were found in strawberry samples taken from eight local markets in six egyptian governorates [39; 40; 38], but not in tomato or strawberry samples taken for this study. the organophosphorus pesticides (thiometon, phorate, and chlorpyrifosmethyl) were found in cucumbers in a different investigation by mansour et al., [41] for the monitoring of pesticides and heavy metals. the levels of pesticides in some vegetables gathered from neighborhood markets in cairo governorate were assessed by farag et al., [42]. according to their findings, strawberries had the highest levels of contamination with various pesticides, with mean contamination levels of 0.034, 0.023, 0.033, 0.024, and 0.050 mg/kg, respectively, for ethion, propargite, permethrin, profenofos, and chlorpyrifos. pepper was found to contain only two different types of insecticides (sulfur, methomyl). pesticides were not found in tomatoes or cucumbers. additionally, ibrahim et al., [43] assessed the pesticide residues in certain vegetables purchased at local marketplaces in eight governorates around egypt. they came to the conclusion that the reported negative samples for pepper and cucumber were 19.4% and 27.9%, respectively. for pepper and cucumber, respectively, the recorded positive samples were 80.6% and 72.1%. according to badr et al., [44] , who assessed the pesticide residues in egyptian crops, profenophos measured pesticide levels in tomato and cucumber at 0.56 and 0.28 mg/kg, respectively. the levels of pesticide residue found in vegetable samples by loutfy et al., [45] and ahmed et al., [46] are consistent with our findings. according to dogheim et al., [38] , the relatively limited amount of pesticides used in the research areas and the widespread awareness and usage of integrated pest management (ipm) programs may be responsible for this low contamination level. conhighlights in bioscience page 5 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 2. pesticide residues levels found in strawberry fruits, frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu acetamiprid 1 0.039 0.039 0.039 0.5 0.3 r-nri azoxystrobin 7 0.012 0.12 0.054 10 2 r-nri bifenazate 4 0.012 0.25 0.083 2 0.1 1 r-ri bifenazole 3 0.01 0.029 0.016 0.1 nr boscalid 6 <loq 0.11 0.043 0.9 r-nri buprofezin 1 0.081 0.081 0.081 3 3 nr captan 5 0.017 0.294 0.133 15 0.1 2 r-ri carbendazim 2 0.02 0.07 0.045 0.5 0.1 banned, 2018 chlorantraniliprole 1 <loq <loq <loq 0.3 1 r-nri chlorpyrifos 3 <loq <loq <loq 0.3 0.01 r-nri cyproconazole 1 0.017 0.017 0.017 0.01 0.05 nr difenoconazole 4 <loq 0.042 0.031 2 1 r-nri fluopyram 2 0.031 0.44 0.236 0.4 0.1 1* r-ri flutazole 1 <loq <loq <loq na 0 nr flutriafol 1 0.021 0.021 0.021 1.5 1.5 r-nri imidacloprid 2 <loq <loq <loq 0.5 0.5 r-nri indoxacarb (p) 3 0.013 0.06 0.024 0.02 0.6 r-nri iprodion 2 0.012 0.013 0.013 10 0.05 banned, 2018 lambda-cyhalothrin 2 <loq <loq <loq 0.02 0.02 r-nri metalax 3 0.01 0.09 0.033 0.5 0.05 1 r-nri methamidophos 3 0.068 0.097 0.078 0.01 0.01 3* nr, 2007 methoxyfenozide 3 0.038 0.076 0.059 2 2 r-nri paclobuttazol 1 <ioq <ioq <ioq na 0.01 nr penconazole 1 0.018 0.018 0.018 0.5 0.1 r-nri pirimicarb 1 0.11 0.11 0.11 0.5 r-nri propargite 5 <loq 0.095 0.067 2 0.05 2 nr propiconazole 2 0.01 0.046 0.028 3 0.05 nr pyraclostrobin 4 0.01 0.053 0.022 1.5 0.1 r-ri pyrimethanil 3 <loq 0.076 0.048 0.1 0.05 2 r-ri spirodiclofen 2 <loq 0.025 0.013 2 0.5 r-nri tetraconazole 1 <loq <loq <loq na 0.2 r-nri thiophanate.mathyle 2 0.038 0.068 0.053 na 0.1 r-ri * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, r= registered, r1= recommended, nr= not registered, nr1= not recommended, eapc=egyptian agriculture pesticide committee , vc=violated compound table 3. pesticide residues levels found in tomato, frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu abamectin 1 0.010 0.010 0.010 0.05 0.09 r-ri acetamiprid 3 0.010 0.010 0.010 0.2 0.5 r-ri azoxystrobin 3 0.014 0.022 0.015 3 3 r-ri bifenazate 5 <loq 0.16 0.160 0.5 (2007) 0.1 2 r-ri boscalid 2 0.010 0.030 0.020 x 3 r-nri captan 4 0.050 0.368 0.228 5 (2008) 0.1 2 r-nri chlorfenapyr 7 0.010 0.071 0.022 0.4 (2019) 0.01 5 r-ri chlorpyrifos 5 0.021 0.053 0.036 1 0.01 5 r-ri cyfluthrin 1 <loq <loq <loq 0.2 (2008) 0.05 nr difenoconazole 3 <loq <loq <loq 0.6 2 r-ri dimethoate 3 0.010 0.020 0.017 na 0.01 2 r-nri fluopyram 2 0.100 0.410 0.255 0.5 0.1 1* r-nri indoxacarb 6 0.010 0.040 0.022 0.5 0.5 r-ri lambda-cyhalothrin 7 <loq 0.110 0.060 na 0.01 2 r-nri methoxyfenozide 7 0.010 0.062 0.024 2 (2005) 2 r-ri thiofanat-methyle 4 0.080 1.200 0.570 x 1 1 r-nri trifloxystrobin 6 <loq 0.010 0.010 0.7 (2006) 0.01* r * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, r= registered, r1= recommended, nr= not registered, nr1= not recommended, eapc=egyptian agriculture pesticide committee , vc= violated compound table 4. pesticide residues levels found in pepper , frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu chlorofenapyr 1 0.01 0.01 0.01 0.05 0.01 r-nri chloropyrifos 7 0.01 0.07 0.03 2 0.02 5 r-nri dimethoate 1 0.03 0.03 0.03 0.5 0.01 r-nri fenarimol 5 <loq 0.3 0.22 0.5 0.1 3 r-nri fenpropathrin 1 <loq <loq <loq 1 0.01 r-nri fluopyram 2 0.03 0.05 0.04 3 0.1 r-nri imidacloprid 1 0.09 0.09 0.09 1 0.5 r-nri malathion 1 0.2 0.2 0.2 0.1 0.01 1* r-nri metalaxyl 2 <loq <loq <loq 1 1 r-nri methoxyfenozide 1 0.06 0.06 0.06 2 2 r-nri spinetoram 4 0.01 0.07 0.04 0.4 0.5 r-ri * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, eapc=egyptian agriculture pesticide committee , vc=violated compound highlights in bioscience page 6 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 5. pesticide residues levels found in cucumber , frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu abamectin 6 0.01 0.07 0.04 0.03 0.04 2* r-ri acetamiprid 9 0.06 0.1 0.12 0.3 0.3 1* r-ri captan 5 <loq 0.7 0.21 3 0.1 2 r-nri chloropyrifos 3 0.03 0.04 0.03 0.05 0.05 r-nri cypermethrin 2 0.17 0.2 0.18 0.2 0.2 r-nri difenoconazole 3 <loq <loq <loq 0.2 0.2 r-ri fenpropathrin 1 0.03 0.03 0.03 0.5 0.5 r-nri fluopyram 1 0.07 0.07 0.07 0.5 0.1 r-nri l-cyhalothrin 2 0.03 0.04 0.03 0.1 0.1 r-nri malathion 1 0.01 0.01 0.01 0.2 0.02 r-ri metalaxyl 3 0.16 0.3 0.23 0.5 0.5 r-ri penconazole 4 0.01 0.04 0.02 0.06 0.01 2 r-nri * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, r= registered, r1= recommended, nr= not registered, nr1= not recommended , eapc=egyptian agriculture pesticide committee , vc=violated compound trarily, numerous pesticides, including those forbidden in egypt, are nevertheless found in a variety of environmental components, including groundwater, surface water, fished, mussels, medical plants, soils, and sediments [47; 48; 49; 50; 46]. additionally, some pesticides were found in only one crop, as shown in the corresponding tables for the crops of strawberry (13 pesticides), tomato (2, cyfluthrin & trifloxystrobin), and pepper (2, fenarimol & spinetoram), despite the fact that many common pesticides were found in multiple crops in some of the samples used for the current investigation. in light of these findings, a distinction between the types of pesticide residue found on the egyptian samples during the current investigation and those conducted previously was noted. the dominant pesticide group was adjusted in addition to the pesticide itself. a regular pesticide mentoring survey should therefore be recommended because it is related to the evaluation of the safety of fruits used in human food. the ministry of agriculture’s various control programs, which vary from one field and/or location to another depending on the type of injury and the different environmental conditions, may be to blame for the high incidence of the majority of tested pesticides in some vegetable samples of the current investigation. pesticide frequencies (detected pesticides type) according to figure 1, the types of pesticide residues that were found in vegetable samples were insecticides and fungicides, with percentages of 55.75% and 44.25%, respectively. insecticides were found in descending order at frequencies of 21.2, 17.05, 8.75, and 8.75% in tomatoes, strawberries, cucumbers and peppers. in descending order, fungicides with rates of 20.74, 10.60, 9.68, and 3.23% were found in strawberries, tomatoes, cucumbers, and peppers. but compared to pepper and cucumber, strawberry and tomato had the highest frequencies of both insecticides and fungicides. these findings contrasted with those of ozowicka et al., [51] who discovered that fungicides were used four times as frequently as insecticides, and were generally consistent with finding of gad alla et al., [52] . in addition, figure 2 lists the nineteen (19) pesticides that were found most frequently in egyptian vegetables examined in 2020, listed in order of frequency percentages: chlorpyrifos (8.3%), captan (6.5%), acetamiprid (6.0%), lambda-cyhalothrin (5.1%), methoxyfenozide (5.1%), azoxystrobin (4.6%), difenoconazole (4.6%), bifenazate (4.1%), indoxacarb (4.1%), boscalid (3.7%), chlorfenapyr (3.7%), metalaxyl (3.7%), abamectin (3.2%), fluopyram (3.2%), thiophanate.mathyle (2.8%), trifloxystrobin (2.8 %), penconazole (2.3%), propargite (2.3%) and fenarimol (2.3%), in addition to 23 other pesticides (11 insecticides, 9 fungicides, 2 acaricides and 1 herbicide) with a frequently number of 1-4 with a percentage < 2% (table 6). this may indicate a more intense use of insecticides than fungicides, which is inconsistent with pest control behavior in fruit and vegetable, due to fungal diseases that are expected to infect fruits.. however, the usage of numerous types and vast quantities of pesticides caused insects to become resistant, which led to the need of more pesticides overall. detected pesticides groups the detected pesticides could be categorized into a variety of groups, as shown by table 6 and figure 4, with the most frequent groups being organophosphorus ops, followed by dmifungicide, strobilurin, neonicotinoids, pyrethroids, pyridine carboxamides, phthalimides, and diacylhydrazines, with frequencies percentages of 12.44%, 9.68%, 9.22%, 7.37%, 7.3 the most often identified pesticide class in egyptian vegetables in 2020 was op’s (12.44%), which has been the case for a number of years. the majority of ops were found in vegetable samples from 2011 that were previously collected (41.5%; sohair et al., [29]); they were also found in fruit samples from 2007 that were previously collected (29.4%; gadallah et al., [52] ) and they are widely utilized in egypt. the effectiveness and low cost may be the causes of the product’s prolonged use in egyptian markets, and farmers find it difficult to change their usage patterns. concerns are raised by a review conducted by roshini et al., [53] that exposure to op pesticides at levels currently regarded as acceptable may have had a negative impact on human reproductive function and survival. contrarily, ops are hazardous to the nervous system and have been substantially eliminated from agriculture in several nations during the past ten years [52]. however, they are not outlawed and continue to be used on some food crops, according to the ewg’s 2012 list of the "dirty dozen" endocrine disruptor pesticides (the-dirty-dozen-eco-group, 2012). fungicides containing strobilurin have been used extensively in agricultural fields for many years. the frac group 11 fungicides known as strobilurin or qoi are very effective at controlling a variety of common vegetable diseases [54] . stabilurins’ highlights in bioscience page 7 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 6. the detected pesticide frequencies in analyzed egyptian vegetable and fruit samples, 2020. sn substance classified* chemical name type freq. no 1 organophosphate chlorpyrifos ins. 18 2 dimethoate ins. 4 3 malathion ins. 2 4 methamidophos ins. 3 5 pyrethroid cyfluthrin ins. 1 6 cypermethrin ins. 2 7 fenpropathrin ins. 2 8 l-cyhalothrin ins. 11 9 dmi** cyproconazole herb. 1 10 difenoconazole fung. 10 11 flutazole fung. 1 12 flutriafol fung. 1 13 penconazole fung. 5 14 propiconazole fung. 2 15 tetraconazole fung. 1 16 strobilurin azoxystrobin fung. 10 17 pyraclostrobin fung. 4 18 trifloxystrobin fung. 6 19 pyridinecarboxamides boscalid fung. 8 20 fluopyram fung. 7 21 neonicotinoids acetamiprid ins. 13 22 imidacloprid ins. 3 23 benzimidazole carbendazim fung. 2 24 thiophanate.m fung. 6 25 carbamates pirimicarb ins. 1 26 growth regulator paclobuttazol fung. 1 27 buprofezin ins. 1 28 avermectins abamectin ins. 7 29 acramite bifenazate acar, 9 30 anilinopyrimidines pyrimethanil fung. 3 31 arylepyrrole chlorfenapyr mite 8 32 diacylhydrazines methoxyfenozide ins. 11 33 diamides chlorantraniliprole ins. 1 34 dicarboximides iprodion fung. 2 35 imidazole bifenazole acar. 3 36 oxadiazine pesticide indoxacarb (p) ins. 9 37 phenylamide :acrylalanin metalaxyl fung. 8 38 phthalimides captan fung. 14 39 pyrimidines fenarimol fung. 5 40 spinosyns spinetoram ins. 4 41 sulfite ester propargite acar. 5 42 tetronic acids spirodiclofen acar. 2 total 217 *-types; acar, acarecides-ins, insecticidefung, fungicide -herb, herbicide, -substance classified is referred to: pan pesticide database, freq; the total number of frequencies for the positive samples of all vegetable under study, **dmi: demethylation inhibitors unique, non-target-specific fungicidal activity. previously, strobilurins were thought to be less hazardous to mammals [55], but a number of authors have noted that this is not entirely true due to gaps in the toxicological endpoints of fungicides [56]. these pesticides are intended to control fungi, but because of their broad-spectrum method of action, they can also have unintended consequences. therefore, strobilurin toxicity may result in ecosystem imbalance and food-web disturbance. the development of synthetic pyrethroids involved extensive chemical modifications that made them more toxic and less biodegradable in the environment [57; 58]. despite this, pyrethroids still have negative effects because they can lead to endocrine disruption, liver function impairment, and respiratory problems when exposed to them over an extended period of time. figure 4. the most detected pesticides groups in vegetable samples analysed during 2020. health risk assessment because of the potentially harmful effects, dietary exposure to pesticides raises health concerns. food quality and safety must be ensured in order to reduce the increased health risks associated with eating foods that contain pesticide residues. in order to reduce the presence of and exposure to pesticide residue, especially non-authorized pesticides, monitoring programs for the detection of pesticide residues in food should give enhanced health risk estimations for hygienic activities. table 0.4.3 clarifies approved daily intake (adi) and estimated daily intake (edi) for pesticides. strawberry, pepper, and cucumber had the lowest absolute intakes from ingesting the commodity, with 0.47, 0.159, and 0.096 g/kg b.w/day, respectively. tomato had the highest absolute intakes, with 2.89 g/kg bw/day (table 0.4.3 ). once more, to evaluate chronic exposure, the amount of pesticide exposure throughout a lifetime and any potential health impacts of highlights in bioscience page 8 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 7. pesticide estimated daily intake (edi), acceptable daily intake (adi) and hazard index (hi%). c om m odity e stim ated dietary intake (1e d i,(g /kg b.w /day) 2a d i h i p esticide s traw berry tom ato p epper c ucum ber total (g /kg b w /day) (source;year) b ifenazate 0.028 0.315 0.342 10 jm p r -2006 & e f s a -2017 3.424 f luopyram 0.079 0.502 0.009 0.007 0.596 10 jm p r -2010 5.956 d im ethoate 0.033 0.007 0.040 2 jm p r -1995 1.997 a bam ectin 0.02 0.004 0.023 1 jm p r -2015 2.327 t hiophanate.m athyle 0.018 1.121 1.139 30 jm p r -2013 3.796 d ifenoconazole 0.01 0.01 0 0.020 10 jm p r -2007,2010,2013 0.205 c hlorpyrifos 0.002 0.07 0.007 0.003 0.082 10 jm p r -2009 0.819 c yperm ethrin 0.018 0.018 5 e f s a ,2018 0.362 p ropargite 0.022 0.022 10 jm p r -1999 0.222 b oscalid 0.014 0.039 0.054 40 jm p r -2006 0.134 indoxacarb 0.008 0.043 0.051 10 jm p r -2005 0.507 o thers 0.29 0.738 0.136 0.064 1.226 3.6606 1e d i:e stim ated daily intake based on vegetable consum ption data show n in table a. a d i:a cceptable daily intake,h i:h azard index = (e d i/a d i*100) jm p r is the jointfa o /w h o m eetings on p esticide r esidues and e f s a is e uropean food s afety a uthority. **taking into consideration thatin the absence of a d i,the value of 10 g /kg b w /day is usually setby default such exposure must be taken into account [29]. this assessment method, which was carefully developed, considers average exposure levels in relation to the adi values determined for specific pesticides. the estimated daily intake (edi) for each pesticide was det independently in an exposure assessment. if consumers are exposed to chronic hazardous pesticide residues, their health won’t be at risk unless they consume more food than the adi each day for a long time. hazard index according to data in table 8, the hazard index (hi) for contaminated strawberry samples ranged from 0.0001% for chlorantraniliprole to 0.785% of the adi for fluopyram, while its values ranged from 0.015% for azoxystrobin to 5.015% for fluopyram in tomato samples, ranged from 0.0013% (metalaxyl) to 0.479% (fenarimol) in pepper samples, but ranged from 0.0003 to 0. the data from the risk assessment of pesticide residues in the tested commodities show that there is no risk associated with eating these vegetable. these findings are consistent with those made by akoto et al., [33] , who stated that when the hi value is less than 100%, the concerned commodity is regarded acceptable. if the hi value is larger than 100%, the concerned food is considered to pose a risk to consumers. according to recent statistics, fluopyram has a tomato hi value of 5.015 percent, which was the highest. due to the hi values, all tested pesticides had no individual potential health harm because the edi did not exceed (below the cut-off limit of 100). according to hossain et al., [59] , pesticide exposure in vegetables has minimal effects on consumers both individually and collectively, although it is possible that eating raw foods like cucumbers that haven’t been washed could increase the risk of pesticide exposure. additionally, seo et al., [60] revealed that the danger of exposure to pesticide residues found in dried vegetables gathered from seoul, korea, was minimal. the current findings demonstrated that there is no link between egyptian consumers’ longterm exposure to pesticide residues from eating raw vegetable and health risks. it should be noted that the current study is restricted to a select few vegetable. furthermore, rather than assessing the cumulative exposure to several pesticide residues in crops, the predicted risk assessment via long-term exposure is based on toxicological evaluation of the individual chemicals. contributed pesticides in total hazard index in order of contribution percentages (via consumption of a single commodity) in the analyzed egyptian vegetables during 2020, figure 5 lists the 13 high contributed pesticides, which include eight insecticides, one acaricide, and four fungicides. in order of decreasing contribution to the hi, the following pesticides were used: fluopyram (25.4%), thiophanate-mathyle (16.2%), bifenazate (14.6%), abamectin (9.9%), dimethoate (8.5%), chlorpyrifos (3.5%), methamidophos (2.8%), lambda-cyhalothrin (2.6%), captan (2.2%), indoxacarb (2.2%), and fenarimol (2.0%), cypermethrin (1.5%) and buprofezin (1.3%) in descending order, while each of the remaining 29 pesticides contributed less than 1% of the hi. figure 5. the most contributed pesticides in total hazard index (hi%). contributed commodities in total hazard index the calculated contributions of each commodity to the his are shown in figure 6, along with the commodities that make highlights in bioscience page 9 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 8. the estimated intake (edi) and the hazard index (hi) of pesticide residues in egyptian vegetable samples. pesticides detected strawberry tomato pepper cucumber edi hi edi hi edi hi edi hi abamectin 0.02 1.97 0.0036 0.361 acetamiprid 0.013 0.019 0.02 0.03 0.0118 0.017 azoxystrobin 0.018 0.009 0.03 0.02 bifenazate 0.028 0.278 0.31 3.15 bifenazole 0.005 0.054 boscalid 0.014 0.036 0.04 0.1 buprofezin 0.027 0.3 captan 0.044 0.044 0.45 0.45 0.0204 0.02 carbendazim 0.015 0.05 chlorantraniliprole 0.002 0.0001 chlorfenapyr 0.04 0.15 0.002 0.01 chlorpyrifos 0.002 0.017 0.07 0.7 0.007 0.07 0.0031 0.031 cyfluthrin 0.01 0.02 cypermethrin 0.0181 0.362 cyproconazole 0.006 0.028 difenoconazole 0.01 0.102 0.01 0.1 0.0005 0.005 dimethoate 0.03 1.67 0.007 0.33 fenarimol 0.048 0.48 fenpropathrin 0.001 0.003 0.003 0.01 fluopyram 0.079 0.785 0.5 5.02 0.009 0.09 0.0069 0.069 flutazole 0.002 0.017 flutriafol 0.007 0.07 imidacloprid 0.002 0.003 0.02 0.03 indoxacarb (p) 0.008 0.081 0.04 0.43 iprodion 0.004 0.007 lambda-cyhalothrin 0.002 0.008 0.12 0.59 0.0033 0.017 malathion 0.043 0.01 0.001 0.0003 metalaxyl 0.011 0.014 0.001 0.00125 0.0223 0.028 methamidophos 0.026 0.65 methoxyfenozide 0.02 0.02 0.05 0.05 0.013 0.01 paclobuttazol 0.002 0.008 penconazole 0.006 0.02 0.002 0.007 pirimicarb 0.037 0.183 propargite 0.022 0.222 propiconazole 0.009 0.013 pyraclostrobin 0.007 0.024 pyrimethanil 0.016 0.008 spinetoram 0.008 0.02 spirodiclofen 0.004 0.043 tetraconazole 0.002 0.042 thiophanate.mathyle 0.018 0.059 1.12 3.74 trifloxystrobin 0.02 0.05 edi: estimated daily intake, adi: acceptable daily intake, hi: hazard index = (edi/adi*100) jmpr is the joint fao/who meetings on pesticide residues and efsa is european food safety authority.**taking into consideration that in the absence of adi, the value of 10 g /kg bw/day is usually set by default up the majority of those contributions. tomatoes were the commodities that contributed the greatest to hazard (hi). as a result, they posed some risks to consumers and had exposures that were above acceptable limits, but generally, home processing methods including washing, heating, and frying may lower the levels of residues in vegetables. figure 6. the most contributed commodities in total hazard index (hi %). conclusion with the exception of levels of several insecticides, such as lambada-cyhalothrin (tomato), methamidophos (strawberry), and malathion (pepper), most pesticide residues were present in the most frequently consumed foods at below detectable levels, and those present above detection levels were in trace amounts. generally speaking, coordinated efforts are required to improve food quality and safety as well as to manage pesticide residues in both the environment and humans. the presence of prohibited pesticides in local markets needs to be addressed by the government authorities. implementing ipm programs correctly within the gap framework, creating new pesticide application methods, and shifting toward the production of organic foods will all assist sustainable development. further studies should be conducted to provide further evidence on the long-term health effects of pesticide exposure through diet. abbreviations adi: acceptable daily intake bw: body weight cccf: codex committee on contaminants in foods cifocoss: chronic individual food consumption database summary statistics efsa: european food safety authority 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nutrition and food science. 2013;18(2):145. highlights in bioscience page 13 of 13 january 2023|volume 6 http://www.epa.gov/oppsrrd1/reds/cypermethrin_red.pdf. http://www.epa.gov/oppsrrd1/reds/cypermethrin_red.pdf. http://www.epa.gov/oppsrrd1/reds/factsheets/permethrin_fs.htm.5/26/09 http://www.epa.gov/oppsrrd1/reds/factsheets/permethrin_fs.htm.5/26/09 http://bioscience.highlightsin.org/ abstract introduction material and methods samples sample preparation and analysis calculation of adi and hi results and discussion pesticide frequencies (detected pesticides type) detected pesticides groups health risk assessment hazard index contributed pesticides in total hazard index contributed commodities in total hazard index conclusion abbreviations highlights in bioscience; paper format highlights in bioscience issn: 2682-4043 highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 1 of 6 citation: zaimah n. s. (2019) plant resistance gene, snp and genome annotation an overview. highlights in bioscience, volume 2.article id 20192, dio:10.36462/ h.biosci.20192 received: june 19, 2019 accepted: july 13, 2019 published: august 12, 2019 copyright:© 2019 zaimah .this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. plant resistance gene, snp and genome annotation an overview nor s. zaimah sciences research institute, selangor, malaysia abstract plants have developed systems of effective and passive protection to safeguard themselves from pathogens. active processes include kinds of immune responses that are adaptive and intrinsic. adaptive immunity is focused on reaction type rnai and works primarily against viruses. biological immunity is more general and allows the plant to protect itself against a wide range of pathogens through bacterial and model resistance receptors (pprs) and forms of resistance (r proteins). pprs identify molecular models associated with microbes or pathogen that are preserved in a specific category of pathogens. with such big marker amounts, it has become feasible to scan the entire genome for interactions of individual markers with particular quantitatively hereditary traits called wholegenome studies (wgs), genome-wide association trials (gwas), or association genetics at exceptionally elevated marker densities. several snp recognition methods have been used in a specified plant to identify significant amounts of snps. these include: identification of snp based on est feature information; identification of snp from sequenced genomes; re-sequencing of amplicons; identification of snp using sequencing techniques of the next generation and identification of snp based on cluster tests. pathologic recognizes parts of genbank completely annotated genome and metacyc has been used as a database for the reference pathway. in contrast to sequence similarity information used in other systems, pathologic uses genbank annotation information and the ec assignment as evidence of the presence of each pathway in the genome of interest reference database. when the matching task is finished, pathologic will infer a number of reactions expected to take place in the destination genome and will determine which one of those pathways in the target genome are likely to exist. keywords: resistance proteins, genome-wide association, snp, biological pathways, genome annotation . review article open access plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 2 of 6 introduction proteins of resistance, in turn, identify distinctive elements of avirulence (avr) which are not conserved between many pathogens. induced-signaling resistance protein contributes to the manufacturing of reactive oxygen species and the induction of a particular form of programmed cell death, called the hypersensitive response, which damages the impacted cells (1). also called gene-to-gene resistance is the resistance of protein-mediated innate immunity, as each r gene reacts to a particular pathogenic avr gene (1). as a result, a big variety of r genes per plant genome are anticipated to be prepared to confer resistance to a wide range of pathogens. r genes are also under selection diversification to maintain pace with the fast pathogen evolution. although distinct r genes react to very unique pathogens, they share multiple regions (domains) that have been conserved. r proteins can be split into four subclasses on the basis of these domains. snp markers have acquired a great deal of concern in the science and breeding society (2) over the past two centuries. they happen in nearly infinite amounts as variations between individual nucleotides and each snp is a possibly helpful marker in single copy dna. in the study of human genome, the potential of snp markers is obviously illustrated. several million snp markers have been recognized (3) and techniques have been created concurrently (mainly up to 1 million) to evaluate big amounts of snp markers based on huge study attempts and the complete description of the human genome. as more genome sequences became accessible, comparative assessment of various genomes became a very significant research method. compared to various genomes of selection, biological pathway assessment is conducted using a range of computational techniques and databases (4,5). there are a range of enzymes and their substrates and products involved in a biological pathway. pathways also communicate with each other. thus, comparison pathway assessment is quite complex and can hardly be achieved without well-designed software systems for pathway assessment (4). we review the concept of plant resistance genes, plant snp markers and plant genome annotation. resistance genes most r proteins comprise a key nucleotide binding site (nbs) that functions as a molecular switch to regulate the protein's activity status, and a c-terminal, leucine-rich repeat region (lrr) needed to recognize avr factor. thus, the ranking of r proteins is focused on n-terminal domain variety (1). drosophila toll and human interleukin receptors categorized as tir-nb-lrr proteins are homologous to nbs-lrr form r proteins with n-terminals. non-tir nbslrr proteins are known to as cc-nbs-lrr proteins since some non-tir proteins in their n terminus form a coiled coil (cc) domain (6). furthermore, there are two categories of r proteins in their n terminus that contain an extracellular lrr. one of these groups, called kinases (rlks) receptor, includes a kinase domain of cytoplasmic protein (7). this cytoplasmic protein kinase domain is also lacking in receptors such as proteins (rlps). since r genes from distinct plant species combine conserved domains, they could be used to monitor plant genomes for r genes and putative r genes (e.g., analogs of resistance genes, rgas) and to generate molecular markers (7). meyers et al. (8) investigated a genome-wide assessment of 149 nbs-lrr processing genes in arabidopsis and verified either 55 cc-nbs-lrr (cnl) or 94 tir-nbs-lrr (tnl) proteins in two significant groups. eight significant motifs varied in their deviation within and between cnl and tnl clusters, and in the model found in particular for plant r protein homologues. introns may be more prevalent in cereals in the nbs region than in dicots. only participants of the arabidopsis rpp8/hrt gene class contain introns in the nbs domain in 20 named dicot nbslrr genes. nevertheless, in their nbs region, pib (9), pi-ta (10) and mla1 (11), three distinguished plant resistance genes have introns. repeats rich in leucine (lrrs) consist of redundant incomplete amino acid sections that fold into solvent-exposed β-cell β-loop constructions and this domain is believed to be engaged in ligand binding and disease identification (12). alternating conservation motifs and hyper variance marked lrr areas. the variation is highest for codons (x) situated in the lrr consensus xxlxlxx around the two preserved aliphatic amino acids, and the number of lrr repeats ranges among family members. there were approximately 65 amino acids in tnl proteins between the nbs and lrr domains in the genome-wide assessment of arabidopsis lrr domains (12). in arabidopsis, in cnl proteins, lrrs represent about half of the c-terminal region in the tnl proteins and almost the entire c-terminal region. the median domain of tnl lrr and cnl lrr comprised an average of 14 lrrs with ~10 separate meme patterns spanning as many as 350 amino acids as possible. in the rice proteins, a total of 25 distinct lrr motifs have been recognized. in any one gene, the amount of lrr clusters varied from 3 to 40 (13) . there are more than 150 genes of nbs-lrr in the arabidopsis thaliana dna. a sum of 166 nbs-lrr sequences were mentioned in (13), along with 33 truncated sequences. these nbs-lrr genes happen in their chromosome layout as 51 plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 3 of 6 singletons and 40 clusters. meyers et al. (2003) identified more nbs-lrr mutations by using comprehensive manual re-annotation of the same species genomic structure. they identified 149 nbs-lrr genes as well as 58 truncated genes; spread as 40 singletons and 43 clusters were the 149 non-truncated genes. in arabidopsis thaliana, tir-nbs-lrr genes family was detected out cc-nbs-lrr genes by approximately two to one, showing either a latest amplification of the former group or reduction of the latter gene group (13–15). nbslrr gene loci arabidopsis are often not dispersed uniformly in the chromosomal genome. there are super groups on chromosomes 1 and 5 whereas chromosomes 2 and 3 were comparatively lacking in the genes of nbs-lrr (13,14). the clusters are believed to be engaged in both r-gene diversity generation and conservation. meyers et al. (14) noted that the nbs gene chromosomal allocation is substantially non-random in maize: chromosome 11 includes about one-quarter of the nbs genes. five hundred and thirty-five nbs encoding genes have been recognized in rice, including 480 non-tir nbs-lrr genes. tir-nbs-lrr genes have been not detected in the rice genomic dna. in 44 gene clusters, two hundred and sixty-three genes (51 percent) occurred. in the "clustered" allocation category, there are 40 pairs and 17 triplets, 394 genes. a total of 125 singletons of nbs have been distributed across all chromosomes. in the plant genome, the proportion of singletons to the complete amount of nbs chromosomes (24.1%) was comparable to that of arabidopsis (26.8%). baumgarten et al. (16) proposed that most of the genomic dispersion of nbs-lrr genes was produced by duplication and translocation of whole chromosome sections (segmental duplication), rather than by small-scale ectopic duplication. most of the dramatic variation happens within local chromosomal areas of the nbs-lrr gene copy number. zhou et al. (17) revealed that 51% of nbs genes in rice originated in 44 gene clusters, where a cluster is an area with four and sometimes more genes within 200 kb or less. many surveys of nbs-lrr sequences or analogs of resistance genes have shown that r genes or nbs-lrr sequences are also structured in big clusters in other plant species. high-throughput genomic studies and plant genome sequences accessible in international databases provide unprecedented possibilities to recognize novel r-genes, investigate their role and method of diversification, find new genes for resistance, and eventually elucidate their interaction processes between pathogens and their crop hosts (18). in 2009, sanseverino et al. (18) launched the plant disease resistance gene database (prgdb), a comprehensive repository of r-genes across hundreds of plant species, with the intention of facilitating research on this agriculturally important gene family. a total of 16,844 gene records were included in prgdb version 1.0. of these 73 r-genes (e.g. the ' reference ' data collection) were recognized and manually curated, 6,308 were putative r-genes recovered from ncbi genbank, and 10,463 were putative r-genes computationally anticipated from ncbi unigene information. many plant genome-sequencing initiatives have developed quickly over the past few years. for example, potato (19), tomato (20), and melon (21) genomes have been finished, providing an chance to find extra r-genes (22). sanseverino et al. (22) presented an overview of the prgdb database of crop resistance genes. this database has been extended to include more than 6-fold valuable biological data from 233 plant species on a total of 104,459 r-genes. of these papers, 112 are defined in the literature as manualcurated r-genes to confer resistance to 122 distinct pathogens. all other genes were predicted gene. snp identification based on est sequence data large amounts of expressed sequence tags (ests) were produced for many plant species (23). the number of accessible ests in the ncbi est database ranges from less than 10,000 to over one million ests in significant crop plants and 1.5 million ests for the arabidopsis model plant. these ests were obtained in many cases within the framework of international efforts and were accumulated from a limited set of different lines and could therefore represent as a source for snp identification. in some instances, ests were specifically produced for snp detection from distinct lines as in arabidopsis thaliana (24) and in other instances, using bioinformatics assessment techniques (25), ests from heterozygous extremely polymorphic samples were used to identify snp. snp identification from sequenced genomes currently, many crop species genomic sequences have been released. sequenced genomes can be used to identify big amounts of snps in several respects. snps can be mined immediately in the genomic sequence in the event of heterozygous species such as grape or poplar, since two genome models have actually been produced. two distinct specimens were sequenced in the scenario of rice (26) and grape (27) so that snps can be mined by comparing the two genome models (28). for arabidopsis thaliana , genome resequencing panels were built based on the col-0 chromosome sequences and used among 20 genotypes to identify snps based on hybridization (29,30). plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 4 of 6 as a public database (https://www.ncbi.nlm.nih.gov/), the single nucleotide polymorphism database (dbsnp) was created. this database contains 1,648,103,041 snps for many organisms, e.g., brassica napus (901.5 thousand snps); arabidopsis thaliana (1.1 million snps); cicer arietinum (519.1 thousand snps); phoenix dactylifera (3.5 million snps); glycine max (16.9 million snps) and zea mays (54.3 million). genome annotation karp et al. (31) established pathway methods that use the pathological algorithm to assess the enzymatic responses catalyzed for each gene product in a query genome and then combine the identified response list from a reference database against all accessible processes. thompson et al. (32) produced the kegg automatic annotation server (kaas) towards the manually mapped kegg/genes registry database (33) providing functional gene annotation through blast comparisons, single best hit (sbh) and bidirectional best scores (bbh). as yield were produced ko tasks to genes and anticipated kegg processes. overbeek et al. (2005) launched the seed platform that offers a webbased,' subsystems'-based, relative genome annotation method. subsystems were a collection of functional features observed in any prevalent biological system, including cellular processes, phenotypes, or complicated multi-subunit constructions. haft et al. (35) established the tigr comprehensive microbial resources (cmr) to enable users from accessing all completed sequences of bacterial genomes. cmr offers two kinds of assets for annotation: main annotation from the genome sequencing centre and tigr annotation produced through an interactive tigr annotation method. the cmr pathway tool kit comprises of three classifications of pathway assessment tools: ' genome properties ' offers information on the features of species extracted from genomic data and literature references ; ' genome properties detailed comparison ' offers direct step-by-step information for selected clients of a collection of genomes ; and ' kegg pathway display ' shows kegg's presence-based pathway measures. conesa et al. (36) developed the blast2go (b2g), a universal guide for go annotation, visualization and stats that provides sophisticated functional evaluation for nonmodel organism genomics studies. b2g is intended to enable annotation of instant and high-throughput succession and incorporate annotation-based data mining features. b2g utilizes blast (37) to discover homologs for fastaformatted sequences of inputs. the program excerpts go conditions by referring to existing annotation associations for each hit acquired. finally, an annotation rule gives go conditions to the sequence of queries. it is possible to visualize annotation and functional analysis in a graph form that reconstructs the go interactions and highlights the most appropriate regions. in an embedded genome framework, markowitz et al. (38) introduced the integrated microbial genomes (jgi) for comparative microbial genome assessment. the data model comprising the img scheme includes main genomic sequence data, algorithmically forecast and ordered gene models, pre-computed sequence resemblance data, functional annotation, and pathway data. microbial gene statistical analysis in img is conducted in a relative framework of various microbial genomes where a range of instruments can be used to assess genomes in terms of genome-specific statistics, genes and sequence conserved. the kegg orthology-based annotation system (kobas) was implemented by wu et al. (39) that can provide statistical significance examinations for anticipated pathways. ko terms have been chosen based either on sequence resemblance to kegg/genes entries or on kegg/genes cross-database links when a list of sequence classifications is accessible in the databases. compared to the background model, regularly occurring or statistically significantly enriched query sequence pathways are recognized forward to ko assignment. pireddu et al. (40) created a path-a (pathway analyst) software that uses a number of query protein samples from a genome and defines which sequences are probable to occur using multiple sequence evaluation methods (e.g. svm, blast and hmm) in any of its endorsed model pathways. the approach of the model pathway allows the pathway prediction algorithm to predict instances of a pathway with pathway structure variations that have never been observed in the training pathway set. at present, path-a offers abstract models for 10 pathways, covering 125 cases of genome-specific processes. choi and kim (4) established a comparative pathway workbench system (compath) that enables researchers to compare biological processes in multiple genomes using a spreadsheet-style online interface where different sequencebased analyzes can be conducted either to match enzymes (e.g. sequence clustering) or processes (e.g. pathway hole recognition) to search for a de novo enzyme forecast algorithm. https://www.ncbi.nlm.nih.gov/ plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 5 of 6 references 1. van ooijen g, van den burg ha, cornelissen bjc, takken flw. structure and function of resistance proteins in solanaceous plants. annu rev phytopathol. 2007;45:43–72. 2. rafalski a. applications of single nucleotide polymorphism in crop genetics. curr opin plant biol. 2002;5:94–100. 3. frazer ka, ballinger dg, cox dr, hinds da, stuve ll, gibbs ra, et al. a second generation human haplotype map of over 3.1 million snps. nature. 2007; 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