Search results for: pseudo-random sequence

Authors: Parveen Kumar

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This article discusses institute information resources. Information, in its most restricted technical sense, is a sequence of symbols that can be interpreted as message information can be recorded as signs, or transmitted as signals. Information is any kind of event that affects the state of a dynamic system. Conceptually, information is the message being conveyed. This concept has numerous other meanings in different contexts. Moreover, the concept of information is closely related to notions of constraint, communication, control, data, form, instruction, knowledge, meaning, mental stimulus, pattern, perception, representation, and especially entropy.

Keywords: institutions, information institutions, information services for mission-oriented institute, pattern

Procedia PDF Downloads 376

Authors: Sa’adu Mafara Abubakar, Muhammad Nuraddeen Danjuma, Adewole Tomiwa Adetunji, Richard Mundembe, Salisu Mohammed, Francis Bayo Lewu, Joseph I. Kiok

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Pearl millet is the most drought tolerant of all domesticated cereals, is cultivated extensively to feed millions of people who mainly live in hash agroclimatic zones. It serves as a major source of food for more than 40 million smallholder farmers living in the marginal agricultural lands of Northern Nigeria. Pearl millet grain is more nutritious than other cereals like maize, is also a principal source of energy, protein, vitamins, and minerals for millions of poorest people in the regions where it is cultivated. Pearl millet has recorded relatively little research attention compared with other crops and no sufficient work has analyzed its genetic diversity in north-western Nigeria. Therefore, this study was undertaken with the objectives to analyze the genetic diversity of pearl millet accessions using SSR marker and to analyze the extent of evolutionary relationship among pearl millet accessions at the molecular level. The result of the present study confirmed diversity among accessions of pearl millet in the study area. Simple Sequence Repeats (SSR) markers were used for genetic analysis and evolutionary relationship of the accessions of pearl millet. To analyze the level of genetic diversity, 8 polymorphic SSR markers were used to screen 69 accessions collected based on three maturity periods. SSR markers result reveal relationships among the accessions in terms of genetic similarities, evolutionary and ancestral origin, it also reveals a total of 53 alleles recorded with 8 microsatellites and an average of 6.875 per microsatellite, the range was from 3 to 9 alleles in PSMP2248 and PSMP2080 respectively. Moreover, both the factorial analysis and the dendrogram of phylogeny tree grouping patterns and cluster analysis were almost in agreement with each other that diversity is not clustering according to geographical patterns but, according to similarity, the result showed maximum similarity among clusters with few numbers of accessions. It has been recommended that other molecular markers should be tested in the same study area.

Keywords: pearl millet, genetic diversity, simple sequence repeat (SSR)

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Authors: Monika Soni, Shovonlal Bhowmick, Chandra Bhattacharya, Jitendra Sharma, Prafulla Dutta, Jagadish Mahanta

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Aedes aegypti and Aedes albopictus are considered as two major vectors to transmit dengue virus. In North-east India, two states viz. Assam and Arunachal Pradesh are known to be high endemic zone for dengue and Chikungunya viral infection. The taxonomical classification of medically important vectors are important for mapping of actual evolutionary trends and epidemiological studies. However, misidentification of mosquito species in field-collected mosquito specimens could have a negative impact which may affect vector-borne disease control policy. DNA barcoding is a prominent method to record available species, differentiate from new addition and change of population structure. In this study, a combined approach of a morphological and molecular technique of DNA barcoding was adopted to explore sequence variation in mitochondrial cytochrome c oxidase subunit I (COI) gene within dengue vectors. The study has revealed the map distribution of the dengue vector from two states i.e. Assam and Arunachal Pradesh, India. Approximate five hundred mosquito specimens were collected from different parts of two states, and their morphological features were compared with the taxonomic keys. The analysis of detailed taxonomic study revealed identification of two species Aedes aegypti and Aedes albopictus. The species aegypti comprised of 66.6% of the specimen and represented as dominant dengue vector species. The sequences obtained through standard DNA barcoding protocol were compared with public databases, viz. GenBank and BOLD. The sequences of all Aedes albopictus have shown 100% similarity whereas sequence of Aedes aegypti has shown 99.77 - 100% similarity of COI gene with that of different geographically located same species based on BOLD database search. From dengue prevalent different geographical regions fifty-nine sequences were retrieved from NCBI and BOLD databases of the same and related taxa to determine the evolutionary distance model based on the phylogenetic analysis. Neighbor-Joining (NJ) and Maximum Likelihood (ML) phylogenetic tree was constructed in MEGA6.06 software with 1000 bootstrap replicates using Kimura-2-Parameter model. Data were analyzed for sequence divergence and found that intraspecific divergence ranged from 0.0 to 2.0% and interspecific divergence ranged from 11.0 to 12.0%. The transitional and transversional substitutions were tested individually. The sequences were deposited in NCBI: GenBank database. This observation claimed the first DNA barcoding analysis of Aedes mosquitoes from North-eastern states in India and also confirmed the range expansion of two important mosquito species. Overall, this study insight into the molecular ecology of the dengue vectors from North-eastern India which will enhance the understanding to improve the existing entomological surveillance and vector incrimination program.

Keywords: COI, dengue vectors, DNA barcoding, molecular identification, North-east India, phylogenetics

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Authors: O. O. Fola-Matthews, O. O. Soyinka, D. N. Bitalo

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Nigeria is one of the major shark fishing nations in Africa, but its fisheries managers still record catch data in aggregates ‘sharks’ with no species-specific details. This is because most of the shark specimens look identical in morphology, and field identification of some closely related species is tricky. This study uses DNA barcoding as a method to identify shark species from five different landing areas off the Nigerian Coast. 100 dorsal fins were sampled in order to provide a Chondrichthyan sequence that would be matched to reference specimens in a DNA barcode database

Keywords: BOLD, DNA barcoding, nigeria, sharks

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Authors: Nwogiji Cletus Olando, Oselebe Happiness Ogba, Enoch Achigan-Dako

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Digitaria iburua, commonly known as black fonio, is a cereal crop native to Africa and extensively cultivated by smallholder farmers in Northern Benin, Togo, and Nigeria. This crop holds immense nutritional and socio-cultural value. Unfortunately, limited knowledge about its genetic diversity exists due to a lack of scientific attention. As a result, its potential for improvement in food and agriculture remains largely untapped. To address this gap, a study was conducted using 41 accessions of D. iburua stored in the genebank of the Laboratory of Genetics, Biotechnology, and Seed Science at Abomey-Calavi University, Benin. The study employed both morphological and simple sequence repeat (SSR) markers to evaluate the genetic variability of the accessions. Agro-morphological assessments were carried out during the 2020 cropping season, utilizing an alpha lattice design with three replications. The collected data encompassed qualitative and quantitative traits. Additionally, molecular variability was assessed using eleven SSR markers. The results revealed significant phenotypic variability among the evaluated accessions, leading to their classification into three main clusters. Furthermore, the eleven SSR markers identified a total of 50 alleles, averaging 4.55 alleles per locus. The primers exhibited an average polymorphic information content value of 0.43, with the DE-ARC019 primer displaying the highest value (0.59). These findings suggest a substantial degree of genetic heterogeneity within the evaluated accessions, and the SSR markers employed in the study proved highly effective in detecting and characterizing this genetic variability. In conclusion, this study highlights the presence of significant genetic diversity in black fonio and provides valuable insights for future efforts aimed at its genetic improvement and conservation.

Keywords: genetic diversity, digitaria iburua, genetic improvement, simple sequence repeat markers, Nigeria, conservation

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Authors: Iftikhar Tayubi, Tabrej Khan, Rayan Alsulmi, Abdulrahman Labban

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Spider produces a special kind of substance. This special kind of substance is called a toxin. The toxin is composed of many types of protein, which differs from species to species. Spider toxin consists of several proteins and non-proteins that include various categories of toxins like myotoxin, neurotoxin, cardiotoxin, dendrotoxin, haemorrhagins, and fibrinolytic enzyme. Protein Sequence information with references of toxins was derived from literature and public databases. From the previous findings, the Spider toxin would be the best choice to treat different types of tumors and cancer. There are many therapeutic regimes, which causes more side effects than treatment hence a different approach must be adopted for the treatment of cancer. The combinations of drugs are being encouraged, and dramatic outcomes are reported. Spider toxin is one of the natural cytotoxic compounds. Hence, it is being used to treat different types of tumors; especially its positive effect on breast cancer is being reported during the last few decades. The efficacy of this database is that it can provide a user-friendly interface for users to retrieve the information about Spiders, toxin and toxin protein of different Spiders species. SPIDTOXD provides a single source information about spider toxins, which will be useful for pharmacologists, neuroscientists, toxicologists, medicinal chemists. The well-ordered and accessible web interface allows users to explore the detail information of Spider and toxin proteins. It includes common name, scientific name, entry id, entry name, protein name and length of the protein sequence. The utility of this database is that it can provide a user-friendly interface for users to retrieve the information about Spider, toxin and toxin protein of different Spider species. The database interfaces will satisfy the demands of the scientific community by providing in-depth knowledge about Spider and its toxin. We have adopted the methodology by using A MySQL and PHP and for designing, we used the Smart Draw. The users can thus navigate from one section to another, depending on the field of interest of the user. This database contains a wealth of information on species, toxins, and clinical data, etc. This database will be useful for the scientific community, basic researchers and those interested in potential pharmaceutical Industry.

Keywords: siptoxd, php, mysql, toxin

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Authors: Nora Geczy

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In today’s architectural practice, during the process of designing public, educational, healthcare and cultural space, human-centered architectural designs helping spatial orientation, safe space usage and the appropriate spatial sequence of actions are gaining increasing importance. Related to the methodology of designing public buildings, several scientific experiments in spatial recognition, spatial analysis and spatial psychology with regard to the components of space producing mental and physiological effects have been going on at the Department of Architectural Design and the Interdisciplinary Student Workshop (IDM) at the Széchenyi István University, Győr since 2013. Defining the creation of preventive, anticipated spatial design and the architectural tools of spatial comfort of public buildings and their practical usability are in the limelight of our research. In the experiments applying eye-tracking cameras, we studied the way public spaces are used, especially concentrating on the characteristics of spatial behaviour, orientation, recognition, the sequence of actions, and space usage. Along with the role of mental maps, human perception, and interaction problems in public spaces (at railway stations, galleries, and educational institutions), we analyzed the spatial situations influencing psychological and ergonomic factors. We also analyzed the eye movements of the experimental subjects in dynamic situations, in spatial procession, using stairs and corridors. We monitored both the consequences and the distorting effects of the ocular dominance of the right eye on spatial orientation; we analyzed the gender-based differences of women and men’s orientation, stress-inducing spaces, spaces affecting concentration and the spatial situation influencing territorial behaviour. Based on these observations, we collected the components of creating public interior spaces, which -according to our theory- contribute to the optimal usability of public spaces. We summed up our research in criteria for design, including 10 points. Our further goals are testing design principles needed for optimizing orientation and space usage, their discussion, refinement, and practical usage.

Keywords: architecture, eye-tracking, human-centered spatial design, public interior spaces, visual perception

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Authors: Hwang Ho Kim, Do Gyun Kim, Jin Young Choi, Sang Chul Park

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Since tire production process is very complicated, company-wide management of it is very difficult, necessitating considerable amounts of capital and labors. Thus, productivity should be enhanced and maintained competitive by developing and applying effective production plans. Among major processes for tire manufacturing, consisting of mixing component preparation, building and curing, the mixing process is an essential and important step because the main component of tire, called compound, is formed at this step. Compound as a rubber synthesis with various characteristics plays its own role required for a tire as a finished product. Meanwhile, scheduling tire mixing process is similar to flexible job shop scheduling problem (FJSSP) because various kinds of compounds have their unique orders of operations, and a set of alternative machines can be used to process each operation. In addition, setup time required for different operations may differ due to alteration of additives. In other words, each operation of mixing processes requires different setup time depending on the previous one, and this kind of feature, called sequence dependent setup time (SDST), is a very important issue in traditional scheduling problems such as flexible job shop scheduling problems. However, despite of its importance, there exist few research works dealing with the tire mixing process. Thus, in this paper, we consider the scheduling problem for tire mixing process and suggest an efficient particle swarm optimization (PSO) algorithm to minimize the makespan for completing all the required jobs belonging to the process. Specifically, we design a particle encoding scheme for the considered scheduling problem, including a processing sequence for compounds and machine allocation information for each job operation, and a method for generating a tire mixing schedule from a given particle. At each iteration, the coordination and velocity of particles are updated, and the current solution is compared with new solution. This procedure is repeated until a stopping condition is satisfied. The performance of the proposed algorithm is validated through a numerical experiment by using some small-sized problem instances expressing the tire mixing process. Furthermore, we compare the solution of the proposed algorithm with it obtained by solving a mixed integer linear programming (MILP) model developed in previous research work. As for performance measure, we define an error rate which can evaluate the difference between two solutions. As a result, we show that PSO algorithm proposed in this paper outperforms MILP model with respect to the effectiveness and efficiency. As the direction for future work, we plan to consider scheduling problems in other processes such as building, curing. We can also extend our current work by considering other performance measures such as weighted makespan or processing times affected by aging or learning effects.

Keywords: compound, error rate, flexible job shop scheduling problem, makespan, particle encoding scheme, particle swarm optimization, sequence dependent setup time, tire mixing process

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Authors: A. Kyprianou, A. Tjirkallis

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Condition monitoring of structures in service is very important as it provides information about the risk of damage development. One of the essential constituents of structural condition monitoring is the damage detection methodology. In the context of condition monitoring of in service structures a damage detection methodology analyses data obtained from the structure while it is in operation. Usually, this means that the data could be affected by operational and environmental conditions in a way that could mask the effects of a possible damage on the data. This, depending on the damage detection methodology, could lead to either false alarms or miss existing damages. In this article a damage detection methodology that is based on the Spatio-temporal continuous wavelet transform (SPT-CWT) analysis of a sequence of experimental time responses of a cantilever beam is proposed. The cantilever is subjected to white and pink noise excitation to simulate different operating conditions. In addition, in order to simulate changing environmental conditions, the cantilever is subjected to heating by a heat gun. The response of the cantilever beam is measured by a high-speed camera. Edges are extracted from the series of images of the beam response captured by the camera. Subsequent processing of the edges gives a series of time responses on 439 points on the beam. This sequence is then analyzed using the SPT-CWT to identify damage. The algorithm proposed was able to clearly identify damage under any condition when the structure was excited by white noise force. In addition, in the case of white noise excitation, the analysis could also reveal the position of the heat gun when it was used to heat the structure. The analysis could identify the different operating conditions i.e. between responses due to white noise excitation and responses due to pink noise excitation. During the pink noise excitation whereas damage and changing temperature were identified it was not possible to clearly identify the effect of damage from that of temperature. The methodology proposed in this article for damage detection enables the separation the damage effect from that due to temperature and excitation on data obtained from measurements of a cantilever beam. This methodology does not require information about the apriori state of the structure.

Keywords: spatiotemporal continuous wavelet transform, damage detection, data normalization, varying temperature

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Authors: Salman Akrokayan

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Synthetic cDNA (ScDNA) of granulocyte colony-stimulating factor (G-CSF) was constructed using a DNA synthesizer with the aim to increase its expression level. 5' end of the ScDNA of G-CSF coding region was modified by decreasing the GC content without altering the predicted amino acids sequence. The identity of the resulting protein from ScDNA was confirmed by the highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, the commercial utilization of this methodology requires rigorous validation and quality control.

Keywords: synthetic cDNA, recombinant G-CSF, cloning, gene expression

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Authors: M. Y. Abubakar, Ipinjolu J. K., Yuzine B. Esa, Magawata I., Hassan W. A., Turaki A. A.

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Catfish (Heterobranchus spp.) is a major freshwater fish that are widely distributed in Nigeria waters and are gaining rapid aquaculture expansion. However, indiscriminate artificial crossbreeding of the species with others poses a threat to their biodiversity. There is a paucity of information about the genetic variability, hence this insight on the genetic variability is badly needed, not only for the species conservation but for aquaculture expansion. In this study, we tested the level of Genetic diversity, population differentiation and phylogenetic relationship analysis on 35 individuals of two populations of Heterobranchus bidorsalis and 29 individuals of three populations of Heterobranchus longifilis using the mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene sequence. Nucleotide sequences of 650 bp fragment of the COI gene of the two species were compared. In the whole 4 and 5 haplotypes were distinguished in the populations of H. bidorsalis & H. longifilis with accession numbers (MG334168 - MG334171 & MG334172 to MG334176) respectively. Haplotypes diversity indices revealed a range of 0.59 ± 0.08 to 0.57 ± 0.09 in H. bidorsalis and 0.000 to 0.001051 ± 0.000945 in H. longifilis population, respectively. Analysis of molecular variance (AMOVA) revealed no significant variation among H. bidorsalis population of the Niger & Benue Rivers, detected significant genetic variation was between the Rivers of Niger, Kaduna and Benue population of H. longifilis. Two main clades were recovered, showing a clear separation between H. bidorsalis and H. longifilis in the phylogenetic tree. The mtDNA COI genes studied revealed high gene flow between populations with no distinct genetic differentiation between the populations as measured by the fixation index (FST) statistic. However, a proportion of population-specific haplotypes was observed in the two species studied, suggesting a substantial degree of genetic distinctiveness for each of the population investigated. These findings present the description of the species character and accessions of the fish’s genetic resources, through gene sequence submitted in Genetic database. The data will help to protect their valuable wild resource and contribute to their recovery and selective breeding in Nigeria.

Keywords: AMOVA, genetic diversity, Heterobranchus spp., mtDNA COI, phylogenetic tree

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Authors: Benjamin Lee Warren

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Many problems exist in additive number theory which is essential to determine the primes that are the sum of two elements from a given single-variable polynomial sequence, and most of them are unattackable in the present day. Here, we determine solutions for this problem to a few certain sequences (certain binomial coefficients and power sums) using only elementary algebra and some algebraic factoring methods (as well as Euclid’s Lemma and Faulhaber’s Formula). In particular, we show that there are finitely many primes as sums of two of these types of elements. Several cases are fully illustrated, and bounds are presented for the cases not fully illustrated.

Keywords: binomial coefficients, power sums, primes, algebra

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Authors: Vernie C. Convicto, Henry P. Aringa, Wilson I. Barredo

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This study presents a conformational model of the helical structures of globular protein particularly ferritin in the framework of white noise path integral formulation by using Associated Legendre functions, Bessel and convolution of Bessel and trigonometric functions as modulating functions. The model incorporates chirality features of proteins and their helix-turn-helix sequence structural motif.

Keywords: globular protein, modulating function, white noise, winding probability

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Authors: Rajkumar Ghosh

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Jhakri Thrust (JT), Sarahan Thrust (ST), and Chaura Thrust (CT) are the three OOST along Jakhri-Chaura segment along the Sutlej river valley in Himachal Pradesh. CT is deciphered only by Apatite Fission Track dating. Such geochronological information is not currently accessible for the Jhakri and Sarahan thrusts. JT was additionally validated as OOST without any dating. The described rock types include ductile sheared gneisses and upper greenschist-amphibolite facies metamorphosed schists. Locally, the Munsiari (Jutogh) Thrust is referred to as the JT. Brittle shear, the JT, borders the research area's southern and ductile shear, the CT, and its northern margins. The JT has a 50° western dip and is south-westward verging. It is 15–17 km deep. A progressive rise in strain towards the JT zone based on microstructural tests was observed by previous researchers. The high-temperature ranges of the MCT root zone are cited in the current work as supportive evidence for the ductile nature of the OOST. In Himachal Pradesh, the lithological boundaries for OOST are not set. In contrast, the Sarahan thrust is NW-SE striking and 50-80 m wide. ST and CT are probably equivalent and marked by a sheared biotite-chlorite matrix with a top-to-SE kinematic indicator. It is inferred from cross-section balancing that the CT is folded with this anticlinorium. These thrust systems consist of several branches, some of which are still active. The thrust system exhibits complex internal geometry consisting of box folds, boudins, scar folds, crenulation cleavages, kink folds, and tension gashes. Box folds are observed on the hanging wall of the Chaura thrust. The ductile signature of CT represents steepen downward of the thrust. After the STDSU stopped deformation, out-of-sequence thrust was initiated in some sections of the Higher Himalaya. A part of GHC and part of the LH is thrust southwestward along the Jutogh Thrust/Munsiari Thrust/JT as also the Jutogh Nappe. The CT is concealed beneath Jutogh Thrust sheet hence the basal part of GHC is unexposed to the surface in Sutlej River section. Fieldwork and micro-structural studies of the Greater Himalayan Crystalline (GHC) along the Sutlej section reveal (a) initial top-to-SW sense of ductile shearing (CT); (b) brittle-ductile extension (ST); and (c) uniform top-to-SW sense of brittle shearing (JT). A group of samples of schistose rock from Jutogh Group of Greater Himalayan Crystalline and Quartzite from Rampur Group of Lesser Himalayan Crystalline were analyzed. No such physiographic transition in that area is to determine a break in the landscape due to OOST. OOSTs from GHC are interpreted mainly from geochronological studies to date, but proper field evidence is missing. Apart from minimal documentation in geological mapping for OOST, there exists a lack of suitable exposure of rock to generalize the features of OOST in the field in NW Higher Himalaya. Multiple sets of thrust planes may be activated within this zone or a zone along which OOST is engaged.

Keywords: out-of-sequence thrust, main central thrust, grain boundary migration, South Tibetan detachment system, Jakhri Thrust, Sarahan Thrust, Chaura Thrust, higher Himalaya, greater Himalayan crystalline

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Authors: Rosa Figueroa, Christopher Flores

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Text categorization is the problem of assigning a new document to a set of predetermined categories, on the basis of a training set of free-text data that contains documents whose category membership is known. To train a classification model, it is necessary to extract characteristics in the form of tokens that facilitate the learning and classification process. In text categorization, the feature extraction process involves the use of word sequences also known as N-grams. In general, it is expected that documents belonging to the same category share similar features. The Smith-Waterman (SW) algorithm is a dynamic programming algorithm that performs a local sequence alignment in order to determine similar regions between two strings or protein sequences. This work explores the use of SW algorithm as an alternative to feature extraction in text categorization. The dataset used for this purpose, contains 2,610 annotated documents with the classes Obese/Non-Obese. This dataset was represented in a matrix form using the Bag of Word approach. The score selected to represent the occurrence of the tokens in each document was the term frequency-inverse document frequency (TF-IDF). In order to extract features for classification, four experiments were conducted: the first experiment used SW to extract features, the second one used unigrams (single word), the third one used bigrams (two word sequence) and the last experiment used a combination of unigrams and bigrams to extract features for classification. To test the effectiveness of the extracted feature set for the four experiments, a Support Vector Machine (SVM) classifier was tuned using 20% of the dataset. The remaining 80% of the dataset together with 5-Fold Cross Validation were used to evaluate and compare the performance of the four experiments of feature extraction. Results from the tuning process suggest that SW performs better than the N-gram based feature extraction. These results were confirmed by using the remaining 80% of the dataset, where SW performed the best (accuracy = 97.10%, weighted average F-measure = 97.07%). The second best was obtained by the combination of unigrams-bigrams (accuracy = 96.04, weighted average F-measure = 95.97) closely followed by the bigrams (accuracy = 94.56%, weighted average F-measure = 94.46%) and finally unigrams (accuracy = 92.96%, weighted average F-measure = 92.90%).

Keywords: comorbidities, machine learning, obesity, Smith-Waterman algorithm

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Authors: M. Gogniashvili, I. Maisaia, A. Kotorashvili, N. Kotaria, T. Beridze

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Three types of plasmon (A, B and G) is typical for genus Triticum. In polyploid species - Triticum turgidum L. and Triticum aestivum L. plasmon B is detected. In the forthcoming paper, complete nucleotide sequence of chloroplast DNA of 11 representatives of Georgian wheat polyploid species, carrying plasmon B was determined. Sequencing of chloroplast DNA was performed on an Illumina MiSeq platform. Chloroplast DNA molecules were assembled using the SOAPdenovo computer program. All contigs were aligned to the reference chloroplast genome sequence using BLASTN. For detection of SNPs and Indels and phylogeny tree construction computer programs Mafft and Blast were used. Using Triticum aestivum L. subsp. macha (Dekapr. & Menabde) Mackey var. paleocolchicum Dekapr. et Menabde as a reference, 5 SNPs can be identified in chloroplast DNA of Georgian endemic polyploid wheat. The number of noncoding substitutions is 2, coding substitutions - 3. In comparison with reference DNA two - 38 bp and 56 bp inversions were observed in paleocolchicum subspecies. There were six 1 bp indels detected in Georgian polyploid wheats, all of them at microsatellite stretches. The phylogeny tree shows that subspecies macha, carthlicum and paleocolchicum occupy different positions. According to the simplified scheme based on SNP and indel data, the ancestral, female parent of the all studied polyploid wheat is unknown X predecesor, from which four lines were formed. 1 SNP and two inversions (38 bp and 56 bp) caused the formation of subsp. paleocolchicum. Three other lines are macha, durum and carthlicum lines. Macha line is further divided into two sublines (M_1 and M_4). Carthlicum line includes subsp.carthlicum and T.aestivum - C_1 - C_2 - A_1. One of the central question of wheat domestication is which people(s) participated in wheat domestication? It is proposed that the predecessors of Georgian peoples (Proto-Kartvelians) must be placed, on the evidence of archaic lexical and toponymic data, in the mountainous regions of the western and central part of the Little Caucasus (the Transcaucasian foothills) at least 4,000 years ago. One of the possibility to explain the ‘wheat puzzle’ is that Kartvelian speakers brought domesticated wheat species and subspecis from Fertile Crescent further north to South Caucasus.

Keywords: chloroplast DNA, sequencing, SNP, triticum

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Authors: Krishnendra Shekhawat

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Given a graph G = (V, E), a rectangular dual of G represents the vertices of G by a set of interior-disjoint rectangles such that two rectangles touch if and only if there is an edge between the two corresponding vertices in G. Rectangular duals do not exist for every graph, so we can define maximal rectangular duals. A maximal rectangular dual is a rectangular dual of a graph G such that there exists no graph G ′ with a rectangular dual where G is a subgraph of G ′. In this paper, we enumerate all maximal rectangular duals (or, to be precise, the corresponding planar graphs) up to six nodes and presents a necessary condition for the existence of a rectangular dual. This work allegedly has applications in integrated circuit design and architectural floor plans.

Keywords: adjacency, degree sequence, dual graph, rectangular dual

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Authors: Narayan Moger, Divya B., Preethi Jambagi, Krishnaveni C. K., Apsana M. R., B. R. Patil, Basvaraj Bagewadi

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Salinity is one of the major threats to world food security. Considering the requirement for salt tolerant crop plants in the present study was undertaken to clone and transferred the salt tolerant ectA gene from marine ecosystem into agriculture crop system to impart salinity tolerance. Ectoine is the compatible solute which accumulates in the cell membrane, is known to be involved in salt tolerance activity in most of the Halophiles. The present situation is insisting to development of salt tolerant transgenic lines to combat abiotic stress. In this background, the investigation was conducted to develop transgenic tomato lines by cloning and transferring of ectA gene is an ectoine derivative capable of enzymatic action for the production of acetyl-diaminobutyric acid. The gene ectA is involved in maintaining the osmotic balance of plants. The PCR amplified ectA gene (579bp) was cloned into T/A cloning vector (pTZ57R/T). The construct pDBJ26 containing ectA gene was sequenced by using gene specific forward and reverse primers. Sequence was analyzed using BLAST algorithm to check similarity of ectA gene with other isolates. Highest homology of 99.66 per cent was found with ectA gene sequences of isolates Halomonas elongata with the available sequence information in NCBI database. The ectA gene was further sub cloned into pRI101-AN plant expression vector and transferred into E. coli DH5α for its maintenance. Further pDNM27 was mobilized into A. tumefaciens LBA4404 through tri-parental mating system. The recombinant Agrobacterium containing pDNM27 was transferred into tomato plants through In planta plant transformation method. Out of 300 seedlings, co-cultivated only twenty-seven plants were able to well establish under the greenhouse condition. Among twenty-seven transformants only twelve plants showed amplification with gene specific primers. Further work must be extended to evaluate the transformants at T1 and T2 generations for ectoine accumulation, salinity tolerance, plant growth and development and yield.

Keywords: salinity, computable solutes, ectA, transgenic, in planta transformation

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Authors: Cristian Chacha, David Asiain, Jesús Ponce de León, José Ramón Beltrán

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This work explores and characterizes the behavior of the AFE AD5941 in impedance measurement using an embedded algorithm with a constant current AC source. The main aim of this research is to improve the exact measurement of impedance values for their application in EDA-focused wearable devices. Through comprehensive study and characterization, it has been observed that employing a measurement sequence with a constant current source produces results with increased dispersion but higher accuracy. As a result, this approach leads to a more accurate system for impedance measurement.

Keywords: EDA, constant current AC source, wearable, precision, accuracy, impedance

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Authors: Benjamin A. Ha, Marco Morselli, Xinhui Paige Zhang, Elizabeth A. C. Heath-Heckman, Jonathan B. Puritz, David K. Jacobs

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Next-generation sequencing has enhanced the ability to acquire massive amounts of sequence data to address classic population genetic questions for non-model organisms. Targeted approaches allow for cost effective or more precise analyses of relevant sequences; although, many such techniques require a known genome and it can be costly to purchase probes from a company. This is challenging for non-model organisms with no published genome and can be expensive for large population genetic studies. Expressed exome capture sequencing (EecSeq) synthesizes probes in the lab from expressed mRNA, which is used to capture and sequence the coding regions of genomic DNA from a pooled suite of samples. A normalization step produces probes to recover transcripts from a wide range of expression levels. This approach offers low cost recovery of a broad range of genes in the genome. This research project expands on EecSeq to investigate if mRNA from one taxon may be used to capture relevant sequences from a series of increasingly less closely related taxa. For this purpose, we propose to use the endangered Northern Tidewater goby, Eucyclogobius newberryi, a non-model organism that inhabits California coastal lagoons. mRNA will be extracted from E. newberryi to create probes and capture exomes from eight other taxa, including the more at-risk Southern Tidewater goby, E. kristinae, and more divergent species. Captured exomes will be sequenced, analyzed bioinformatically and phylogenetically, then compared to previously generated phylogenies across this group of gobies. This will provide an assessment of the utility of the technique in cross-species studies and for analyzing low genetic variation within species as is the case for E. kristinae. This method has potential applications to provide economical ways to expand population genetic and evolutionary biology studies for non-model organisms.

Keywords: coastal lagoons, endangered species, non-model organism, target capture method

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Authors: Rowan Moore, Kai Scheffler, Eugen Damoc, Jennifer Sutton, Aaron Bailey, Stephane Houel, Simon Cubbon, Jonathan Josephs

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Purpose: MS analysis of monoclonal antibodies (mAbs) at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to various modifications which may affect efficacy. Intact proteins analyzed by MS are detected in higher charge states that also provide more complexity in mass spectra. Protein analysis in native or native-like conditions with zero or minimal organic solvent and neutral or weakly acidic pH decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. Methods: Three commercially available mAbs were used for all experiments. Intact proteins were desalted online using size exclusion chromatography (SEC) or reversed phase chromatography coupled on-line with a mass spectrometer. For streamlined use of the LC- MS platform we used a single SEC column and alternately selected specific mobile phases to perform separations in either denaturing or native-like conditions: buffer A (20 % ACN, 0.1 % FA) with Buffer B (100 mM ammonium acetate). For peptide analysis mAbs were proteolytically digested with and without prior reduction and alkylation. The mass spectrometer used for all experiments was a commercially available Thermo Scientific™ hybrid Quadrupole-Orbitrap™ mass spectrometer, equipped with the new BioPharma option which includes a new High Mass Range (HMR) mode that allows for improved high mass transmission and mass detection up to 8000 m/z. Results: We have analyzed the profiles of three mAbs under reducing and native conditions by direct infusion with offline desalting and with on-line desalting via size exclusion and reversed phase type columns. The presence of high salt under denaturing conditions was found to influence the observed charge state envelope and impact mass accuracy after spectral deconvolution. The significantly lower charge states observed under native conditions improves the spatial resolution of protein signals and has significant benefits for the analysis of antibody mixtures, e.g. lysine variants, degradants or sequence variants. This type of analysis requires the detection of masses beyond the standard mass range ranging up to 6000 m/z requiring the extended capabilities available in the new HMR mode. We have compared each antibody sample that was analyzed individually with mixtures in various relative concentrations. For this type of analysis, we observed that apparent native structures persist and ESI is benefited by the addition of low amounts of acetonitrile and formic acid in combination with the ammonium acetate-buffered mobile phase. For analyses on the peptide level we analyzed reduced/alkylated, and non-reduced proteolytic digests of the individual antibodies separated via reversed phase chromatography aiming to retrieve as much information as possible regarding sequence coverage, disulfide bridges, post-translational modifications such as various glycans, sequence variants, and their relative quantification. All data acquired were submitted to a single software package for analysis aiming to obtain a complete picture of the molecules analyzed. Here we demonstrate the capabilities of the mass spectrometer to fully characterize homogeneous and heterogeneous therapeutic proteins on one single platform. Conclusion: Full characterization of heterogeneous intact protein mixtures by improved mass separation on a quadrupole-Orbitrap™ mass spectrometer with extended capabilities has been demonstrated.

Keywords: disulfide bond analysis, intact analysis, native analysis, mass spectrometry, monoclonal antibodies, peptide mapping, post-translational modifications, sequence variants, size exclusion chromatography, therapeutic protein analysis, UHPLC

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Authors: Lara Dutil-Fafard, Caroline Rhéaume, Patrick Archambault, Daniel Lafond, Neal W. Pollock

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Improving resources for medical autonomy of astronauts in prolonged space missions, such as a Mars mission, requires not only technology development, but also decision-making support systems. The Advanced Crew Medical System - Medical Condition Requirements study, funded by the Canadian Space Agency, aimed to create knowledge content and a scenario-based query capability to support medical autonomy of astronauts. The key objective of this study was to create a prototype tool for identifying medical infrastructure requirements in terms of medical knowledge, skills and materials. A multicriteria decision-making method was used to prioritize the highest risk medical events anticipated in a long-term space mission. Starting with those medical conditions, event sequence diagrams (ESDs) were created in the form of decision trees where the entry point is the diagnosis and the end points are the predicted outcomes (full recovery, partial recovery, or death/severe incapacitation). The ESD formalism was adapted to characterize and compare possible outcomes of medical conditions as a function of available medical knowledge, skills, and supplies in a given mission scenario. An extensive literature review was performed and summarized in a medical condition database. A PostgreSQL relational database was created to allow query-based evaluation of health outcome metrics with different medical infrastructure scenarios. Critical decision points, skill and medical supply requirements, and probable health outcomes were compared across chosen scenarios. The three medical conditions with the highest risk rank were acute coronary syndrome, sepsis, and stroke. Our efforts demonstrate the utility of this approach and provide insight into the effort required to develop appropriate content for the range of medical conditions that may arise.

Keywords: decision support system, event-sequence diagram, exploration mission, medical autonomy, scenario-based queries, space medicine

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Authors: Lien Gysens, Bert Vanmechelen, Maarten Haspeslagh, Piet Maes, Ann Martens

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Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in a clinical presentation on the one hand and the host-dependent clinical outcome of BPV-1 infection, on the other hand, indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of the cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. By generating a significant amount of complete-length BPV genomes, we succeeded in introducing next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

Keywords: Bovine papillomavirus, equine sarcoid, horse, nanopore sequencing, phylogenetic analysis

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Authors: Salina Yahya Saddick

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Ovarian cancer remains the most lethal gynecological malignancy due to the lack of highly sensitive and specific screening tools for detection of early-stage disease. The OSE provides the progenitor cells for 90% of human ovarian cancers. Recent morphologic, immunohistochemical and molecular genetic studies have led to the development of a new paradigm for the pathogenesis and origin of epithelial ovarian cancer (EOC) based on a ualistic model of carcinogenesis that divides EOC into two broad categories designated Types I and II which are characterized by specific mutations, including KRAS, BRAF, ERBB2, CTNNB1, PTEN PIK3CA, ARID1A, and PPPR1A, which target specific cell signaling pathways. Type 1 tumors rarely harbor TP53. type I tumors are relatively genetically stable and typically display a variety of somatic sequence mutations that include KRAS, BRAF, PTEN, PIK3CA CTNNB1 (the gene encoding beta catenin), ARID1A and PPP2R1A but very rarely TP53 . The cancer stem cell (CSC) hypothesis postulates that the tumorigenic potential of CSCs is confined to a very small subset of tumor cells and is defined by their ability to self-renew and differentiate leading to the formation of a tumor mass. Potential protein biomarker miRNA, are promising biomarkers as they are remarkably stable to allow isolation and analysis from tissues and from blood in which they can be found as free circulating nucleic acids and in mononuclear cells. Recently, genomic anaylsis have identified biomarkers and potential therapeutic targets for ovarian cancer namely, FGF18 which plays an active role in controlling migration, invasion, and tumorigenicity of ovarian cancer cells through NF-κB activation, which increased the production of oncogenic cytokines and chemokines. This review summarizes update information on epithelial ovarian cancers and point out to the most recent ongoing research.

Keywords: epithelial ovarian cancers, somatic sequence mutations, cancer stem cell (CSC), potential protein, biomarker, genomic analysis, FGF18 biomarker

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Authors: Don Anushka Sandaruwan Elvitigala, P. D. S. U. Wickramasinghe, Jehee Lee

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Cystatins are a large superfamily of proteins which act as reversible inhibitors of cysteine proteases. Papain proteases and cysteine cathepsins are predominant substrates of cystatins. Cystatin superfamily can be further clustered into three groups as Stefins, Cystatins, and Kininogens. Among them, stefines are also known as family 1 cystatins which harbors cystatin Bs and cystatin As. In this study, a homologue of family one cystatins more close to cystatin Bs was identified from Korean black rockfish (Sebastes schlegeli) using a prior constructed cDNA (complementary deoxyribonucleic acid) database and designated as RfCyt1. The full-length cDNA of RfCyt1 consisted of 573 bp, with a coding region of 294 bp. It comprised a 5´-untranslated region (UTR) of 55 bp, and 3´-UTR of 263 bp. The coding sequence encodes a polypeptide consisting of 97 amino acids with a predicted molecular weight of 11kDa and theoretical isoelectric point of 6.3. The RfCyt1 shared homology with other teleosts and vertebrate species and consisted conserved features of cystatin family signature including single cystatin-like domain, cysteine protease inhibitory signature of pentapeptide (QXVXG) consensus sequence and N-terminal two conserved neighboring glycine (⁸GG⁹) residues. As expected, phylogenetic reconstruction developed using the neighbor-joining method showed that RfCyt1 is clustered with the cystatin family 1 members, in which more closely with its teleostan orthologues. An SYBR Green qPCR (quantitative polymerase chain reaction) assay was performed to quantify the RfCytB transcripts in different tissues in healthy and immune stimulated fish. RfCyt1 was ubiquitously expressed in all tissue types of healthy animals with gill and spleen being the highest. Temporal expression of RfCyt1 displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCyt1 showed concentration-dependent papain inhibitory activity. Collectively these findings evidence for detectable protease inhibitory and immunity relevant roles of RfCyt1 in Sebastes schlegeli.

Keywords: Sebastes schlegeli, family 1 cystatin, immune stimulation, expressional modulation

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Authors: Norhidayu Muhamad Zain

Abstract:

Gelatin, a purified protein derived mostly from porcine and bovine sources, is used widely in food manufacturing, pharmaceutical, and cosmetic industries. However, the presence of any porcine-related products are strictly forbidden for Muslim and Jewish consumption. Therefore, analytical methods offering reliable results to differentiate the sources of gelatin are needed. The aim of this study was to differentiate the sources of gelatin (porcine and tilapia fish) using Fourier transform infrared- attenuated total reflection (FTIR-ATR) combined with two dimensional infrared (2DIR) correlation analysis. Porcine gelatin (PG) and tilapia fish gelatin (FG) samples were diluted in distilled water at concentrations ranged from 4-20% (w/v). The samples were then analysed using FTIR-ATR and 2DIR correlation software. The results showed a significant difference in the pattern map of synchronous spectra at the region of 1000 cm⁻¹ to 1100 cm⁻¹ between PG and FG samples. The auto peak at 1080 cm⁻¹ that attributed to C-O functional group was observed at high intensity in PG samples compared to FG samples. Meanwhile, two auto peaks (1080 cm⁻¹ and 1030 cm⁻¹) at lower intensity were identified in FG samples. In addition, using 2D correlation analysis, the original broad water OH bands in 1D IR spectra can be effectively differentiated into six auto peaks located at 3630, 3340, 3230, 3065, 2950 and 2885 cm⁻¹ for PG samples and five auto peaks at 3630, 3330, 3230, 3060 and 2940 cm⁻¹ for FG samples. Based on the rule proposed by Noda, the sequence of the spectral changes in PG samples is as following: NH₃⁺ amino acid > CH₂ and CH₃ aliphatic > OH stretch > carboxylic acid OH stretch > NH in secondary amide > NH in primary amide. In contrast, the sequence was totally in the opposite direction for FG samples and thus both samples provide different 2D correlation spectra ranged from 2800 cm-1 to 3700 cm⁻¹. This method may provide a rapid determination of gelatin source for application in food, pharmaceutical, and cosmetic products.

Keywords: 2 dimensional infrared (2DIR) correlation analysis, Fourier transform infrared- attenuated total reflection (FTIR-ATR), porcine gelatin, tilapia fish gelatin

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Authors: Abu S. Mustafa, Mohammad W. Khan, Faraz Shaheed Khan, Nazima Habibi

Abstract:

Brucellosis is a zoonotic disease of worldwide prevalence. There are at least four species and several strains of Brucella that cause human disease. Brucella genomes have very limited variation across strains, which hinder strain identification using classical molecular techniques, including PCR and 16 S rDNA sequencing. The aim of this study was to perform whole genome sequencing of clinical isolates of Brucella and perform bioinformatics and comparative genomics analyses to determine the existence of genetic differences across the isolates of a single Brucella species and strain. The draft sequence data were generated from 15 clinical isolates of Brucella melitensis (biovar 2 strain 63/9) using MiSeq next generation sequencing platform. The generated reads were used for further assembly and analysis. All the analysis was performed using Bioinformatics work station (8 core i7 processor, 8GB RAM with Bio-Linux operating system). FastQC was used to determine the quality of reads and low quality reads were trimmed or eliminated using Fastx_trimmer. Assembly was done by using Velvet and ABySS softwares. The ordering of assembled contigs was performed by Mauve. An online server RAST was employed to annotate the contigs assembly. Annotated genomes were compared using Mauve and ACT tools. The QC score for DNA sequence data, generated by MiSeq, was higher than 30 for 80% of reads with more than 100x coverage, which suggested that data could be utilized for further analysis. However when analyzed by FastQC, quality of four reads was not good enough for creating a complete genome draft so remaining 11 samples were used for further analysis. The comparative genome analyses showed that despite sharing same gene sets, single nucleotide polymorphisms and insertions/deletions existed across different genomes, which provided a variable extent of diversity to these bacteria. In conclusion, the next generation sequencing, bioinformatics, and comparative genome analysis can be utilized to find variations (point mutations, insertions and deletions) across different genomes of Brucella within a single strain. This information could be useful in surveillance and epidemiological studies supported by Kuwait University Research Sector grants MI04/15 and SRUL02/13.

Keywords: brucella, bioinformatics, comparative genomics, whole genome sequencing

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Authors: Nurulhikma Md Isa, Intan Elya Suka, Nur Farhana Roslan, Chew Bee Lynn

Abstract:

The evolutionarily conserved N-end rule pathway marks proteins for degradation by the Ubiquitin Proteosome System (UPS) based on the nature of their N-terminal residue. Proteins with a destabilizing N-terminal residue undergo a series of condition-dependent N-terminal modifications, resulting in their ubiquitination and degradation. Intensive research has been carried out in Arabidopsis previously. The group VII Ethylene Response Factor (ERFs) transcription factors are the first N-end rule pathway substrates found in Arabidopsis and their role in regulating oxygen sensing. ERFs also function as central hubs for the perception of gaseous signals in plants and control different plant developmental including germination, stomatal aperture, hypocotyl elongation and stress responses. However, nothing is known about the role of this pathway during fruit development and ripening aspect. The plant model system Arabidopsis cannot represent fleshy fruit model system therefore tomato is the best model plant to study. PROTEOLYSIS6 (PRT6) is an E3 ubiquitin ligase of the N-end rule pathway. Two homologs of PRT6 sequences have been identified in tomato genome database using the PRT6 protein sequence from model plant Arabidopsis thaliana. Homology search against Ensemble Plant database (tomato) showed Solyc09g010830.2 is the best hit with highest score of 1143, e-value of 0.0 and 61.3% identity compare to the second hit Solyc10g084760.1. Further homology search was done using NCBI Blast database to validate the data. The result showed best gene hit was XP_010325853.1 of uncharacterized protein LOC101255129 (Solanum lycopersicum) with highest score of 1601, e-value 0.0 and 48% identity. Both Solyc09g010830.2 and uncharacterized protein LOC101255129 were genes located at chromosome 9. Further validation was carried out using BLASTP program between these two sequences (Solyc09g010830.2 and uncharacterized protein LOC101255129) to investigate whether they were the same proteins represent PRT6 in tomato. Results showed that both proteins have 100 % identity, indicates that they were the same gene represents PRT6 in tomato. In addition, we used two different RNAi constructs that were driven under 35S and Polygalacturonase (PG) promoters to study the function of PRT6 during tomato developmental stages and ripening processes.

Keywords: ERFs, PRT6, tomato, ubiquitin

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Authors: Pranjali Sharma, Swati Neogi

Abstract:

Composite pressure vessels are low weight structures mainly used in a variety of applications such as automobiles, aeronautics and chemical engineering. Fiber reinforced polymer (FRP) composite materials offer the simplicity of design and use, high fuel storage capacity, rapid refueling capability, excellent shelf life, minimal infrastructure impact, high safety due to the inherent strength of the pressure vessel, and little to no development risk. Apart from these preliminary merits, the subsidized weight of composite vessels over metallic cylinders act as the biggest asset to the automotive industry, increasing the fuel efficiency. The result is a lightweight, flexible, non-explosive, and non-fragmenting pressure vessel that can be tailor-made to attune with specific applications. The winding pattern of the composite over-wrap is a primary focus while designing a pressure vessel. The critical stresses in the system depend on the thickness, angle and sequence of the composite layers. The composite over-wrap is wound over a plastic liner, whose geometry can be varied for the ease of winding. In the present study, we aim to optimize the FRP vessel geometry that provides an ease in winding and also aids in weight reduction for enhancing the vessel performance. Finite element analysis is used to study the effect of dome geometry, yielding a design with maximum value of burst pressure and least value of vessel weight. The stress and strain analysis of different dome ends along with the cylindrical portion is carried out in ANSYS 19.2. The failure is predicted using different failure theories like Tsai-Wu theory, Tsai-Hill theory and Maximum stress theory. Corresponding to a given winding sequence, the optimum dome geometry is determined for a fixed internal pressure to identify the theoretical value of burst pressure. Finally, this geometry is used to decrease the number of layers to reach the set value of safety in accordance with the available safety standards. This results in decrease in the weight of the composite over-wrap and manufacturing cost of the pressure vessel. An improvement in the overall weight performance of the pressure vessel gives higher fuel efficiency for its use in automobile applications.

Keywords: Compressed Gas Storage, Dome geometry, Theoretical Analysis, Type-4 Composite Pressure Vessel, Improvement in Vessel Weight Performance

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Authors: Milda Nainyte, Viktoras Masevicius

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Biological methylation is a methyl group transfer from S-adenosyl-L-methionine (AdoMet) onto N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules. The reaction is catalyzed by enzymes called AdoMet-dependent methyltransferases (MTases), which represent more than 3 % of the proteins in the cell. As a general mechanism, the methyl group from AdoMet replaces a hydrogen atom of nucleophilic center producing methylated DNA and S-adenosyl-L-homocysteine (AdoHcy). Recently, DNA methyltransferases have been used for the sequence-specific, covalent labeling of biopolymers. Two types of MTase catalyzed labeling of biopolymers are known, referred as two-step and one-step. During two-step labeling, an alkylating fragment is transferred onto DNA in a sequence-specific manner and then the reporter group, such as biotin, is attached for selective visualization using suitable chemistries of coupling. This approach of labeling is quite difficult and the chemical hitching does not always proceed at 100 %, but in the second step the variety of reporter groups can be selected and that gives the flexibility for this labeling method. In the one-step labeling, AdoMet analog is designed with the reporter group already attached to the functional group. Thus, the one-step labeling method would be more comfortable tool for labeling of biopolymers in order to prevent additional chemical reactions and selection of reaction conditions. Also, time costs would be reduced. However, effective AdoMet analog appropriate for one-step labeling of biopolymers and containing cleavable bond, required for reduction of PCR interferation, is still not known. To expand the practical utility of this important enzymatic reaction, cofactors with activated sulfonium-bound side-chains have been produced and can serve as surrogate cofactors for a variety of wild-type and mutant DNA and RNA MTases enabling covalent attachment of these chains to their target sites in DNA, RNA or proteins (the approach named methyltransferase-directed Transfer of Activated Groups, mTAG). Compounds containing hex-2-yn-1-yl moiety has proved to be efficient alkylating agents for labeling of DNA. Herein we describe synthetic procedures for the preparation of N-biotinoyl-N’-(pent-4-ynoyl)cystamine starting from the coupling of cystamine with pentynoic acid and finally attaching the biotin as a reporter group. The synthesis of the first AdoMet based cofactor containing a cleavable reporter group and appropriate for one-step labeling was developed.

Keywords: adoMet analogs, DNA alkylation, cofactor, methyltransferases

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