Search results for: genome sequencing and assembly

Authors: Zhaopeng Xuan, Yuan Bian, C. Cailleaux, Jing Qin, S. Traore

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Traditional software engineering allows engineers to propose to their clients multiple specialized software distributions assembled from a shared set of software assets. The management of these assets however requires a trade-off between client satisfaction and software engineering process. Clients have more and more difficult to find a distribution or components based on their needs from all of distributed repositories. This paper proposes a software engineering for a user-driven software product line in which engineers define a feature model but users drive the actual software distribution on demand. This approach makes the user become final actor as a release manager in software engineering process, increasing user product satisfaction and simplifying user operations to find required components. In addition, it provides a way for engineers to manage and assembly large software families. As a proof of concept, a user-driven software product line is implemented for eclipse, an integrated development environment. An eclipse feature model is defined, which is exposed to users on a cloud-based built platform from which clients can download individualized Eclipse distributions.

Keywords: software product line, model-driven development, reverse engineering and refactoring, agile method

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Authors: A. Alyami, S. Christmas, K. Neeltje, G. Pollakis, B. Paxton, Z. Al-Bayati

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The Human leukocyte antigen-G (HLA-G) molecule plays an important role in immunomodulation. To date, 16 untranslated regions (UTR) HLA-G haplotypes have been previously defined by sequenced SNPs in the coding region. From these, UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-6 and UTR-7 are the most frequent 3’UTR haplotypes at the global level. UTR-1 is associated with higher levels of soluble HLA-G and HLA-G expression, whereas UTR-5 and UTR-7 are linked with low levels of soluble HLA-G and HLA-G expression. Human immunodeficiency virus type 1 (HIV-1) infection results in the progressive loss of immune function in infected individuals. The virus escape mechanism typically includes T lymphocytes and NK cell recognition and lyses by classical HLA-A and B down-regulation, which has been associated with non-classical HLA-G molecule up-regulation, respectively. We evaluated the haplotypes of the HLA-G 3′ untranslated region frequencies observed in three HIV-1 groups from the Netherlands and their susceptibility to develop infection. The three groups are made up of mainly men who have sex with men (MSM), injection drug users (IDU) and a high-risk-seronegative (HRSN) group. DNA samples were amplified with published primers prior sequencing. According to our results, the low expresser frequencies show higher in HRSN compared to other groups. This is indicating that 3’UTR polymorphisms may be identified as potential prognostic biomarkers to determine susceptibility to HIV.

Keywords: Human leukocyte antigen-G (HLA-G) , men who have sex with men (MSM), injection drug users (IDU), high-risk-seronegative (HRSN) group, high-untranslated region (UTR)

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Authors: Pui Shan Wong, Kosuke Tashiro, Satoru Kuhara, Sachiyo Aburatani

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Functional genomics and gene regulation inference has readily expanded our knowledge and understanding of gene interactions with regards to expression regulation. With the advancement of transcriptome sequencing in time-series comes the ability to study the sequential changes of the transcriptome. This method presented here works to augment existing regulation networks accumulated in literature with transcriptome data gathered from time-series experiments to construct a sequential representation of transcription factor activity. This method is applied on a time-series RNA-Seq data set from Escherichia coli as it transitions from growth to stationary phase over five hours. Investigations are conducted on the various metabolic activities in gene regulation processes by taking advantage of the correlation between regulatory gene pairs to examine their activity on a dynamic network. Especially, the changes in metabolic activity during phase transition are analyzed with focus on the pagP gene as well as other associated transcription factors. The visualization of the sequential transcriptional activity is used to describe the change in metabolic pathway activity originating from the pagP transcription factor, phoP. The results show a shift from amino acid and nucleic acid metabolism, to energy metabolism during the transition to stationary phase in E. coli.

Keywords: Escherichia coli, gene regulation, network, time-series

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Authors: Gao Qin, Hu Liwei, Dong Xiangzhou, Zhu Qifa, Cheng Tingming, Zhao Limei, Yang Mengmeng, Zhai Zhen, Dai Huaxin, Liang Taibo, Zhang Shixiang, Xue Chaoqun

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The functional strain T976 for starch degradation was isolated from Nicotiana tabacum L. Yunyan 97 tobacco leaves, the ratio of starch hydrolysis transparent circle diameter to colony diameter of the strain was 4.14, 16S rDNA sequencing identified these strains as Enterobacter ludwigii. Then Enterobacter ludwigii T976 was fermented and spaying Yunyan 97 plant in vigorous growing stage. The results of once spraying fermentation broth of Enterobacter ludwigii T976 showed that starch content of upper leaves decreased slightly, from 3.77% to 3.1%, the reducing sugar content increased from 4.39% to 5.53%, and the total sugar content increased from 5.82% to 7.39%. The chemical content was also checked after three time spraying. The starch content of middle leaves decreased from 5.63% to 3.74%, while the content of total sugar and reducing sugar decreased slightly. And the starch content of upper leaves decreased from 7.62% to 4.78%, the total sugar and reducing sugar decreased slightly, and starch content of middle leaf decreased from 6.27% to 3.62%, the total sugar and reducing sugar did not change much, and other chemical components were in a suitable range.

Keywords: nicotiana tabacum, yunyan 97, leaf, starch, degradation, enterobacter ludwigii

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Authors: Abeer A. Q. Ahmed, Tracey McKay

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The increasing demand for renewable fuels and chemicals is pressuring manufacturing industry toward finding more sustainable cost-effective resources. Lignocellulose, such as agro-food wastes, is a suitable equivalent to petroleum for fine chemicals and fuels production. The complex structure of lignocellulose, however, requires a variety of enzymes in order to degrade its components into their respective building blocks that can be used further for the production of various value added products. This study aimed to isolate bacterial strain with the ability to produce a variety of lignocellulosic enzymes. One bacterial isolate was identified by 16S rRNA gene sequencing and phylogenetic analysis as Bacillus safensis LCX found to have CMCase, xylanase, manganese peroxidase, lignin peroxidase, and laccase activities. The enzymes production was induced by growing Bacillus safensis LCX in solid state fermentation using wheat straw, wheat bran, and corn stover. The activities of enzymes were determined by specific colorimetric assays. This study presents Bacillus safensis LCX as a promising source for lignocellulosic enzymes. These findings can extend the knowledge on agro-food wastes valorization strategies toward a sustainable production of fuels and chemicals.

Keywords: Bacillus safensis LCX, high valued chemicals, lignocellulosic enzymes, solid state fermentation

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Authors: Alieh Farshbaf, Tayyeb Bahrami

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Introduction: Cc-chemokine receptor-5 (CCR5) is known as a main co-receptor in human immunodeficiency virus type-1 (HIV-1) infection. Many studies showed 32bp deletion (Δ32) in CCR5 gene, provide natural resistance to HIV-1 infection in homozygous individuals. Inducing the resistance mechanism by CCR5 in HIV-1 infected patients eliminated many problems of highly-active-anti retroviral therapy (HAART) drugs like as low safety, side-effects and virus rebounding from latent reservoirs. New treatments solved some restrictions that are based on gene modification and cell therapy. Literature review: The stories of the “Berlin and Boston patients” showed autologous hematopoietic stem cells transplantation (HSCT) could provide effective cure of HIV-1 infected patients. Furthermore, gene modification by zinc finger nuclease (ZFN) demonstrated another successful result again. Despite the other studies for gene therapy by ∆32 genotype, there is another mutation -CCR5 ∆32/m303- that provides HIV-1 resistant. It is a heterozygote genotype for ∆32 and T→A point mutation at nucleotide 303. These results approved the key role of CCR5 gene. Conclusion: Recent studies showed immune gene therapy and cell therapy could provide effective cure for refractory disease like as HIV. Eradication of HIV-1 from immune system was not observed by HAART, because of reloading virus genome from latent reservoirs after stopping them. It is showed that CCR5 could induce natural resistant to HIV-1 infection by the new approaches based on stem cell transplantation and gene modifying.

Keywords: CCR5, HIV-1, stem cell, immune gene therapy, gene modification

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Authors: Robson C. Santos, Julio C. P. Ribeiro, Iorran M. de Castro, Luan C. F. Rodrigues, Sandro R. L. Silva, Diego M. Quesada

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The work aims to develop a robot in the form of autonomous vehicle to detect, inspection and mapping of underground pipelines through the ATmega328 Arduino platform. Hardware prototyping very similar to C / C ++ language that facilitates its use in robotics open source, resembles PLC used in large industrial processes. The robot will traverse the surface independently of direct human action, in order to automate the process of detecting buried pipes, guided by electromagnetic induction. The induction comes from coils that sends the signal to the Arduino microcontroller contained in that will make the difference in intensity and the treatment of the information, then this determines actions to electrical components such as relays and motors, allowing the prototype to move on the surface and getting the necessary information. The robot was developed by electrical and electronic assemblies that allowed test your application. The assembly is made up of metal detector coils, circuit boards and microprocessor, which interconnected circuits previously developed can determine, process control and mechanical actions for a robot (autonomous car) that will make the detection and mapping of buried pipelines plates.

Keywords: robotic, metal detector, embedded system, pipeline inspection

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Authors: H.-H. Doh, J.-M. Yu, Y.-J. Kwon, J.-H. Shin, H.-W. Kim, S.-H. Nam, D.-H. Lee

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This paper suggests a decision tree based approach for flexible job shop scheduling with multiple process plans, i. e. each job can be processed through alternative operations, each of which can be processed on alternative machines. The main decision variables are: (a) selecting operation/machine pair; and (b) sequencing the jobs assigned to each machine. As an extension of the priority scheduling approach that selects the best priority rule combination after many simulation runs, this study suggests a decision tree based approach in which a decision tree is used to select a priority rule combination adequate for a specific system state and hence the burdens required for developing simulation models and carrying out simulation runs can be eliminated. The decision tree based scheduling approach consists of construction and scheduling modules. In the construction module, a decision tree is constructed using a four-stage algorithm, and in the scheduling module, a priority rule combination is selected using the decision tree. To show the performance of the decision tree based approach suggested in this study, a case study was done on a flexible job shop with reconfigurable manufacturing cells and a conventional job shop, and the results are reported by comparing it with individual priority rule combinations for the objectives of minimizing total flow time and total tardiness.

Keywords: flexible job shop scheduling, decision tree, priority rules, case study

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Authors: Ling Ai, Ziyu Zhao, Zeyu Zhou, Xiaochen Yang, Heng Zhai, Stuart Holmes

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Optimizing the catalyst layer structure is crucial for enhancing the performance of proton exchange membrane fuel cells (PEMFCs) with low Platinum (Pt) loading. Current works focused on the utilization, durability, and site activity of Pt particles on support, and performance enhancement has been achieved by loading Pt onto porous support with different morphology, such as graphene, carbon fiber, and carbon black. Some schemes have also incorporated cost considerations to achieve lower Pt loading. However, the design of the catalyst layer (CL) structure in the membrane electrode assembly (MEA) must consider the interactions between the layers. Addressing the crucial aspects of water management, low contact resistance, and the establishment of effective three-phase boundary for MEA, multi-walled carbon nanotubes (MWCNTs) are promising CL support due to their intrinsically high hydrophobicity, high axial electrical conductivity, and potential for ordered alignment. However, the drawbacks of MWCNTs, such as strong agglomeration, wall surface chemical inertness, and unopened ends, are unfavorable for Pt nanoparticle loading, which is detrimental to MEA processing and leads to inhomogeneous CL surfaces. This further deteriorates the utilization of Pt and increases the contact resistance. Robust chemical oxidation or nitrogen doping can introduce polar functional groups onto the surface of MWCNTs, facilitating the creation of open tube ends and inducing defects in tube walls. This improves dispersibility and load capacity but reduces length and conductivity. Consequently, a trade-off exists between maintaining the intrinsic properties and the degree of functionalization of MWCNTs. In this work, MWCNTs were modified based on the operational requirements of the MEA from the viewpoint of interlayer interactions, including the search for the optimal degree of oxidation, N-doping, and micro-arrangement. MWCNT were functionalized by oxidizing, N-doping, as well as micro-alignment to achieve lower contact resistance between CL and proton exchange membrane (PEM), better hydrophobicity, and enhanced performance. Furthermore, this work expects to construct a more continuously distributed three-phase boundary by aligning MWCNT to form a locally ordered structure, which is essential for the efficient utilization of Pt active sites. Different from other chemical oxidation schemes that used HNO3:H2SO4 (1:3) mixed acid to strongly oxidize MWCNT, this scheme adopted pure HNO3 to partially oxidize MWCNT at a lower reflux temperature (80 ℃) and a shorter treatment time (0 to 10 h) to preserve the morphology and intrinsic conductivity of MWCNT. The maximum power density of 979.81 mw cm-2 was achieved by Pt loading on 6h MWCNT oxidation time (Pt-MWCNT6h). This represented a 59.53% improvement over the commercial Pt/C catalyst of 614.17 (mw cm-2). In addition, due to the stronger electrical conductivity, the charge transfer resistance of Pt-MWCNT6h in the electrochemical impedance spectroscopy (EIS) test was 0.09 Ohm cm-2, which was 48.86% lower than that of Pt/C. This study will discuss the developed catalysts and their efficacy in a working fuel cell system. This research will validate the impact of low-functionalization modification of MWCNTs on the performance of PEMFC, which simplifies the preparation challenges of CL and contributing for the widespread commercial application of PEMFCs on a larger scale.

Keywords: carbon nanotubes, electrocatalyst, membrane electrode assembly, proton exchange membrane fuel cell

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Authors: B. S. Yadav, A. Mani, S. Srivastava

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Chickpea (Cicer arietinum L.) is an annual, self-pollinating, diploid (2n = 2x = 16) pulse crop that ranks second in world legume production after common bean (Phaseolus vulgaris). ICC 4958 flowers approximately 39 days after sowing under peninsular Indian conditions and the crop matures in less than 90 days in rained environments. The estimated collective yield losses due to abiotic stresses (6.4 million t) have been significantly higher than for biotic stresses (4.8 million t). Most legumes are known to be salt sensitive, and therefore, it is becoming increasingly important to produce cultivars tolerant to high-salinity in addition to other abiotic and biotic stresses for sustainable chickpea production. Our aim was to identify the genes that are involved in the defence mechanism against heavy metal toxicity in chickpea and establish the biological network of heavy metal toxicity in chickpea. ICC4958 variety of chick pea was taken and grown in normal condition and 150µM concentration of different heavy metal salt like CdCl₂, K₂Cr2O₇, NaAsO₂. At 15th day leave samples were collected and stored in RNA Later solution microarray was performed for checking out differential gene expression pattern. Our studies revealed that 111 common genes that involved in defense mechanism were up regulated and 41 genes were commonly down regulated during treatment of 150µM concentration of CdCl₂, K₂Cr₂O₇, and NaAsO₂. Biological network study shows that the genes which are differentially expressed are highly connected and having high betweenness and centrality.

Keywords: abiotic stress, biological network, chickpea, microarray

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Authors: Xiaoping Su, Gabriel G. Malouf

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Introduction: Breast cancer is a heterogeneous disease that can be classified in 4 subgroups using transcriptional profiling. The role of lncRNA expression in human breast cancer biology, prognosis, and molecular classification remains unknown. Methods and results: Using an integrative comprehensive analysis of lncRNA, mRNA and DNA methylation in 900 breast cancer patients from The Cancer Genome Atlas (TCGA) project, we unraveled the molecular portraits of 1,700 expressed lncRNA. Some of those lncRNAs (i.e, HOTAIR) are previously reported and others are novel (i.e, HOTAIRM1, MAPT-AS1). The lncRNA classification correlated well with the PAM50 classification for basal-like, Her-2 enriched and luminal B subgroups, in contrast to the luminal A subgroup which behaved differently. Importantly, estrogen receptor (ESR1) expression was associated with distinct lncRNA networks in lncRNA clusters III and IV. Gene set enrichment analysis for cis- and trans-acting lncRNA showed enrichment for breast cancer signatures driven by breast cancer master regulators. Almost two third of those lncRNA were marked by enhancer chromatin modifications (i.e., H3K27ac), suggesting that lncRNA expression may result in increased activity of neighboring genes. Differential analysis of gene expression profiling data showed that lncRNA HOTAIRM1 was significantly down-regulated in basal-like subtype, and DNA methylation profiling data showed that lncRNA HOTAIRM1 was highly methylated in basal-like subtype. Thus, our integrative analysis of gene expression and DNA methylation strongly suggested that lncRNA HOTAIRM1 should be a tumor suppressor in basal-like subtype. Conclusion and significance: Our study depicts the first lncRNA molecular portrait of breast cancer and shows that lncRNA HOTAIRM1 might be a novel tumor suppressor.

Keywords: lncRNA profiling, breast cancer, HOTAIRM1, tumor suppressor

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Authors: Vivek Vaishnav, Shamim Akhtar Ansari

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Teak (Tectona grandis L.f.) belonging to plant family Lamiaceae and the most commercialized timber species is endemic to South-Asia. The adaptive fitness of the species metapopulation was evaluated through its genetic differentiation and assessing the influence of geo-climatic conditions. 290 genotypes were sampled from 29 locations of its natural distribution and the genetic data was incorporated with geo-climatic parameters. Through Bayesian approach based analysis of 43 highly polymorphic ISSR markers, six homogeneous clusters (0.8% genetic variability) were identified. The six clusters were found with the various regimes of the temperature range, i.e., I - 9.10±1.35⁰C, II -6.35±0.21⁰C, III -12.21±0.43⁰C, IV - 10.8±1.06⁰C, V - 11.67±3.04⁰C, and VI - 12.35±0.21⁰C. The population had a very high percentage of LD (21.48%) among the amplified loci possibly due to experiencing restricted gene flow as well as co-adaptation and association of distant/diverse loci/alleles as a result of the stabilized climatic conditions and countless cycles of historical recombination events on a large geological timescale. The same possibly accounts for the narrow distribution of teak as a climax species in the tropical deciduous forests of the country. The regions of strong LD in teak genome significantly associated with climatic parameters also reflect that the species is tolerant to the wide regimes of the temperature range and may possibly withstand global warming and climate change in the coming millennium.

Keywords: Bayesian analysis, inter simple sequence repeat, linkage disequilibrium, marker-geoclimatic association

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Authors: Saleh Alshehri

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Thermoelectric technology is currently being used in many industrial applications for cooling, heating and generating electricity. This research mainly focuses on using thermoelectric to cool down high-speed computer chips at different operating conditions. A previously developed and validated three-dimensional model for optimizing and assessing the performance of cascaded thermoelectric and non-cascaded thermoelectric is used in this study to investigate the possibility of decreasing the hotspot temperature of computer chip. Additionally, a test assembly is built and tested at steady-state and transient conditions. The obtained optimum thermoelectric current at steady-state condition is used to conduct a number of pulsed tests (i.e. transient tests) with different shapes to cool the computer chips hotspots. The results of the steady-state tests showed that at hotspot heat rate of 15.58 W (5.97 W/cm²), using thermoelectric current of 4.5 A has resulted in decreasing the hotspot temperature at open circuit condition (89.3 °C) by 50.1 °C. Maximum and minimum hotspot temperatures have been affected by ON and OFF duration of the electrical current pulse. Maximum hotspot temperature was resulted by longer OFF pulse period. In addition, longer ON pulse period has generated the minimum hotspot temperature.

Keywords: thermoelectric generator, TEG, thermoelectric cooler, TEC, chip hotspots, electronic cooling

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Authors: Abhishikta Ghosh Roy

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Breast cancer is the most common malignancy among women globally, including India. Human breast cancer results from the genetic and environmental interaction. The present study attempts to understand the molecular heterogeneity of BRCA1 and BRCA2 genes, as well as to understand the association of various lifestyle and reproductive variables for the Breast Cancer risk. The study was conducted amongst 110 patients and 128 controls with total DNA sequencing of flanking and coding regions of BRCA1 BRCA2 genes that revealed ten Single Nucleotide Polymorphisms (SNPs) (6 novels). The controls selected for the study were age, sex and ethnic group matched. After written and informed consent biological samples were collected from the subjects. After detailed molecular analysis, significant (p < 0.005) molecular heterogeneity is revealed in terms of SNPs in BRCA1 (4 Exonic & 1 Intronic) and BRCA2 (2exonic and 3 Intronic) genes. The augmentation study investigated significant (p < 0.05) association with positive family history, early age at menarche, irregular menstrual periods, menopause, prolong contraceptive use, nulliparity, history of abortions, consumption of alcohol and smoking for breast cancer risk. To the best of authors knowledge, this study is the first of its kind, envisaged that the identification of the SNPs and modification of the lifestyle factors might aid to minimize the risk among the Bengalee Hindu females.

Keywords: breast cancer, BRCA, lifestyle, India

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Authors: Simona Pileviciene

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On 24th January 2014, Lithuania notified two primary cases of African swine fever (ASF) in wild boars. The animals were tested positive for ASF virus (ASFV) genome by real-time PCR at the National Reference Laboratory for ASF in Lithuania (NRL), results were confirmed by the European Union Reference Laboratory for African swine fever (CISA-INIA). Intensive wild and domestic animal monitoring program was started. During the period of 2014-2017 ASF was confirmed in two large commercial pig holding with the highest biosecurity. Pigs were killed and destroyed. Since 2014 ASF outbreak territory from east and south has expanded to the middle of Lithuania. Diagnosis by PCR is one of the highly recommended diagnostic methods by World Organization for Animal Health (OIE) for diagnosis of ASF. The aim of the present study was to compare singleplex real-time PCR assays to a duplex assay allowing the identification of ASF and internal control in a single PCR tube and to compare primers, that target the p72 gene (ASF 250 bp and ASF 75 bp) effectivity. Multiplex real-time PCR assays prove to be less time consuming and cost-efficient and therefore have a high potential to be applied in the routine analysis. It is important to have effective and fast method that allows virus detection at the beginning of disease for wild boar population and in outbreaks for domestic pigs. For experiments, we used reference samples (INIA, Spain), and positive samples from infected animals in Lithuania. Results show 100% sensitivity and specificity.

Keywords: African swine fewer, real-time PCR, wild boar, domestic pig

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Authors: Syed M. Shahid, Rozeena Shaikh, Syeda N. Nawab, Abid Azhar

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Diabetes mellitus (DM) is a prevalent non-communicable disease worldwide. DM may lead to many vascular complications like hypertension, nephropathy, retinopathy, neuropathy and foot infections. Pathogenesis of diabetic nephropathy (DN) is implicated by the polymorphisms in genes encoding the specific components of renin angiotensin aldosterone system (RAAS) which include angiotensinogen (AGT), angiotensin-II receptor and angiotensin converting enzyme (ACE) genes. This study was designed to explore the possible association of AG (M268T) polymorphism in the patients of diabetes and nephropathy in Pakistan. Study subjects included 100 controls, 260 diabetic patients without renal insufficiency and 190 diabetic nephropathy patients with persistent albuminuria. Fasting blood samples were collected from all the subjects after getting institutional ethical approval and informed consent. The biochemical estimations, PCR amplification and direct sequencing for the specific region of AGT gene was carried out. A significantly high frequency of TT genotype and T allele of AGT (M268T) was observed in the patients of diabetes with nephropathy as compared to controls and diabetic patients without any known renal impairment. The TT genotype and T allele of AGT (M268T) polymorphism may be considered as a genetic risk factor for the development and progression of nephropathy in diabetes. Further cross sectional population studies would be of help to establish and confirm the observed possible association of AGT gene variations with development of nephropathy in diabetes.

Keywords: RAAS, AGT (M268T), diabetes, nephropathy

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Authors: Dalia. G. Aseel

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Begomoviruses are economically important plant viruses that infect dicotyledonous plants and exclusively transmitted by the whitefly Bemisia tabaci. Here, replicative form was isolated from Okra, Cotton, Tomato plants and whitefly infected with Begomoviruses. Using coat protein specific primers (AV1), the viral infection was verified with amplicon at 450 bp. The sequence of OLCuV-AV1 gene was recorded and received an accession number (FJ441605) from Genebank. The phylogenetic tree of OLCuV was closely related to Okra leaf curl virus previously isolated from Cameroon and USA with nucleotide sequence identity of 92%. The protein purification was carried out using His-Tag methodology by using Affinity Chromatography. The purified protein was separated on SDS-PAGE analysis and an enriched expected size of band at 30 kDa was observed. Furthermore, RAPD and SDS-PAGE were used to detect genetic variability between different hosts of okra leaf curl virus (OLCuV), cotton leaf curl virus (CLCuV), tomato yellow leaf curl virus (TYLCuV) and the whitefly vector. Finally, the present study would help to understand the relationship between the whitefly and different economical crops in Egypt.

Keywords: okra leaf curl virus, AV1 gene, sequencing, phylogenetic, cloning, purified protein, genetic diversity and viral proteins

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Authors: Shuofeng Yuan, Bojian Zheng

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Assembly of the heterotrimeric polymerase complex of influenza virus from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the N-terminal of PB1 (PB1N) and the C terminal of PA (PAC) may be a desired target for antiviral development. In this study, we first compared the feasibility of high throughput screening by enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization (FP) assay. Among the two, ELISA was demonstrated to own broader dynamic range so that it was used for screening inhibitors, which blocked PA and PB1 interaction. Several binding inhibitors of PAC-PB1N were identified and subsequently tested for the antiviral efficacy. Apparently, 3-(2-chlorophenyl)-6-ethyl-7-methyl[1,2,4]triazolo[4,3-a]pyrimidin-5-ol, designated ANA-1, was found to be a strong inhibitor of PAC-PB1N interaction and act as a potent antiviral agent against the infections of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 subtypes, in cell cultures. Intranasal administration of ANA-1 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted that ANA-1 bound to an allosteric site of PAC, which would cause conformational changes thereby disrupting the PAC-PB1N interaction. Overall, our study has identified a novel compound with potential to be developed as an anti-influenza drug.

Keywords: influenza, antiviral, viral polymerase, compounds

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Authors: Christian Röling, Elena Y. Poimanova, Vladimir V. Bruevich

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Here we demonstrate a non-destructive optical technique to localize and characterize single crystals of semiconductive organic materials – Spectroscopic Imaging Ellipsometry. With a combination of microscopy and ellipsometry, it is possible to characterize even micro-sized thin film crystals on plane surface regarding anisotropy, optical properties, crystalline domains and thickness. The semiconducting thiophene-phenylene co-oligomer 1,4-bis(5'-hexyl-[2,2'-bithiophen]-5-yl)benzene (dHex-TTPTT) crystals were grown by solvent based self-assembly technique on silicon substrate with 300 nm thermally silicon dioxide. The ellipsometric measurements were performed with an Ep4-SE (Accurion). In an ellipsometric high-contrast image of the complete sample, we have localized high-quality single crystals. After demonstrating the uniaxial anisotropy of the crystal by using Müller-Matrix imaging ellipsometry, we determined the optical axes by rotating the sample and performed spectroscopic measurements (λ = 400-700 nm) in 5 nm intervals. The optical properties were described by using a Lorentz term in the Ep4-Model. After determining the dispersion of the crystals, we converted a recorded Delta and Psi-map into a 2D thickness image. Based on a quantitative analysis of the resulting thickness map, we have calculated the height of a molecular layer (3.49 nm).

Keywords: anisotropy, ellipsometry, SCFET, thin film

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Authors: Chenyu Huang, Minrong Cui, Hua Fang, Luqing Zhang, Yunlong Yu

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The emergence and spread of antibiotic resistance genes (ARGs) have become a pressing issue in global agricultural production. However, understanding how these ARGs spread across different spatial scales, especially when exposed to both pesticides and antibiotics, has remained a challenge. Here, metagenomic assembly and binning methodologies were used to determine the mechanism of ARG propagation within soil-lettuce systems exposed to both fungicides and antibiotics. The results of our study showed that the presence of fungicide and antibiotic stresses had a significant impact on certain bacterial communities. Notably, we observed that ARGs were primarily transferred from the soil to the plant through plasmids. The selective pressure exerted by fungicides and antibiotics contributed to an increase in unique ARGs present on lettuce leaves. Moreover, ARGs located on chromosomes and plasmids followed different transmission patterns. The presence of diverse selective pressures, a result of compound treatments involving antibiotics and fungicides, amplifies this phenomenon. Consequently, there is a higher probability of bacteria developing multi-antibiotic resistance under the combined pressure of fungicides and antibiotics. In summary, our findings highlight that combined fungicide and antibiotic treatments are more likely to drive the acquisition of ARGs within the soil-plant system and may increase the risk of human ingestion.

Keywords: soil-lettuce system, fungicide, antibiotic, ARG, transmission

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Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

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PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provides better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

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Authors: Alaa Koaik, Bruno Jurkiewiez, Sylvain Bel

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Recently, the use of GFRP pultruded profiles increased in the domain of civil engineering especially in the construction of sandwiched slabs and footbridges. However, under heavy loads, the risk of using these profiles increases due to their high deformability and instability as a result of their weak stiffness and orthotropic nature. A practical solution proposes the assembly of these profiles with concrete slabs to create a stiffer hybrid element to support higher loads. The connection of these two elements is established either by traditional means of steel studs (bolting in our case) or bonding technique. These two techniques have their advantages and disadvantages regarding the mechanical behavior and in-situ implementation. This paper presents experimental results of interface characterization and bending behavior of two hybrid beams, PB7 and PB8, designed and constructed using both connection techniques. The results obtained are exploited to design and build a hybrid footbridge BPBP1 which is tested within service limits (elastic domain). Analytical methods are also developed to analyze the behavior of these structures in the elastic range and the ultimate phase. Comparisons show acceptable differences mainly due to the sensitivity of the GFRP moduli as well as the non-linearity of concrete elements.

Keywords: analytical model, concrete, flexural behavior, GFRP pultruded profile, hybrid structure, interconnection slip, push-out

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Authors: Hyo-Min Kim, Seokjin Ham, Mi-Joung Yoo, Minseon Kim, Tae-Young Roh

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The gastrointestinal (GI) tract of most animals, including murine, is highly compartmentalized epithelia which also provide distinct different functions of its own tissue. Nevertheless, these epithelia share certain characteristics that enhance immune responses to infections and maintain the barrier function of the intestine. GI tract epithelia also undergo regeneration not only in homeostatic conditions but also in a response to the damage. A full turnover of the murine gastrointestinal epithelium occurs every 4-5 day, a process that is regulated and maintained by a minor population of Lgr5+ adult stem cell that commonly conserved in the bottom of crypts through GI tract. Maintenance of the stem cell is somehow regulated by epigenetic factors according to recent studies. Chromatin vacancy, remodelers, histone variants and histone modifiers could affect adult stem cell fate. In this study, Lgr5-EGFP reporter mouse was used to take advantage of exploring the epigenetic dynamics among Lgr5 positive mutual stem cell in GI tract. Cells were isolated by fluorescence-activated cell sorting (FACS), gene expression levels, chromatin accessibility changes and histone modifications were analyzed. Some notable chromatin structural related epigenetic variants were detected. To identify the overall cell-cell interaction inside the stem cell niche, an extensive genome-wide analysis should be also followed. According to the results, nevertheless, we expected a broader understanding of cellular niche maintaining stem cells and epigenetic barriers through conserved stem cell in GI tract. We expect that our study could provide more evidence of adult stem cell plasticity and more chances to understand each stem cell that takes parts in certain organs.

Keywords: adult stem cell, epigenetics, LGR5 stem cell, gastrointestinal tract

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Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provide better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

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Authors: S. Hallal, Z. Chami, A. Hadji-Lehtihet, S. Sokhal-Boudella, A. Berhoune, L. Yargui

Abstract:

Gaucher disease is the most common lysosomal storage in our population, it is due to a deficiency of β –glucosidase acid. The enzyme deficiency causes a pathological accumulation of undegraded substrate in lysosomes. This metabolic overload is responsible for a multisystemic disease with hepatosplenomegaly, anemia, thrombocytopenia, and bone involvement. Neurological involvement is rare. The laboratory diagnosis of Gaucher disease consists of phenotypic diagnosis by determining the enzymatic activity of β - glucosidase by fluorimetric method, a study by genotypic diagnosis in the GBA gene, limiting the search recurrent mutations (N370S, L444P, 84 GG); PCR followed by an enzymatic digestion. Abnormal profiles were verified by sequencing. Monitoring of treated patients is provided by the determination of chitotriosidase. Our experience spaning a period of 6 years (2007-2014) has enabled us to diagnose 78 patients out of a total of 328 requests from the various departments of pediatrics, internal medicine, neurology. Genotypic diagnosis focused on the entire family of 9 children treated at pediatric CHU Mustapha, which help define the clinical form; or 5 of them had type III disease, carrying the L444P mutation in the homozygous state. Three others were composite (N370/L444P) (N370S/other unintended mutation in our study), and only in one family no recurrent mutation has been found. This molecular study permits screening of heterozygous essential for genetic counseling.

Keywords: Gaucher disease, mutations, N370S, L444P

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Authors: O. H. Gebril, N. A. Meguid

Abstract:

Background: Iron had been a focus of interest recently as a main exaggerating factor for oxidative stresses in the central nervous system and a link to various neurological disorders is suspected. Many studies with various techniques showed evidence of disturbed iron-related proteins in the cell in human and animal models of neurodegenerative disorders. Also, linkage to significant pathological changes had been evidenced e.g. apoptosis and cell signaling. On the other hand, the role of iron in neurodevelopmental disorders is still unclear. With increasing prevalence of autism worldwide, some changes in iron parameters and its stores were documented in many studies. This study includes Haemochromatosis HFE gene polymorphisms (p.H63D and p.C282Y) and ferroportin gene (SLC40A1) Q248H polymorphism in autism and control children. Materials and Methods: Whole genome DNA was extracted; p.H63D and p.C282Y genotyping was studied using specific sequence amplification followed by restriction enzyme digestion on a sample of autism patients (25 cases) and twenty controls. Results: The p.H63D is seen more than the C282Y among both autism and control samples, with no significant association of p.H63D or p.C282Y polymorphism and autism was revealed. Also, no association with Q248H polymorphism was evidenced. Conclusion: The study results do not prove the role of cellular iron genes polymorphisms as risk factors for neurodevelopmental disorders, and in turn highlights the specificity of cellular iron related pathways in neurodegeneration. These results demand further gene expression studies to elucidate the main pathophysiological pathways that are disturbed in autism and other neurodevelopmental disorders.

Keywords: iron, neurodevelopmental, oxidative stress, haemohromatosis, ferroportin, genes

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Authors: Isaac Quiros-Fernandez, Angel Cid-Arregui

Abstract:

Tumor-reactive T cell receptors (TCRs) are being subject of intense investigation since they offer great potential in adoptive cell therapies against cancer. However, the identification of tumor-specific TCRs has proven challenging, for instance, due to the limited expansion capacity of tumor-infiltrating T cells (TILs) and the extremely low frequencies of tumor-reactive T cells in the repertoire of patients and healthy donors. We have developed an approach for rapid identification and characterization of neoepitope-reactive TCRs from the T cell repertoire of healthy donors. CD8 T cells isolated from multiple donors are subjected to a first sorting step after staining with HLA multimers carrying the peptide of interest. The isolated cells are expanded for two weeks, after which a second sorting is performed using the same peptide-HLA multimers. The cells isolated in this way are then processed for single-cell sequencing of their TCR alpha and beta chains. Newly identified TCRs are cloned in appropriate expression vectors for functional analysis on Jurkat, NK92, and primary CD8 T cells and tumor cells expressing the appropriate antigen. We have identified TCRs specifically binding HLA-A2 presenting epitopes of tumor antigens, which are capable of inducing TCR-mediated cell activation and cytotoxicity in target cancer cell lines. This method allows the identification of tumor-reactive TCRs in about two to three weeks, starting from peripheral blood samples of readily available healthy donors.

Keywords: cancer, TCR, tumor antigens, immunotherapy

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Authors: Muna A. Eissawi, Safaa Abed Elfataah, Haytham Hago, Fatima E Abukunna, Ibtisam Amin Goreish, Nahid Gornas

Abstract:

The study examined and analyzed the genetic diversity of DRB1locus of exon 2 of major histocompatibility complex of Sudanese desert sheep using PCR-RFLP and DNA sequencing. Five hundred samples belonging to five ecotypes of Desert Sudanese sheep (Abrag (Ab), Ashgar (Ash), Hamari (H), Kabashi (K) and Watish (W) were included. Amplification of exon 2 of the DRB1 gene yielded (300bp) amplified product in different ecotypes. Nine different digestion patterns corresponding to Five distinct alleles were observed with Rsa1 digestion. Genotype (ag) was the most common among all ecotypes, with a percentage comprised (40.4 %). The Hardy-Weinberg equilibrium (HWE) test showed that the studied ecotypes have significantly deviated from the theoretical proportions of Rsa1 patterns; probability values of the Chi-square test for HWE for MHC-DRB1 gene in SDS were 0.00 in all ecotypes. The constructed phylogenetic tree revealed the relation of 22 Sudanese isolates with each other and showed the shared sequences with 47 published foreign sequences randomly selected from different geographic regions. The results of this study highlight the effect of heterozygosity of MHC genes of the Desert sheep of Sudan which may clarify some of genetic back ground of their disease resistance and adaptation to environment.

Keywords: desert sheep, MHC, Ovar-DRB1, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)

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Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh

Abstract:

Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

Procedia PDF Downloads 241

Authors: M. Błoch, P. Karpiński, P. Gasperowicz, R. Płoski, A. Lebioda, P. Skiba, A. Rozensztrauch, D. Patkowski, R. Śmigiel

Abstract:

Introduction: The cause of the isolated form of esophageal atresia has been yet unknown. Objectives: The primary objective of this study was to indicate epigenetic factors which may play an important role in the etiopathogenesis of esophageal atresia. Methods: We recruited a group of 6 pairs of twins, among whom one of the twins developed EA. The selection of such a group for testing allows for excluding external factors (e.g., infections, drugs, toxins) as the cause of the birth defect. The analyzes were performed with the use of genetic material isolated from the whole blood and esophagus tissue of a patient with EA. The reduced representation bisulphite sequencing (RRBS) technique was used to study the change in the genomic imprinting -a change in the expression of genes, which may be the epigenetic cause of EA. Results: In the course of the analyzes, significant hypomethylation and hypermethylation regions were identified. 65 genes with probably increased expression and 65 with decreased expression were selected. These genes have not been marked in literature as possibly pathogenic in esophageal atresia. However, their participation in the pathogenesis of esophageal atresia cannot be clearly excluded. Conclusion: We suggest a role of hypomethylation or hypermethylation of selected genes as one of the possible epigenetic factors in EA pathogenesis. The use of the RRBS technique in the search for the cause of EA is pioneer research; therefore, it seems necessary to extend the research group to new patients with EA. Acknowledgment: The work was supported by the National Science Centre, Poland, under research project 2016/21/N/NZ5/01927.

Keywords: esophageal atresia, epigenetics, embryonic development, surgery, genes expression, twins

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