Multiple Sequence Alignment Analysis Research Articles

H1N1 influenza virus epitopes classified by monoclonal antibodies.

Epitopes serve an important role in influenza infection. It may be useful to screen universal influenza virus vaccines, analyzing the epitopes of multiple subtypes of the hemagglutinin (HA) protein. A total of 40 monoclonal antibodies (mAbs) previously obtained from flu virus HA antigens (development and characterization of 40 mAbs generated using H1N1 influenza virus split vaccines were previously published) were used to detect and classify mAbs into distinct flu virus sub-categories using the ELISA method. Following this, the common continuous amino acid sequences were identified by multiple sequence alignment analysis with the GenBank database and DNAMAN software, for use in predicting the epitopes of the HA protein. Synthesized peptides of these common sequences were prepared, and used to verify and determine the predicted linear epitopes through localization and distribution analyses. With these methods, nine HA linear epitopes distributed among different strains of influenza virus were identified, which included three from influenza A, four from 2009 H1N1 and seasonal influenza, and two from H1. The present study showed that considering a combination of the antigen-antibody reaction specificity, variation in the influenza virus HA protein and linear epitopes may present a useful approach for designing effective multi-epitope vaccines. Furthermore, the study aimed to clarify the cause and pathogenic mechanism of influenza virus HA-induced flu, and presents a novel idea for identifying the epitopes of other pathogenic microorganisms.

The expression of natriuretic peptide receptors in developing zebrafish embryos

There are three isoforms of natriuretic peptide (NP) specific cell surface receptor: NP receptor-A (NPRA), receptor-B (NPRB), and receptor-C (NPRC). They are also known as NPR1, NPR2 and NPR3, respectively. NPs and their receptors were revealed to involve in diverse cellular and physiological processes including renal, cardiovascular, neuronal, and immunological aspects. However, the systematic analysis of the expression of these receptors in non-mammalian vertebrates is thus far lacking. In this study, two versions of the npr1 gene (npr1a and npr1b) in zebrafish was identified. Multiple sequences alignment analysis showed that zebrafish NPRs shared high homologies with NPRs of other species and possessed a typical signature domain of NPRs. The results of whole mount in situ hybridization and reverse transcription polymerase chain reaction analysis revealed that at embryonic stages, npr1a was mainly expressed in tectal ventricle, brian, heart and retina, whereas npr1b was broadly present in anterior pronephric duct. Unlike npr1, npr2 mainly expressed in branchial arches and neural tube during embryonic development. However, npr3 was expressed in pronephric ducts and corpuscle of stannius in zebrafish embryos at 72 hpf. In adults, we demonstrated that all the three NP receptors were highly existed in brain and kidney. Overall, these findings will provide an important basis for the functional analysis of NPs and its receptor during embryonic development.

Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated Mr of 44.8 kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4 h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT50) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p < 0.01). A 3.5-fold increase in E. coli colony forming units occurred in larval hemolymph of the OfPAP knockdown larvae, as compared with that of the control larvae not injected with dsRNA. There were notable decreases in PO and IEARase activities in hemolymph of the OfPAP knockdown larvae. In summary, we have demonstrated that OfPAP is a component of the PPO activation system, likely by functioning as a PPO activating protease in O. furnacalis larvae.

Genome-Wide Identification and Expression Analysis of Heat Shock Transcription Factor Family in Chenopodium quinoa Willd

Heat shock transcription factor (Hsf) is one of the conserved gene families in plants, playing a crucial role in growth and development, as well as in response to diverse stresses. Although it has been systematically studied in many species, little is known about the Hsf gene family in Chenopodium quinoa, especially those involved in the regulatory network of stress processes. In this study, we identified 23 Hsf genes in quinoa (CqHsfs) through a genome-wide search method based on the latest available genome information. Phylogenetic analysis classified them into three groups, and group A was further divided into nine subgroups, which was supported by conserved domain organizations. Gene structure and multiple sequence alignment analysis revealed that all of the CqHsfs possessed a similar structure organization and were highly conserved in BDB domain. Interaction network analysis identified 13 CqHsfs involved in the network pathway to regulate diverse biological processes. Expression profiles of these CqHsfs were further investigated using the RNA-seq data, and tissue-specific and stress-responsive candidates were identified. Finally, four heat-responsive CqHsfs were selected to validate their expression level through semi-quantitative RT-PCR analysis. This study reported the organization, structure, and expression profiles of the Hsf gene family in quinoa, which will contributes to further functional analysis, and helps to better understand the roles and regulatory mechanism of heat shock factors playing in quinoa and beyond.

Purification, developmental expression, and in silico characterization of α-amylase inhibitor from Echinochloa frumentacea.

Barnyard (Echinochloa frumentacea) and finger (Eleusine coracana) millet growing at northwestern Himalaya were explored for the α-amylase inhibitor (α-AI). The mature seeds of barnyard millet variety PRJ1 had maximum α-AI activity which increases in different developmental stage. α-AI was purified up to 22.25-fold from barnyard millet variety PRJ1. Semi-quantitative PCR of different developmental stages of barnyard millet seeds showed increased levels of the transcript from 7 to 28days. Sequence analysis revealed that it contained 315bp nucleotide which encodes 104 amino acid sequence with molecular weight 10.72kDa. The predicted 3D structure of α-AI was 86.73% similar to a bifunctional inhibitor of ragi. In silico analysis of 71 α-AI protein sequences were carried out for biochemical features, homology search, multiple sequence alignment, phylogenetic tree construction, motif, and superfamily distribution of protein sequences. Analysis of multiple sequence alignment revealed the existence of conserved regions NPLP[S/G]CRWYVV[S/Q][Q/R]TCG[V/I] throughout sequences. Superfam analysis revealed that α-AI protein sequences were distributed among seven different superfamilies.

Transcription Factor SmWRKY1 Positively Promotes the Biosynthesis of Tanshinones in Salvia miltiorrhiza.

Tanshinones, one group of bioactive diterpenes, were widely used in the treatment of cardiovascular diseases. WRKYs play important roles in plant metabolism, but their regulation mechanism in Salvia miltiorrhiza remains elusive. In this study, one WRKY transcription factor SmWRKY1 was isolated and functionally characterized from S. miltiorrhiza. Multiple sequence alignment and phylogenetic tree analysis showed SmWRKY1 shared high homology with other plant WRKYs such as CrWRKY1. SmWRKY1 was found predominantly expressed in leaves and stems, and was responsive to salicylic acid (SA), methyl jasmonate (MeJA), and nitric oxide (NO) treatment. Subcellular localization analysis found that SmWRKY1 was localized in the nucleus. Over-expression of SmWRKY1 significantly elevated the transcripts of genes coding for enzymes in the MEP pathway especially 1-deoxy-D-xylulose-5-phosphate synthase (SmDXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (SmDXR), resulted in over fivefold increase in tanshinones production in transgenic lines (up to 13.7 mg/g DW) compared with the control lines. A dual-luciferase (Dual-LUC) assay showed that SmWRKY1 can positively regulate SmDXR expression by binding to its promoter. Our work revealed that SmWRKY1 participated in the regulation of tanshinones biosynthesis and acted as a positive regulator through activating SmDXR in the MEP pathway, thus provided a new insight to further explore the regulation mechanism of tanshinones biosynthesis.

Design of Novel Lectins by Computer‐Guided Directed Evolution

Changes in the level and type of glycosylation are implicated in mediating countless physiological and disease processes, yet the glycome is poorly defined. To overcome this barrier, we integrate state of the art computational methods with guided directed evolution for the design of selective glycan binding reagents based on a natural lectin, cyanovirin.Cyanovirin‐N (CV‐N) is a small (11 kDa) lectin that displays potent antiviral activity by binding to the oligomannosides of gp120. The solution structure shows two quasi‐symmetric domains, defined as A (residues 1‐38/90‐101) and B (residues 39–89), which are connected on each side by a short helical linker. Each domain contains a carbohydrate binding pocket, which binds selectively Mana(1®2)Mana termini in branched high‐mannose oligosaccharides. We assessed the binding affinity of WT CV‐N through ITC titrations with dimannose revealing a high‐affinity binding site (Kd=15.3 mM) and a low‐affinity site (Kd=0.400 mM). To increase the binding affinity for glycosylated gp120, we exploited avidity effects by designing a dimeric CV‐N in which a CV‐N sequence is spliced into another. The construct contains four glycan binding domains in two sets (A, A′, B, B′), and binds dimannose with Kd=24.5 mM and 0.95 mM respectively. Four indistinguishable binding events, with Kd=1.1 mM, are observed with Man9.We recently developed computational tools to predicted protein‐glycan interactions accurately by using multiple sequence alignment (MSA) and flexibility analysis to identify co‐evolved positions, and coupling this analysis to our flexible docking tool (BP‐Dock). These tools were validated by assessing the energetic contribution of polar residues within the binding pocket, docking dimannose to single‐point CVN mutant models. We found that the E41A/G and T57A mutations led to a significant decrease in binding energy scores due to rearrangements of the hydrogen‐bond network that reverberated throughout the binding cavity. N42A decreased binding by affecting the integrity of the local protein structure. In contrast, N53S resulted in a high binding energy score. Experimental characterization of the five mutants by NMR and ITC confirmed the binding affinity pattern predicted computationally.Computational analysis also yielded a set of mutations at several positions beyond the immediate binding pocket that impact glycan recognition. These predictions were validated by directed evolution in yeast display; screening resulted in mutants with increased affinity and selectivity for dimannose. We are currently using the same library to identify mutants with high affinity and selectivity towards glycan biomarkers.Support or Funding InformationThis work is supported by NIH‐IR21AI129895 grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gills specific type 2 crustin isoforms: Its molecular cloning and characterization from kuruma shrimp Marsupenaeus japonicus

Crustins are diverse group of antimicrobial peptides (AMPs) that have numerous isoforms mainly identified from hemocytes in decapods crustacean. However, little is known about its presence solely in gills tissue. In this study, we found two new crustin isoforms MjCRS8 and MjCRS9 by using transcriptome analysis from gills. Open reading frame of MjCRS8 and MjCRS9 were 593 bp and 459 bp encoding 197aa and 152aa, respectively. Tissue distribution analysis indicated that both MjCRS8 and MjCRS9 are expressed only in gills tissue. Multiple sequence alignment and phylogenetic analysis with previously reported crustin suggested that both MjCRS8 and MjCRS9 belong to type 2 crustin family. Experimental infection was conducted against Vibrio parahaemolyticus and white spot syndrome virus (WSSV) by immersion test. However, no significant upregulation was observed.

Molecular characterisation of plant parasitic nematode Longidorus poessneckensis Altherr, 1974 (Nematoda: Longidoridae)

The plant parasitic nematode Longidorus poessneckensis found in Austria, the Czech Republic, Germany, Poland and the Slovak Republic was molecularly characterized. Mitochondrial genes encoding cytochrome c oxidase subunit I (COI) and nicotinamide dehydrogenase subunit 4 (nad4), the D2 and D3 expansion segments of 28S rRNA and Internal transcribed spacer 1 (ITS1) rRNA were sequenced for 16 L. poessneckensis populations. Six haplotypes of COI and five haplotypes of nad4 were determined. Nucleotide intraspecific variation was up to 17.1% for the partial sequenced COI gene and up to 17.7% for the partial sequenced nad4 gene, the latter being the highest up to date known intraspecific variation in this genus. The analyses of multiple amino acid sequence alignments of mitochondrial genes revealed low variability (0–2.4%) for COI gene and high divergence (0–7.6%) for nad4 gene. Intraspecific sequence diversity for the D2-D3 of 28S rRNA gene was up to 1.2% and for ITS1 rRNA gene was up to 1.6%. It has been hypothesized, that during the Last Glacial Maximum, L. poessneckensis populations probably persisted in refuge areas in the Carpathian Mountains and subsequently expanding from these areas and colonizing other European regions.

Cloning and prokaryotic expression of Cryptocaryon irritans α-tubulin gene and its mRNA expression levels in different life history stages

PDF HTML阅读 XML下载 导出引用 引用提醒 刺激隐核虫α-微管蛋白基因的克隆、原核表达及在生活史不同阶段的mRNA表达 DOI: 作者: 作者单位: 1. 集美大学水产学院, 福建 厦门 361021;2. 福建农林大学动物科学学院, 福建 福州 350002;3. 莆田市水产科学研究所, 福建 莆田 351100;4. 福建省水产研究所, 福建 厦门 361013 作者简介: 孙嘉阳(1992-),女,硕士研究生,从事分子生物学研究.E-mail:344156662@qq.com 通讯作者: 中图分类号: S917 基金项目: 福建省海洋与渔业厅五新项目（201209200023）；福建省科技厅高校产学合作项目（2017N5002）. Cloning and prokaryotic expression of Cryptocaryon irritans α-tubulin gene and its mRNA expression levels in different life history stages Author: Affiliation: 1. Fisheries College, Jimei University, Xiamen 361021, China;2. Animal Science College, Fujian University of Agriculture, Fuzhou 350002, China;3. Putian Institute of Fishery Sciences, Putian, 351100, China;4. Fujian Fisheries Research Institute, Xiamen, 361013, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:从本实验室已有刺激隐核虫（）转录组数据中筛选获得α-微管蛋白基因片段，利用5'RACE和3'RACE技术首次克隆获得其cDNA，全长为1602 bp，包含1356 bp的开放阅读框，编码451个氨基酸，预测蛋白质分子量为49.78 kD。生物信息学分析表明α-微管蛋白为亲水性非跨膜蛋白，氨基酸序列的第142~148位有特异且保守的GTP核苷酸结合位点（GGGTGSG）。对刺激隐核虫α-微管蛋白氨基酸序列进行同源性比对及进化树分析，发现其与间日疟原虫（Euplotesoct ocarinatus）、尾刺耐格里原虫（）等的序列一致性高达94%~95%，且在系统进化树上聚为一支。采用实时荧光定量PCR技术对α-微管蛋白基因在刺激隐核虫3个生活史阶段的表达进行检测，结果显示α-微管蛋白基因的表达量在纤毛虫时期显著高于包囊和滋养体时期（<0.05）。我们进一步构建了α-微管蛋白重组表达载体，并转化至大肠杆菌表达菌株BL21（DE3）和Rosetta （DE3）中进行原核表达，SDS-PAGE分析表明，诱导表达的重组蛋白分子量约为50 kD，与预测的结果一致，即成功诱导表达α-微管蛋白。本实验结果为后续制备α-微管蛋白有效亚单位疫苗防治刺激隐核虫病奠定了基础。 Abstract:is an extremely destructive ciliate parasite of marine fishes, which results in severe economic costs in mariculture. Its life history is divided into three stages:trophont, tomont, and theront. Tubulin is a major component of the cytoskeleton, cilia, and flagella, which plays an important role in maintaining cell morphology, intracellular trafficking, cell division, ciliary, and flagella motility. At the same time, many studies showed that tubulin has immunogenicity and can be used as a target antigen vaccine. In this study, the α-tubulin gene fragment was obtained from the transcriptome data in our laboratory. Full-length cDNA of α-tubulin was cloned for the first time by 5'RACE and 3'RACE. The results showed that full-length cDNA of α-tubulin is 1602 bp containing a 1356 bp open reading frame, which encodes proteins with 451 amino acids. The predicted molecular mass of α-tubulin is 49.78 kD. Bioinformatics analysis showed that it is a hydrophilic non-transmembrane protein. The amino acid sequence at positions 142-148 has a unique conserved GTP nucleotide binding site (GGGTGSG). Multiple sequence alignment analysis and phylogenetic analysis revealed that the α-tubulin in was integrated with other protozoa in the phylogenetic tree and had 94%-95% sequence identity with Naegleria gruberi, and . The real-time quantitative PCR technique was used to detect the expression of α-tubulin gene in the life history of . The results showed that α-tubulin gene expression was significantly higher in the theront stage than in the tomont and trophont stages ( < 0.05). Furthermore, the α-tubulin recombinant expression vector was constructed and transformed into the BL21 (DE3) and Rosetta (DE3) of the expression strain for prokaryotic expression. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was approximately 50 kD, which was consistent with the predicted result, indicating that α-tubulin protein induced expression successfully. The results of this study laid the foundation for the subsequent preparation of an effective subunit vaccine. 参考文献 相似文献 引证文献

Cloning and characterization of a gene encoding lysophosphatidylethanolamine acyltransferase (LPEAT) in the green microalga Myrmecia incisa Reisigl

PDF HTML阅读 XML下载 导出引用 引用提醒 缺刻缘绿藻溶血磷脂酰乙醇胺酰基转移酶(LPEAT)的基因克隆与特征分析 DOI: 作者: 作者单位: 1. 水产种质资源发掘与利用教育部重点实验室, 上海海洋大学, 上海 201306;2. 海洋生物科学国际联合研究中心, 上海海洋大学, 上海 201306;3. 水产科学国家级实验教学示范中心, 上海海洋大学, 上海 201306 作者简介: 周志刚(1964-),博士,教授,主要从事藻类学及藻类生物技术的研究.E-mail:zgzhou@shou.edu.cn 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31772821，31402274）；国家海洋局可再生能源专项基金项目（SHME2011SW02）；上海高校水产科学高峰学科建设项目. Cloning and characterization of a gene encoding lysophosphatidylethanolamine acyltransferase (LPEAT) in the green microalga Myrmecia incisa Reisigl Author: Affiliation: 1. The Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Conferred by Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;2. International Research Center for Marine Biosciences Conferred by Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;3. National Demonstration Center for the Experimental Teaching of Fisheries Science, Shanghai Ocean University, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:缺刻缘绿藻三酰甘油（TAG）中花生四烯酸（ArA）占其总脂肪酸含量的68.0%。为了弄清ArA是如何被优先地用于合成TAG，鉴于Lands循环是通过改变膜脂的脂肪酸组成进而影响TAG的脂肪酸组成，本研究选择在Lands循环中起关键作用的溶血磷脂酰乙醇胺酰基转移酶（LPEAT）作为突破口。运用反转录PCR与3'-及5'-cDNA末端快速扩增技术，自缺刻缘绿藻（；它的cDNA序列全长1303 bp，其中，5'-非翻译区（UTR）长129 bp，3'-UTR长193 bp，开放阅读框长981 bp，编码326个氨基酸残基；以缺刻缘绿藻基因组DNA为模板扩增得到该基因长为1871 bp的DNA序列；这两序列的比对结果显示，含有6个内含子，将其开放阅读框（ORF）分割成7个外显子。通过多序列比对及生物信息学分析，发现MiLPEAT存在一个磷酸酰基转移酶结构域PlsC，并含有LPEAT家族所具有的NH（x）4D、FPEGT等4个特征性模体；Wolfpsort等在线预测结果以及MiLPEAT羧基端存在的双赖氨酸模体"KKxx"，暗示它可能位于内质网并参与分泌途径。基于不同植物LPEAT的氨基酸序列所构建的邻接聚类图表明，MiLPEAT因与2型LPEAT有不同的序列特征而形成不同的分支，但却与1型LPEAT聚类在一起，推测它们的功能可能更近似。通过荧光定量PCR检测，发现MiLPEAT的基因转录量在氮饥饿8 h时显著（<0.01）减少49%，但磷脂酰乙醇胺（PE）相对丰度的增加却不显著；推测在氮饥饿过程中增加的磷脂酰乙醇胺（PE）可能被磷脂：二酰甘油酰基转移酶作用以合成为TAG，致使缺刻缘绿藻TAG含量的增加。 Abstract:Reisigl, a coccoid green microalga, is an oleaginous alga that can accumulate an unprecedentedly high level of arachidonic acid (ArA, 20:4w6)-rich triacylglycerols (TAG) under the growth stress of nitrogen starvation. ArA accounts for 68.0% of total fatty acids in TAG. How is ArA preferentially utilized for the biosynthesis of TAG? The Lands' cycle plays an important role in the composition change of fatty acids of phospholipids, thus altering the composition of fatty acids in TAG. Lysophosphatidylethanolamine acyltransferase (LPEAT), a key enzyme in the Lands' cycle, was the focus of the present study. was cloned using reverse transcription-PCR and 3°-and 5'-cDNA rapid amplification of cDNA ends technique. The full-length cDNA was 1303 bp long, and contained a 129-bp 5'-untranslated region (UTR) and 193-bp 3'-UTR. The length of the open read frame was 981 bp that encoded a 326-amino acid protein. The DNA sequence of with the isolated algal genomic DNA as a template, and it was 1871 bp long. Comparison of the cDNA and DNA sequences showed that possessed 6 introns that separated the coding sequence into 7 exons. Multiple sequence alignment and bioinformatics analysis of LPEATs from different species demonstrated that MiLPEAT possessed a phosphate acyltransferase domain, PlsC, thus suggesting that it was one member of the lysophospholipid acyltransferase (LPLAT) superfamily. MiLPEAT also had the 4 typical motifs, NH(x)4D, GCxYVxR, FPEGT, and PVxPVx, which are characterized in the LPLAT superfamily. Both the prediction, as analyzed online by Wolfsport and Protein Prowler, and the presence of a dilysine motif at the carboxyl terminus of MiLPEAT, implied that MiLPEAT might reside at the algal endoplasmic reticulum and possibly participate in the secretion pathway. A neighbor-joining phylogeny was constructed on the basis of deduced amino acids of LPEATs from different species of plants. It illustrated that MiLPEAT was so different from LPEAT2 that they located at different clades due to their various characteristics of sequences. MiLPEAT was clustered phylogenetically with LPEAT1, suggesting their similar functions in the acylation of phospholipids. Quantitative real-time PCR detection pointed out that <0.05) at 8 h after treatment with nitrogen starvation in . By coincidence, the relative abundance of lysophosphatidylethanolamine (LPE) in the microalgal cells reduced by approximately 49% at an extreme significance level (<0.01) under the nitrogen starvation stress. The corresponding phosphatidylethanolamine (PE) generated from LPE as catalyzed by MiLPEAT, however, did not increase significantly. It is assumed that the net increase of PE under nitrogen starvation in was possibly utilized for TAG biosynthesis by the phospholipid DAG acyltransferase (PDAT), so that the content of TAG has been reported to increase. This research lays a foundation for us to understand the TAG and phospholipid biosynthetic pathway and how to regulate TAG synthesis in . 参考文献 相似文献 引证文献

LeptoDB: an integrated database of genomics and proteomics resource of Leptospira.

Leptospirosis is a potentially fatal zoo-anthroponosis caused by pathogenic species of Leptospira belonging to the family of Leptospiraceae, with a worldwide distribution and effect, in terms of its burden and risk to human health. The ‘LeptoDB’ is a single window dedicated architecture (5 948 311 entries), modeled using heterogeneous data as a core resource for global Leptospira species. LeptoDB facilitates well-structured knowledge of genomics, proteomics and therapeutic aspects with more than 500 assemblies including 17 complete and 496 draft genomes encoding 1.7 million proteins for 23 Leptospira species with more than 250 serovars comprising pathogenic, intermediate and saprophytic strains. Also, it seeks to be a dynamic compendium for therapeutically essential components such as epitope, primers, CRISPR/Cas9 and putative drug targets. Integration of JBrowse provides elaborated locus centric description of sequence or contig. Jmol for structural visualization of protein structures, MUSCLE for interactive multiple sequence alignment annotation and analysis. The data on genomic islands will definitely provide an understanding of virulence and pathogenicity. Phylogenetics analysis integrated suggests the evolutionary division of strains. Easily accessible on a public web server, we anticipate wide use of this metadata on Leptospira for the development of potential therapeutics.Database URL: http://leptonet.org.in

Molecular Characterization of Bovine Papillomaviruses Obtained From Cattle Cutaneous Warts in Nigeria

Bovine papillomatosis is a benign, self-limiting tumor in cattle that is caused by a wide diverse of bovine papillomaviruses (BPVs 1-23). The disease is of high economic importance to the beef and hide industries in Nigeria. To date, there is no information on the molecular characterization of BPVs in Nigeria. Consequently, we collected cutaneous wart lesions from 31 cows in Ibadan, Nigeria and screened them for BPV using a degenerate primer that amplify partial L1 gene sequence of the virus in PCR. Six samples (19.4%) were positive for bovine papillomavirus. Thereafter, amplicons were purified and sequenced. BLAST search, pairwise sequence alignments, pairwise genetic distance calculations, multiple sequence alignment and phylogenetic analysis revealed the viruses to be bovine papillomavirus-1 belonging to the genus Deltapapillomavirus.

Molecular cloning and characterization of (β -1, 3-Dextran gene (Glu) in endangered tropic medicinal plant Dendrobium officinale

Objective: Dendrobium officinale (Orchidaceae) (D. officinale) is one of the world’s most endangered plants with great medicinal value. It is mainly distributed in south China, and is often used as auxiliary treatment for a variety of tropical diseases. The strictly demanding for growing environment and climate conditions making its wild resources endangered. In nature, the relationship between seeds of D. officinale and fungi must be established by symbiotic system. Our purpose is to analyze the molecular events involved in the interaction between fungus and plant during this process, and provide scientific basis for the protection of its germplasm resources. Methods: Beta-1,3-glucan (Glu) is a key enzyme in the biosynthesis of polyamines, which is essential to the basic physiological and biochemical processes of plants. In this study, the full-length cDNA of Glu gene was obtained from symbiotic germinating seeds of medicinal plants by rapid amplified cDNA terminal (RACE) - PCR, and its expression characteristics were analyzed for the first time. Results: Glu has a full length cDNA of 1 317 bp, with one open reading frame (ORF). The deduced protein was 356 amino acids (aa) with molecular weight of 37.24 KDa and a theoretical isoelectric point of 5.17. The deduced β-1,3-Dextran protein, without signal peptide, had three transmembrane domain each contained 23,17 and 20 aa. Multiple sequence alignment and phylogenetic analysis showed that the sequence similarity between beta-1,3-glucan and rice beta-1,3-glucan was higher than that of dicotyledons. The expression pattern analyzed by qPCR showed that Glu transcripts were expressed in four tissues (non-infected fungi) without significant change. In symbiotic germinating seeds, the expression level is 23.67 times higher than that of ungerminated seeds. Conclusion: These results indicated that β-1,3-Dextran was possible involved in the symbiotic seed germination of D. officinale, and played an important role in the symbiosis system of mycorrhizal mutual.

Comparative analysis of phosphoethanolamine transferases involved in polymyxin resistance across 10 clinically relevant Gram-negative bacteria

The rapid emergence of Gram-negative ‘superbugs’ has become a significant threat to human health globally, and polymyxins have become a last-line therapy for these very problematic pathogens. Polymyxins exhibit their antibacterial killing by initial interaction with lipid A in Gram-negative bacteria. Polymyxin resistance can be mediated by phosphoethanolamine (PEA) modification of lipid A, which abolishes the initial electrostatic interaction with polymyxins. Both chromosome-encoded (e.g. EptA, EptB and EptC) and plasmid-encoded (e.g. MCR-1 and MCR-2) PEA transferases have been reported in Gram-negative bacteria; however, their sequence and functional heterogeneity remain unclear. This article reports a comparative analysis of PEA transferases across 10 clinically relevant Gram-negative bacterial species using multiple sequence alignment and phylogenetic analysis. The results show that the pairwise identities among chromosome-mediated EptA, EptB and EptC from Escherichia coli are low, and EptA shows the greatest similarity with MCR-1 and MCR-2. Among PEA transferases from representative strains of 10 clinically relevant species, the catalytic domain is more conserved compared with the transmembrane domain. In particular, PEA acceptor sites and zinc-binding pockets show high conservation between different species, indicating their potential importance for the function of PEA transferases. The evolutionary relationship of MCR-1, MCR-2 and EptA from the 10 selected bacterial species was evaluated by phylogenetic analysis. Cluster analysis illustrates that 325 EptA from 275 strains of 10 species within each individual species are highly conserved, whereas interspecies conservation is low. This comparative analysis provides key bioinformatic information to better understand the mechanism of polymyxin resistance via PEA modification of lipid A.

Identification and Characterisation of Putative Glutathione S-Transferase Genes from Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae).

Glutathione S-transferases (GSTs) in insects are widely known for their role in the detoxification of both endogenous and xenobiotic compounds. Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera: Phylloxeridae) is a serious grape pest, which causes great economic damage in vineyards, and has currently spread throughout the world. In this study, eight putative GST genes were identified by analyzing the transcriptomes of grape phylloxera. Phylogenetic analyses showed that there are seven cytosolic DviGSTs and one microsomal DviGST. These cytosolic DviGSTs are clustered into four different classes including two delta genes, one omega gene, one theta gene, and three sigma genes. Among candidate cytosolic DviGSTs, a conserved N-terminal domain and a less conserved C-terminal domain were identified. For the candidate microsomal DviGST, three transmembrane regions were predicted. Multiple sequence alignment analysis of the candidate microsomal DviGST was conducted with other insect microsomal GSTs and the result showed that there is a conserved sequence pattern. Semiquantitative polymerase chain reaction was used to examine the tissue expression of these transcripts, and the results revealed that DviGSTs were ubiquitously expressed in the head and the body, but DviGSTd1, DviGSTd2, DviGSTs2, and DviGSTs3 were abundantly expressed in the head and body. This is the first study of the molecular characteristics of GST genes in grape phylloxera. Our results will provide a molecular basis for future studies of the detoxification mechanisms in grape phylloxera.

Characterization of 2-Cys peroxiredoxin 3 and 4 in common carp and the immune response against bacterial infection.

Accumulating evidence suggests that peroxiredoxins (Prxs) eliminate excessive cellular H2O2 and are important factors in redox signaling pathways. In this study, we cloned the full-length cDNAs and genomic sequences of Prx3 and Prx4 from common carp. The common carp Prx3 and Prx4 open reading frames were 753 base pairs (bp) and 783bp in length, respectively, and contained seven exons and six introns. Multiple sequence alignment and phylogenetic analyses revealed that the common carp Prx1-4 proteins share high identities and similar characteristics with other known animal Prxs. Prx3 and Prx4 mRNA were constitutively expressed in all tissues, and the highest Prx3 and Prx4 transcript abundances occurred in head kidney. Although the highest Prx4 protein and mRNA expression were also observed in head kidney, many differences were detected between Prx4 mRNA and protein expression levels in other tissues. Prx3 expression increased significantly in the head kidney 12h after an Aeromonas hydrophila challenge. The A. hydrophila challenge upregulated Prx3 mRNA expression in liver and spleen, increased Prx4 mRNA expression levels in liver and spleen excluding at 36h in spleen, but decreased Prx4 mRNA expression level in the head kidney. The mature Prx4 peptide was recombinantly expressed and purified using Dextrin Beads 6FF and it exhibited thioredoxin (Trx)-dependent peroxidase activity. These data suggest that Prx3 and Prx4 are constitutive and inducible proteins that might play important roles in innate immune function. The Trx-dependent peroxidase activity analysis of recombinant Prx4 further verified the important role of Prxs in the redox system of fish.

Polymeric immunoglobulin receptor in dojo loach (Misgurnus anguillicaudatus): Molecular characterization and expression analysis in response to bacterial and parasitic challenge

The polymeric immunoglobulin receptor (pIgR) is an essential component of the mucosal immune system in jawed vertebrates including teleost fish, which mediate transepithelial transport of secretory immunoglobulins (sIgs) to protect organisms against environmental pathogens. In this study, we firstly cloned and identified the pIgR from dojo loach (Misgurnus anguillicaudatus). The full-length cDNA of Ma-pIgR was of 1145 bp, containing an open reading frame (ORF) of 1101 bp encoded a predicted protein of 336 amino acids. The structure of Ma-pIgR is comprised of a signal peptide, a transmembrane region, an intracellular region and an extracellular region with two Ig-like domains (ILDs), which are similar to their counterparts described in other teleosts. Multiple sequence alignment and phylogenetic analysis showed the dojo loach is closely related to the fish family Cyprinidae. The transcriptional level of Ma-pIgR was detected by quantitative real-time PCR (qRT-PCR) in different tissues and high expression was found in liver, skin, kidney, eye, fin and gills. Two infection models of the loach with bacteria (Aeromonas hydrophila) and parasite (Ichthyophthirius multifiliis) were constructed for the first time. Histological studies showed the goblet cells in skin significantly increased and the ratio of gill length to width also significantly changed after challenged with A.hydrophila. Both challenge experiments resulted in the significant up-regulated expression of Ma-pIgR not only in kidney and spleen, but also in skin and gills. Our results suggest that pIgR may play an important role in skin and gill mucosal immunity to protect the loach against bacteria and parasite.

Genome-Wide Comprehensive Analysis the Molecular Phylogenetic Evaluation and Tissue-Specific Expression of SABATH Gene Family in Salvia miltiorrhiza.

The plant SABATH gene family is a group of O-methyltransferases (O-MTs), which belongs to the S-adenosyl-l-methionine-dependent methyltransferases (SAM-MTs). The resulting reaction products of SABATH genes play an important role in various processes of plant development. In this study, a total of 30 SABATH genes were detected in Salvia miltiorrhiza, which is an important medicinal plant, widely used to treat cardiovascular disease. Multiple sequence alignment and phylogenetic analyses showed that SmSABATH genes could be classified into three groups. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates of 11 pairs paralogous of SmSABATH genes revealed that the SmSABATH genes had gone through purifying selection. Positive selection analyses using site models and branch-site models indicated that SmSABATH genes had undergone selective pressure for adaptive evolution. Functional divergence analyses suggested that the SmSABATH subgroup genes were divergent in terms of functions and positive selection sites that contributed to a functional divergence among the subgroups that were detected. Tissue-specific expression showed that the SABATH gene family in S. miltiorrhiza was primarily expressed in stems and leaves.

Molecular cloning, characterization, tissue expression and nutritional regulation of O-GlcNAc transferase gene in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

O-GlcNAc transferase gene (OGT) was considered as the sole rate-limiting enzyme in the O-GlcNAc modification. In the present study, the OGT gene of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned and characterized, and its expression in response to dietary carbohydrate level and acute glucose treatment was investigated. The full-length of OGT (GenBank accession no. KY656469) was 4,063 bp, including a 302 bp 5′untranslated terminal region (UTR), a 3,165 bp coding region that encoded 1,054 amino acids residues and a 596 bp 3′ UTR. The highly conservation of OGT gene between fish and mammals was also observed through multiple sequences alignment and phylogenetic analysis. O-GlcNAc transferase gene was ubiquitously expressed in all detected tissues with highest expressions in brain and liver, to a lesser degree, in eye, heart, kidney and intestine. The increasing dietary carbohydrate from 8.02% to 16.08% had no significant effect on the mRNA expression of OGT. However, the expression of OGT was slightly elevated at 6 hr post-glucose injection, and the elevation became significant at 24 hr time-point. These data may enhance our understanding on the nutritional regulation of OGT and O-GlcNAc modification in fish species.