lncRNA-miRNA-mRNA network in kidney transcriptome of Labeo rohita under hypersaline environment.

The present study describes the kidney transcriptome of Labeo rohita, a freshwater fish, exposed to gradually increased salinity concentrations (2, 4, 6 and 8ppt). A total of 10.25 Gbps data was generated, and a suite of bioinformatics tools, including FEELnc, CPC2 and BLASTn were employed for identification of long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). Our analysis revealed a total of 170, 118, 99, and 269 differentially expressed lncRNA and 120, 118, 99, and 124 differentially expressed miRNAs in 2, 4, 6 and 8 ppt treatment groups respectively. Two competing endogenous RNA (ceRNA) networks were constructed i.e. A* ceRNA network with up-regulated lncRNAs and mRNAs, down-regulated miRNAs; and B* ceRNA network vice versa. 2ppt group had 131 and 83 lncRNA-miRNA-mRNA pairs in A* and B* networks, respectively. 4ppt group featured 163 pairs in A* network and 191 in B* network, while the 6ppt had 103 and 105 pairs. 8ppt group included 192 and 174 pairs. These networks illuminate the intricate RNA interactions in freshwater fish to varying salinity conditions.

Untargeted metabolomics of buffalo urine reveals hydracyrlic acid, 3-bromo-1-propanol and benzyl serine as potential estrus biomarkers.

Buffalo is a silent heat animal and doesn't show prominent signs of estrous like cattle so it becomes difficult for farmers to determine the receptivity of the animal based purely on the animal behaviour. India, having a huge population size, needs to produce more milk for the population. Successful artificial insemination greatly depends on the receptivity of the animal. Hence the present study aimed to identify the changes in the metabolome of the buffalo. GC-MS based mass spectrometric analysis was deployed for the determination of estrous by differential expression of metabolites. It was found that hydracrylic acid, 3-bromo-1-propanol and benzyl serine were significantly upregulated in the estrous phase of buffalo (p.value ≤0.05, FC ≥ 2). The pathway enrichment analysis also supported the same as pathways related to amino acid metabolism and fatty acid metabolism were up regulated along with the Warburg effect which is linked to the rapid cell proliferation which might help prepare animals to meet the energy requirement during the estrous. Further analysis of the metabolic biomarkers using ROC analysis also supported these three metabolites as probable biomarkers as they were identified with AUC values of 0.7 or greater. SIGNIFICANCE: The present study focuses on the untargeted metabolomics studies of buffalo urine with special reference to the estrous phase of reproductive cycle. The estrous signals are more prominent in cattle, where animals show clear estrous signals such as mounting and discharge along with vocal signals. Buffalo is a silent heat animal and it becomes difficult for farmers to detect the estrous based on the physical and behavioral signals. Hence the present study focuses on GC-MS based untargeted metabolomics to identify differentially expressed urine metabolites. In this study, hydracrylic acid, 3-bromo-1-propanol and benzyl serine were found to be significantly upregulated in the estrous phase of buffalo (p-value ≤0.05, FC ≥ 2). Further confirmation of the metabolic biomarkers was done using Receiver operating characteristics (ROC) analysis which also supported these three metabolites as probable biomarkers as they had AUC values of 0.7 or greater. Hence, this study will be of prime importance for the people working in the area of animal metabolomics.

Cloning and characterization of FMN-dependent azoreductases from textile industry effluent identified through metagenomic sequencing.

Azo dyes, when released untreated in the environment, cause detrimental effects on flora and fauna. Azoreductases are enzymes capable of cleaving commercially used azo dyes, sometimes in less toxic by-products which can be further degraded via synergistic microbial cometabolism. In this study, azoreductases encoded by FMN1 and FMN2 genes were screened from metagenome shotgun sequences generated from the samples of textile dye industries' effluents, cloned, expressed, and evaluated for their azo dye decolorization efficacy. At pH 7 and 45°C temperature, both recombinant enzymes FMN1 and FMN2 were able to decolorize methyl red at 20 and 100 ppm concentrations, respectively. FMN2 was found to be more efficient in decolorization/degradation of methyl red than FMN1. This study offers valuable insights into the possible application of azoreductases to reduce the environmental damage caused by azo dyes, with the hope of contributing to sustainable and eco-friendly practices for the environment management. This enzymatic approach offers a promising solution for the bioremediation of textile industrial effluents. However, the study acknowledges the need for further process optimization to enhance the efficacy of these enzymes in large-scale applications.Implications: The study underscores the environmental hazards associated with untreated release of azo dyes into the environment and emphasizes the potential of azoreductases, specifically those encoded by FMN1 and FMN2 genes, to mitigate the detrimental effects. The study emphasizes the ongoing commitment to refining and advancing the enzymatic approach for the bioremediation of azo dye-containing effluents, marking a positive stride toward more sustainable industrial practices.

Oncolytic Activity of Canine Distemper Virus in Human Ductal Breast Carcinoma Cells.

INTRODUCTION: Oncolytic virotherapy is a novel strategy for cancer treatment in humans and companion animals. Canine distemper virus (CDV) is known to induce apoptosis in tumor cells, thus serving as a potential candidate for oncolytic therapy. However, the mechanism of viral oncolytic activity is less studied and varies depending on the type of cancer and cell lines. METHODS: In the present study, the susceptibility of the MCF-7 cell line to CDV infection was assessed using the CDV strain, which was confirmed previously through sequence analysis in the Vero cell line. The impact of CDV infection on cell proliferation and apoptosis was studied by evaluating the expression of four target genes including the myeloid cell leukemia 1 (MCL-1), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), transcription factor (SP1), and DNA (cytosine-5)-methyltransferase 3A (DNMT3A). RESULTS: CDV replication in the cells induced cytopathic effect and decreased in the cell proliferation rates compared to the uninfected control. MCL-1, SP1, and PIK3R1 gene expression was down-regulated, while the expression of DNMT3A was up-regulated 3 days post-infection. The expression levels of the target genes suggest that CDV may be inducing the intrinsic apoptotic pathway in the cancer cell line. CONCLUSION: Overall, the results strongly propose CDV strain as a potential candidate for cancer therapy after detailed studies.

Resistant cumin cultivar, GC-4 counters Fusarium oxysporum f. sp. cumini infection through up-regulation of steroid biosynthesis, limonene and pinene degradation and butanoate metabolism pathways.

Cumin (Cuminum cyminum L.), an important spice crop belonging to the Apiaceae family is infected by Fusarium oxysporum f. sp. cumini (Foc) to cause wilt disease, one of the most devastating diseases of cumin adversely affects its production. As immune responses of cumin plants against the infection of Foc are not well studied, this research aimed to identify the genes and pathways involved in responses of cumin (cv. GC-2, GC-3, GC-4, and GC-5) to the wilt pathogen. Differential gene expression analysis revealed a total of 2048, 1576, 1987, and 1174 differentially expressed genes (DEGs) in GC-2, GC-3, GC-4, and GC-5, respectively. In the resistant cultivar GC-4 (resistant against Foc), several important transcripts were identified. These included receptors, transcription factors, reactive oxygen species (ROS) generating and scavenging enzymes, non-enzymatic compounds, calcium ion (Ca2+) transporters and receptors, R-proteins, and PR-proteins. The expression of these genes is believed to play crucial roles in conferring resistance against Foc. Gene ontology (GO) analysis of the up-regulated DEGs showed significant enrichment of 19, 91, 227, and 55 biological processes in GC-2, GC-3, GC-4, and GC-5, respectively. Notably, the resistant cultivar GC-4 exhibited enrichment in key GO terms such as 'secondary metabolic process', 'response to reactive oxygen species', 'phenylpropanoid metabolic process', and 'hormone-mediated signaling pathway'. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the enrichment of 28, 57, 65, and 30 pathways in GC-2, GC-3, GC-4, and GC-5, respectively, focusing on the up-regulated DEGs. The cultivar GC-4 showed enrichment in pathways related to steroid biosynthesis, starch and sucrose metabolism, fatty acid biosynthesis, butanoate metabolism, limonene and pinene degradation, and carotenoid biosynthesis. The activation or up-regulation of various genes and pathways associated with stress resistance demonstrated that the resistant cultivar GC-4 displayed enhanced defense mechanisms against Foc. These findings provide valuable insights into the defense responses of cumin that could contribute to the development of cumin cultivars with improved resistance against Foc.

Integrative miRNA-mRNA network analysis to identify crucial pathways of salinity adaptation in brain transcriptome of Labeo rohita.

Introduction: Brain being the master regulator of the physiology of animal, the current study focuses on the gene expression pattern of the brain tissue with special emphasis on regulation of growth, developmental process of an organism and cellular adaptation of Labeo rohita against unfavourable environmental conditions. Methods: RNA-seq study was performed on collected brain samples at 8ppt salt concentration and analyzed for differential gene expression, functional annotation and miRNA-mRNA regulatory network. Results: We found that 2450 genes were having significant differential up and down regulation. The study identified 20 hub genes based on maximal clique centrality algorithm. These hub genes were mainly involved in various signaling pathways, energy metabolism and ion transportation. Further, 326 up and 1214 down regulated genes were found to be targeted by 7 differentially expressed miRNAs i.e., oni-miR-10712, oni-miR-10736, ssa-miR-221-3p, ssa-miR-130d-1-5p, ssa-miR-144-5p and oni-miR-10628. Gene ontology analysis of these differentially expressed genes led to the finding that these genes were involved in signal transduction i.e., calcium, FOXO, PI3K-AKT, TGF-ß, Wnt and p53 signalling pathways. Differentially expressed genes were also involved in regulation of immune response, environmental adaptation i.e., neuroactive ligand-receptor interaction, ECM-receptor interaction, cell adhesion molecules and circadian entrainment, osmoregulation and energy metabolism, which are critical for salinity adaptation. Discussion: The findings of whole transcriptomic study on brain deciphered the miRNA-mRNA interaction patterns and pathways associated with salinity adaptation of L. rohita.

Association of NOTCH4 and ACHE gene polymorphism in Alzheimer's disease of Gujarat cohort.

BACKGROUND: Alzheimer's Disease (AD) is the most common form of dementia, affecting cognitive and behavioral functions. AD is a complex disease resulting from the modest effect of gene interaction and environmental factors, as a result of which the exact pathogenesis is still unknown. AIM: The aim of the present study was to investigate the association between variants of 98 targeted genes with Alzheimer's disease phenotype. METHOD: A total of 98 genes from 32 AD cases and 11 controls were genotyped using the Haloplex target enrichment method and the PCR-RFLP approach.Association analysis was performed using the PLINK tool to identify the variant significantly associated with AD. Functional enrichment analysis and network analysis was performed using ClueGo and String database respectively. The Expression Quantitative Trait Loci (eQTL) analysis using the Genotype Tissue Expression (GTEx) dataset to explore the possible implication of the variant on the expression of one or more genes in different brain regions and whole blood. RESULT: Association analysis showed significant association of 19 variant assigned to 16 genes with Alzheimer's with p-value < 0.05 with rs367398/NOTCH4 only variant that passed multiple test corrections. Functional enrichment analysis showed association of these genes with AD. ClueGo and network analysis utilizing the String database suggested that genes are directly and indirectly linked to the AD pathogenesis. eQTL analysis revealed that the rs367398/NOTCH4 and rs1799806/ACHE variant showed significant eQTL for the neighbouring genes. CONCLUSION: The present study showed the possible role of 16 genes in AD pathogenesis, especially highlighting the role of rs367398/NOTCH4 and rs1799806/ACHE. However further investigation with large cohort is required to study and validate the implication of these variants in the AD pathogenesis.

Interplay of gene expression and regulators under salinity stress in gill of Labeo rohita.

BACKGROUND: Labeo rohita is the most preferred freshwater carp species in India. The concern of increasing salinity concentration in freshwater bodies due to climate change may greatly impact the aquatic environment. Gills are one of the important osmoregulatory organs and have direct contact with external environment. Hence, the current study is conducted to understand the gill transcriptomic response of L. rohita under hypersalinity environment. RESULTS: Comprehensive analysis of differentially expressed long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs was performed in gills of L. rohita treated with 2, 4, 6 and 8ppt salinity concentrations. Networks of lncRNA-miRNA-mRNA revealed involvement of 20, 33, 52 and 61 differentially expressed lncRNAs, 11, 13, 26 and 21 differentially expressed miRNAs in 2, 4, 6 and 8ppt groups between control and treatment respectively. These lncRNA-miRNA pairs were regulating 87, 214, 499 and 435 differentially expressed mRNAs (DE mRNAs) in 2, 4, 6 and 8ppt treatments respectively. Functional analysis of these genes showed enrichment in pathways related to ion transportation and osmolyte production to cope with induced osmotic pressure due to high salt concentration. Pathways related to signal transduction (MAPK, FOXO and phosphatidylinositol signaling), and environmental information processing were also upregulated under hypersalinity. Energy metabolism and innate immune response pathways also appear to be regulated. Protein turnover was high at 8ppt as evidenced by enrichment of the proteasome and aminoacyl tRNA synthesis pathways, along with other enriched KEGG terms such as apoptosis, cellular senescence and cell cycle. CONCLUSION: Altogether, the RNA-seq analysis provided valuable insights into competitive endogenous (lncRNA-miRNA-mRNA) regulatory network of L. rohita under salinity stress. L. rohita is adapting to the salinity stress by means of upregulating protein turnover, osmolyte production and removing the damaged cells using apoptotic pathway and regulating the cell growth and hence diverting the essential energy for coping with salinity stress.

Heterologous expression and biochemical characterization of novel multifunctional thermostable α-amylase from hot-spring metagenome.

Hot-springs are regarded as the best source of industrially significant biocules and one of the unique locations for extremophiles. The α-amylase is one of the most important enzymes used in starch consuming industries, where the need of thermostability is paramount. In this study, the full metagenome sequences obtained from the soil of Tuwa hot-spring (Gujarat, India) were examined for the presence of several thermostable enzymes using bioinformatic techniques. The whole gene sequence for α-amylase was found from the metagenome. The α-amylase gene was amplified, cloned, and expressed in Escherichia coli and further characterized in vitro. The rm-α-amylase was found optimally active at 60 °C and at pH 6.0 and showed significantly high activity in 0.1 mM Co2+ as well as in other heavy metal ions without any effect on its thermostability. Apart from α-amylase activity the purified rm-α-amylase was also shown to hydrolyse agar, xylan, pectin, alginate and cellulose. To our knowledge, this is the first report of a new, multifunctional, thermostable amylase that was discovered from the hot-spring metagenomes. Owing to their multifunctionality, resilience towards high temperature and heavy metal ions, stability with solvents, additives and inhibitors, rm-α-amylase can be exploited for a variety of biotechnological applications.

Bioinformatics analysis of miRNA and its associated genes to identify potential biomarkers of oral submucous fibrosis and oral malignancy.

BACKGROUND: MicroRNAs are a group of non-coding RNA that controls the gene expression. The interaction between miRNA and mRNA is thought to be dynamic. Oral cancer "The cancer of mouth" is quite prevailing in developing countries. miRNA has been found associated with oral cancer targeting tumor growth, cell proliferation, metastasis, invasion. The significant association of miRNA with genes could be used as a remarkable tool for diagnosis as well as prognostic analysis of oral cancer. AIM: The aim of the present study is to evaluate common upregulated and downregulated miRNAs in oral submucous fibrosis (OSMF) and oral malignancy (OM) patients that can be used as diagnostic biomarkers, and to find out their interactions with target genes to establish associated networks in cancer pathways. METHODS AND RESULTS: Using miRDeep2 and DESeq analysis, the upregulated and downregulated miRNA in OSMF (Oral Submucous Fibrosis) and OM (Oral Malignancies) samples were compared to GEO (Gene Expression Omnibus) control dataset. There were 50 common downregulated miRNAs and 13 common upregulated miRNAs in OSMF and OM samples. miRNet analysis of common upregulated miRNA and common downregulated miRNA identified 1295 and 5954 genes, respectively connected with cancer pathways. From analysis of Hub genes, HRAS, STAT3, TP53, MYC, PTEN, CTNNB1, CCND1, JUN, VEGFA, KRAS were found associated with downregulated miRNA and VEGFA, TP53, MDM2, PTEN, MYC, ERBB2, CDKN1A, HSP90AA1, CCND1, AKTI were found associated with upregulated miRNA. The gene enrichment analysis of these hub genes were associated with cell communication, metabolic process, cell proliferation, and cellular component organization. Hub Genes linked with upregulated miRNA had an enrichment ratio of 11.828, whereas hub genes linked with downregulated miRNA had an enrichment ratio of 45.912. CONCLUSION: We identified common deregulated miRNAs between OSMF and OM patients, which were further analyzed to find out associations with the genes correlated to cancer pathways. The hub genes identified in this study were found to have a significant impact on tumor growth and carcinogenesis. Also, the enrichment of these genes has revealed that the genes are associated with cellular communication, metabolic processes and various biological regulation. These deregulated miRNAs can be used to make a panel of biomarkers to diagnose oral cancer from blood even before its onset.

Heterologous expression of recombinant nattokinase in Escherichia coli BL21(DE3) and media optimization for overproduction of nattokinase using RSM.

Nattokinase, a serine protease, was discovered in Bacillus subtilis during the fermentation of a soybean byproduct. Nattokinase is essential for the lysis of blood clots and the treatment of cardiac diseases including atherosclerosis, thrombosis, high blood pressure, and stroke. The demand for thrombolytic drugs rises as the prevalence of cardiovascular disease rises, and nattokinase is particularly effective for the treatment of cardiovascular diseases due to its long duration of action. In this study, we cloned the nattokinase gene from the Bacillus subtilis strain into the pET32a vector and expressed the protein in the E. coli BL21(DE3) strain. The active recombinant nattokinase was purified using Ni-NTA affinity chromatography and then evaluated for fibrinolytic and blood clot lysis activity. Physiological parameters for optimizing protein production at optimal pH, temperature, IPTG concentration, and incubation time were investigated. A statistical technique was used to optimize media components for nattokinase overproduction, and Central Composite Design-Response Surface Methodology-based optimization was used to select significant components for protein production. The optimized media produced 1805.50 mg/L of expressed nattokinase and 42.80 gm/L of bacterial mass. The fibrinolytic activity obtained from refolded native protein was 58FU/mg, which was five times higher than the available orokinase drug (11FU/mg). The efficiency with which a statistical technique for media optimization was implemented improved recombinant nattokinase production and provides new information for scale - up nattokinase toward industrial applications.

Defective ORF8 dimerization in SARS-CoV-2 delta variant leads to a better adaptive immune response due to abrogation of ORF8-MHC1 interaction.

In India, during the second wave of the COVID-19 pandemic, the breakthrough infections were mainly caused by the SARS-COV-2 delta variant (B.1.617.2). It was reported that, among majority of the infections due to the delta variant, only 9.8% percent cases required hospitalization, whereas only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections cases were observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Downregulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant resulted in structural instability of ORF8 dimer caused by disruption of hydrogen bonds and salt bridges as revealed by structural analysis and MD simulation studies. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild-type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 of SARS-CoV-2 delta variant may not be hindering the MHC-I expression thereby resulting in a better immune response against the SARS-CoV-2 delta variant, which partly explains the possible reason for sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India.

Kidney transcriptome response to salinity adaptation in Labeo rohita.

The increasing salinization of freshwater resources, owing to global warming, has caused concern to freshwater aquaculturists. In this regard, the present study is aimed at economically important freshwater fish, L. rohita (rohu) adapting to varying degrees of salinity concentrations. The RNA-seq analysis of kidney tissue samples of L. rohita maintained at 2, 4, 6, and 8 ppt salinity was performed, and differentially expressed genes involved in various pathways were studied. A total of 755, 834, 738, and 716 transcripts were downregulated and 660, 926, 576, and 908 transcripts were up-regulated in 2, 4, 6, and 8 ppt salinity treatment groups, respectively, with reference to the control. Gene ontology enrichment analysis categorized the differentially expressed genes into 69, 154, 92, and 157 numbers of biological processes with the p value < 0.05 for 2, 4, 6, and 8 ppt salinity groups, respectively, based on gene functions. The present study found 26 differentially expressed solute carrier family genes involved in ion transportation and glucose transportation which play a significant role in osmoregulation. In addition, the upregulation of inositol-3-phosphate synthase 1A (INO1) enzyme indicated the role of osmolytes in salinity acclimatization of L. rohita. Apart from this, the study has also found a significant number of genes involved in the pathways related to salinity adaptation including energy metabolism, calcium ion regulation, immune response, structural reorganization, and apoptosis. The kidney transcriptome analysis elucidates a step forward in understanding the osmoregulatory process in L. rohita and their adaptation to salinity changes.

Evaluation of immune evasion in SARS-CoV-2 Delta and Omicron variants.

Emerging SARS-CoV-2 variants with higher transmissibility and immune escape remain a persistent threat across the globe. This is evident from the recent outbreaks of the Delta (B.1.617.2) and Omicron variants. These variants have originated from different continents and spread across the globe. In this study, we explored the genomic and structural basis of these variants for their lineage defining mutations of the spike protein through computational analysis, protein modeling, and molecular dynamic (MD) simulations. We further experimentally validated the importance of these deletion mutants for their immune escape using a pseudovirus-based neutralization assay, and an antibody (4A8) that binds directly to the spike protein's NTD. Delta variant with the deletion and mutations in the NTD revealed a better rigidity and reduced flexibility as compared to the wild-type spike protein (Wuhan isolate). Furthermore, computational studies of 4A8 monoclonal antibody (mAb) revealed a reduced binding of Delta variant compared to the wild-type strain. Similarly, the MD simulation data and virus neutralization assays revealed that the Omicron also exhibits immune escape, as antigenic beta-sheets appear to be disrupted. The results of the present study demonstrate the higher possibility of immune escape and thereby achieved better fitness advantages by the Delta and Omicron variants, which warrants further demonstrations through experimental evidences. Our study, based on in-silico computational modelling, simulations, and pseudovirus-based neutralization assay, highlighted and identified the probable mechanism through which the Delta and Omicron variants are more pathogenically evolved with higher transmissibility as compared to the wild-type strain.

Transcriptome profiling reveals upregulation of benzoate degradation and related genes in Pseudomonas aeruginosa D6 during textile dye degradation.

An upsurge in textile dye pollution has demanded immediate efforts to develop an optimum technology for their bioremediation. However, the molecular mechanism underpinning aerobic decolorization of dyes is still in its infancy. Thus, in the current work, the intricacies of aerobic remediation of textile dyes by Pseudomonas aeruginosa D6 were understood via a transcriptomic approach. The bacterium isolated from the sludge sample of a common effluent treatment plant was able to decolorize 54.42, 57.66, 50.84 and 65.86% of 100 mg L-1 of four different dyes i.e., TD01, TD04, TD05, and TD06, respectively. The maximum decolorization was achieved within six days and thus, the first and sixth day of incubation were selected for transcriptome analysis at the early and late phase of the decolorization, respectively. The expression profiles of all samples were compared to gain insight into the dye-specific response of bacterium and it was found that it behaved most uniquely in the presence of the dye TD01. Several genes critical to core metabolic processes like the TCA cycle, glycolysis, pentose phosphate pathway, translation, cell motility etc. Were found to be overexpressed in the presence of dyes. Interestingly, in response to dyes, the benzoate degradation pathway was significantly upregulated in the bacterium as compared to control (i.e., bacterium without dye). Thus, seven genes contributing to the induction of the same were further studied by RT-qPCR analysis. Overall, the involvement of the benzoate pathway implies the appearance of aromatic intermediates during decolorization, which in turn infers dye degradation.

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis.

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source of streptokinase is various strains of Streptococci. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD gene-mediated post-translational modification of streptolysin, an inhibitor of streptokinase expression through the degradation of FasX small RNA (through CoV/RS), which stabilizes streptokinase mRNA. In order to improve the stability of mRNA and increase the expression of streptokinase, which is inhibited by SagA, we used CRISPR-Cas9 to successfully knockout the SagD gene and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type. We have demonstrated the successful gene knockout of SagD using CRISPR-Cas9 in S. equisimilis, where an engineered strain can be further used for overexpression of streptokinase for therapeutic applications.

Bovine adenovirus-3 protein VIII associates with eukaryotic initiation factor-6 during infection.

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein-protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2-hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S-transferase-pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus-3 (BAdV-3) infected cells. The C-terminus amino acids 147 to 174 of protein VIII and N-terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV-3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV-3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.

Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome.

A cellulase encoding gene, Cel PRII, was identified from Mehsani buffalo rumen metagenome, and cloned and expressed in Escherichia coli BL21(DE3)pLysS. The 1170 bp full length gene encodes a 389 residue polypeptide (Cel PRII) containing a catalytic domain belonging to glycosyl hydrolase (GH) 5 family. The fusion protein consisting of the Cel PRII, thioredoxin tag and 6x Histidine tag with predicted molecular weight of 63 kDa when recovered from inclusion bodies under denaturing conditions, exhibited cellulolytic activity against carboxymethyl cellulose (CMC). Recombinant Cel PRII was stable in the pH range 4.0-10.0 with pH optima 6.0. The optimal reaction temperature of Cel PRII was 30 °C with more than 50% of its activity retained at the temperatures ranging from 0 to 50 °C. Cel PRII exhibited enhanced enzymatic activity in the presence of Mn2+ ions and was inhibited in the presence of chelating agent EDTA. The K m and V max values for CMC were found to be 166 mg/mL and 1292 IU/mg, respectively. Cel PRII identified in the present study may act as an excellent candidate for industrial applications, and may aid in lignocellulosic biomass conversion because of its potential cellulolytic activity, thermostability, and excellent pH stability.

Functional gene profiling through metaRNAseq approach reveals diet-dependent variation in rumen microbiota of buffalo (Bubalus bubalis).

Recent advances in next generation sequencing technology have enabled analysis of complex microbial community from genome to transcriptome level. In the present study, metatranscriptomic approach was applied to elucidate functionally active bacteria and their biological processes in rumen of buffalo (Bubalus bubalis) adapted to different dietary treatments. Buffaloes were adapted to a diet containing 50:50, 75:25 and 100:0 forage to concentrate ratio, each for 6 weeks, before ruminal content sample collection. Metatranscriptomes from rumen fiber adherent and fiber-free active bacteria were sequenced using Ion Torrent PGM platform followed by annotation using MG-RAST server and CAZYmes (Carbohydrate active enzymes) analysis toolkit. In all the samples Bacteroidetes was the most abundant phylum followed by Firmicutes. Functional analysis using KEGG Orthology database revealed Metabolism as the most abundant category at level 1 within which Carbohydrate metabolism was dominating. Diet treatments also exerted significant differences in proportion of enzymes involved in metabolic pathways for VFA production. Carbohydrate Active Enzyme(CAZy) analysis revealed the abundance of genes encoding glycoside hydrolases with the highest representation of GH13 CAZy family in all the samples. The findings provide an overview of the activities occurring in the rumen as well as active bacterial population and the changes occurring through different dietary treatments.

Complete genome sequence analysis of chicken astrovirus isolate from India.

OBJECTIVE: Chicken astroviruses have been known to cause severe disease in chickens leading to increased mortality and "white chicks" condition. Here we aim to characterize the causative agent of visceral gout suspected for astrovirus infection in broiler breeder chickens. METHODS: Total RNA isolated from allantoic fluid of SPF embryo passaged with infected chicken sample was sequenced by whole genome shotgun sequencing using ion-torrent PGM platform. The sequence was analysed for the presence of coding and non-coding features, its similarity with reported isolates and epitope analysis of capsid structural protein. RESULTS: The consensus length of 7513 bp genome sequence of Indian isolate of chicken astrovirus was obtained after assembly of 14,121 high quality reads. The genome was comprised of 13 bp 5'-UTR, three open reading frames (ORFs) including ORF1a encoding serine protease, ORF1b encoding RNA dependent RNA polymerase (RdRp) and ORF2 encoding capsid protein, and 298 bp of 3'-UTR which harboured two corona virus stem loop II like "s2m" motifs and a poly A stretch of 19 nucleotides. The genetic analysis of CAstV/INDIA/ANAND/2016 suggested highest sequence similarity of 86.94% with the chicken astrovirus isolate CAstV/GA2011 followed by 84.76% with CAstV/4175 and 74.48%% with CAstV/Poland/G059/2014 isolates. The capsid structural protein of CAstV/INDIA/ANAND/2016 showed 84.67% similarity with chicken astrovirus isolate CAstV/GA2011, 81.06% with CAstV/4175 and 41.18% with CAstV/Poland/G059/2014 isolates. However, the capsid protein sequence showed high degree of sequence identity at nucleotide level (98.64-99.32%) and at amino acids level (97.74-98.69%) with reported sequences of Indian isolates suggesting their common origin and limited sequence divergence. The epitope analysis by SVMTriP identified two unique epitopes in our isolate, seven shared epitopes among Indian isolates and two shared epitopes among all isolates except Poland isolate which carried all distinct epitopes.