Heterologous expression, purification and single step efficient refolding of recombinant tissue plasminogen activator (Reteplase) from E. coli.

Reteplase (recombinant plasminogen activator, rPA) is a mutant non-glycosylated tissue-type plasminogen activator (tPA) containing 355 amino acids with longer half-life and promising thrombolytic activity than its original counterpart, full length tPA. In this study, we aimed to produce and optimize the purification process of recombinant tissue-type plasminogen activator (tPA) known as Reteplase (rPA). Reteplase cDNA synthesized from total mRNA isolated from human placenta was PCR amplified, cloned into a pET-28a(+) E. coli expression vector and expressed in Rosetta-gami 2 E. coli (NovagenⓇ) host. rPA was expressed as an inclusion body in E. coli and its biological activity was achieved after single step solubilization, purification and refolding. We exploited the strategy of Slow Refolding using Gradual Dialysis (SRGD) in which a refolding buffer containing glutathione oxidized (1 mM GSSG) and glutathione reduced (3 mM GSH) and pH 9.0 was used. Using the SRGD method, we were able to successfully obtain the protein in its active form. We obtained 4.26 mg of active refolded protein from a 50 mL culture that was scaled up in a bioreactor. The purity and homogeneity of rPA was evaluated by SDS-PAGE, Western blotting and mass spectrometry. Circular dichroism spectroscopy was conducted to evaluate the refolding and stability of the refolded rPA in comparison to reference standard rPA. The thrombolytic potential of rPA was assessed by fibrin plate assay and In Vitro clot lysis assay. The presented protocol offers a viable approach for enhancing both the yield and refolding efficiency of reteplase, potentially resulting in an increase in yield.

Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination.

Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.

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 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.

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.

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.

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.

Isolation and characterization of novel multifunctional recombinant family 26 glycoside hydrolase from Mehsani buffalo rumen metagenome.

Rumen microbiota harbor a diverse set of carbohydrate-active enzymes (CAZymes), which play a crucial role in the degradation of a complex plant polysaccharide thereby providing metabolic energy to the host animals. Earlier, we reported CAZYme analysis from the buffalo rumen metagenome by high throughput shotgun sequencing. Among the various CAZymes, glycoside hydrolase family 26 (GH26) enzymes have a number of industrial applications including in paper, oil, biofuel, food, feed, pharmaceutical, coffee, and detergent industries. Here, we report isolation and characterization of GH26 enzyme from the buffalo rumen metagenome. A novel GH26 gene composed of 1,119 base pairs was successfully amplified using the gene-specific primers inferred based on the contig generated from metagenome sequence assembly and cloned in a pET32a (+) expression vector as an N-terminal histidine tag fusion protein. A novel GH26 protein from an unknown rumen microorganism shared a maximum of 68% identity with the Prevotella ruminicola 23 encoded carbohydrate esterase family 7 and 46% with Bacteroides sp. 2_1_33B encoded mannan endo-1, 4-ß-mannosidase. The recombinant GH26-histidine tag fusion protein was expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The purified enzyme displayed multifunctional activities against various carbohydrate substrates including locust bean gum, beechwood xylan, pectin, and carboxymethyl cellulose suggesting mannanase, xylanase, pectin esterase, and endoglucanase activities, respectively.

Effect of roughage on rumen microbiota composition in the efficient feed converter and sturdy Indian Jaffrabadi buffalo (Bubalus bubalis).

BACKGROUND: The rumen microbiota functions as an effective system for conversion of dietary feed to microbial proteins and volatile fatty acids. In the present study, metagenomic approach was applied to elucidate the buffalo rumen microbiome of Jaffrabadi buffalo adapted to varied dietary treatments with the hypothesis that the microbial diversity and subsequent in the functional capacity will alter with diet change and enhance our knowledge of effect of microbe on host physiology. Eight adult animals were gradually adapted to an increasing roughage diet (4 animals each with green and dry roughage) containing 50:50 (J1), 75:25 (J2) and 100:0 (J3) roughage to concentrate proportion for 6 weeks. Metagenomic sequences of solid (fiber adherent microbiota) and liquid (fiber free microbiota) fractions obtained using Ion Torrent PGM platform were analyzed using MG-RAST server and CAZymes approach. RESULTS: Taxonomic analysis revealed that Bacteroidetes was the most abundant phylum followed by Firmicutes, Fibrobacter and Proteobacteria. Functional analysis revealed protein (25-30 %) and carbohydrate (15-20 %) metabolism as the dominant categories. Principal component analysis demonstrated that roughage proportion, fraction of rumen and type of forage affected rumen microbiome at taxonomic as well as functional level. Rumen metabolite study revealed that rumen fluid nitrogen content reduced in high roughage diet fed animals and pathway analysis showed reduction in the genes coding enzymes involved in methanogenesis pathway. CAZyme annotation revealed the abundance of genes encoding glycoside hydrolases (GH), with the GH3 family most abundant followed by GH2 and GH13 in all samples. CONCLUSIONS: Results reveals that high roughage diet feed improved microbial protein synthesis and reduces methane emission. CAZyme analysis indicated the importance of microbiome in feed component digestion for fulfilling energy requirements of the host. The findings help determine the role of rumen microbes in plant polysaccharide breakdown and in developing strategies to maximize productivity in ruminants.

Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro.

BACKGROUND: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. METHODS: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. RESULTS: We observed a significant silencing (64-81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16-46% silencing with maximum of 46% by sh6 (p = 0.0318) against endogenous ACVR2B whereas up to 66% (p = 0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r = 0.547; p = 0.102), myogenin (r = 0.517; p = 0.126) and Myf5 (r = 0.262; p = 0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. CONCLUSIONS: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.

Insights into resistome and stress responses genes in Bubalus bubalis rumen through metagenomic analysis.

Buffalo rumen microbiota experience variety of diets and represents a huge reservoir of mobilome, resistome and stress responses. However, knowledge of metagenomic responses to such conditions is still rudimentary. We analyzed the metagenomes of buffalo rumen in the liquid and solid phase of the rumen biomaterial from river buffalo adapted to varying proportion of concentrate to green or dry roughages, using high-throughput sequencing to know the occurrence of antibiotics resistance genes, genetic exchange between bacterial population and environmental reservoirs. A total of 3914.94 MB data were generated from all three treatments group. The data were analysed with Metagenome rapid annotation system tools. At phyla level, Bacteroidetes were dominant in all the treatments followed by Firmicutes. Genes coding for functional responses to stress (oxidative stress and heat shock proteins) and resistome genes (resistance to antibiotics and toxic compounds, phages, transposable elements and pathogenicity islands) were prevalent in similar proportion in liquid and solid fraction of rumen metagenomes. The fluoroquinolone resistance, MDR efflux pumps and Methicillin resistance genes were broadly distributed across 11, 9, and 14 bacterial classes, respectively. Bacteria responsible for phages replication and prophages and phage packaging and rlt-like streptococcal phage genes were mostly assigned to phyla Bacteroides, Firmicutes and proteaobacteria. Also, more reads matching the sigma B genes were identified in the buffalo rumen. This study underscores the presence of diverse mechanisms of adaptation to different diet, antibiotics and other stresses in buffalo rumen, reflecting the proportional representation of major bacterial groups.

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet.

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P < 0.05) between solid and liquid fraction of rumen (in 65 categories), between all three treatments (in 56 categories), and between green and dry roughage (17 categories). Diet treatment also exerted significant difference in environmental gene tags (EGTs) involved in metabolic pathways for production of volatile fatty acids. EGTs for butyrate production were abundant in K2, whereas EGTs for propionate production was abundant during K1. Principal component analysis also demonstrated that diet proportion, fraction of rumen, and type of forage affected rumen microbiome at taxonomic as well as functional level.

Alteration in the expression of exon IIC transcripts of brain-derived neurotrophic factor gene by simvastatin [correction of simvastain] in chronic mild stress in mice: a possible link with dopaminergic pathway.

We have investigated the influence of dopaminergic agents on the expression of brain-derived neurotrophic factor (BDNF) gene in relation with lipid levels in chronic mild stress (CMS). Mice subjected to CMS were treated with simvastatin (10 mg/kg, per os (orally)) along with bromocriptine (2 mg/kg, intraperitoneally (ip)), levodopa (200 mg/kg, ip), or haloperidol (0.1 mg/kg, ip) for 14 days. CMS produced a decrease in sucrose intake and an increase in serum cholesterol and triglycerides levels with a decrease in high-density lipoprotein cholesterol, which were prevented by simvastatin. This was greater when it was combined with bromocriptine or levodopa. Haloperidol significantly prevented the simvastatin-induced increase in sucrose intake but not the alterations in lipids. There was an upregulation in the expression of BDNF exon-IIA and -IIB transcripts by CMS but not the exon-IIC transcripts. Simvastatin could increase expression of exon-IIC transcripts in stressed mice. This was partially increased by bromocriptine. Haloperidol significantly prevented simvastatin-induced increase in expression of BDNF exon-IIC transcripts. The results showed a positive correlation between expression of BDNF exon-IIC transcripts and sucrose intake. In conclusion, our data suggest the involvement of lipid levels and BDNF exon-IIC transcripts in CMS-induced behaviour in mice, possibly through the dopaminergic system.

Identification of novel splice variants in horn cancer by RNA-Seq analysis in Zebu cattle.

Horn cancer accounts for nearly 83% of total tumors found in Indian Zebu cattle, which results in chronic suffering and causes heavy economic losses. Alternative splicing has been frequently implicated in the various types of cancer progression. Utilizing the transcriptome sequence generated by next generation sequencing, we analyzed the transcript data for the presence of alternative splicing using BLAT program and identified 27 alternatively spliced genes, of which 12 spliced variants appeared to be the novel spliced candidates. Protein prediction of these novel spliced variants revealed that splice variation has caused either truncation of protein, insertion/deletion of stretch of amino acids or formation of unique carboxy terminus. The RT-PCR analysis confirmed the expression of 8 of the 12 novel spliced variants observed by transcriptome sequencing. Additionally, altered splicing/expression of these novel candidates between cancer and normal tissues revealed by qPCR suggests their potential involvement in the development of horn cancer.

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.

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.

Human leukocyte antigen alleles, genotypes and haplotypes frequencies in renal transplant donors and recipients from West Central India.

BACKGROUND: Human leukocyte antigen (HLA) is comprised of a highly polymorphic set of genes which determines the histocompatibility of organ transplantation. The present study was undertaken to identify HLA class I and class II allele, genotype and haplotype frequencies in renal transplant recipients and donors from West Central India. MATERIALS AND METHODS: HLA typing was carried out using Polymerase Chain Reaction-Sequence Specific Primer in 552 live related and unrelated renal transplant recipients and donors. RESULTS: The most frequent HLA class I and class II alleles and their frequencies in recipients were HLA-AFNx0101 (0.1685) and AFNx0102 (0.1649), HLA-BFNx0135 (0.1322), and HLA-DR beta 1 (DRB 1)FNx0115 (0.2192), whereas in donors, these were HLA-AFNx0102 (0.1848) and AFNx0101 (0.1667), HLA-BFNx0135 (0.1359), and HLA-DRB1FNx0115 (0.2409). The two-locus haplotype statistical analysis revealed HLA-AFNx0102-B61 as the most common haplotype with the frequency of 0.0487 and 0.0510 in recipients and donors, respectively. Further, among the three locus haplotypes HLA-AFNx0133-BFNx0144-DRB1FNx0107 and HLA-AFNx0102-BFNx0161-DRB1FNx0115 were the most common haplotypes with frequencies 0.0362 and 0.0326, respectively in recipients and 0.0236 and 0.0323, respectively in donors. Genotype frequency revealed a high prevalence of genotype HLA-AFNx0102/AFNx0124 in recipients (0.058) compared to donors (0.0109) whereas low prevalence of HLA-AFNx0101/AFNx0102 in recipients (0.0435) than in donors (0.0797). The phylogenetic and principal component analysis of HLA allele and haplotype frequency distribution revealed genetic similarities of various ethnic groups. Further, case control analysis provides preliminary evidence of association of HLA-A genotype (P < 0.05) with renal failure. CONCLUSION: This study will be helpful in suitable donor search besides providing valuable information for population genetics and HLA disease association analysis.