Tejas M. Shah

Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value<0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value<0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.

Metagenome of Mehsani Buffalo Rumen Microbiota: An Assessment of Variation in Feed-Dependent Phylogenetic and Functional Classification

Aim: To gain a greater understanding of the ecology and metabolic potential of the rumen microbiome with the changes in the animal diet. Methods: Diet composed of varying proportion of green and dry roughages along with grains was given to 8 Mehsani buffaloes, and rumen metagenome was sketched using shotgun semiconductor sequencing. Results: In the present study, the Bacteroidetes were found to be dominant at the phyla level and Prevotella at the genus level. The ratio of Firmicutes to Bacteroidetes was found to be higher in the solid fraction as compared to the liquid fraction. In the solid fraction of the dry roughage group, the significant increment (p < 0.05) in Bacteroidetes abundance was observed with increment of roughage concentration. At the genus level, Clostridium significantly increased with the increment in roughage concentration. A comparison of glycoside hydrolase and cellulosome functional genes revealed more glycoside hydrolase 3 encoding genes with higher fiber diet and significant difference in carbohydrate-active enzymes family composition between green and dry roughage groups of the liquid fraction. Conclusion: The present study provides a base to understand the modulating behavior of microbiota which can be manipulated to improve livestock nutrient utilization efficiency and for targeting the efficient catabolism of complex carbohydrate molecules as well.

Analysis of consequences of non-synonymous SNP in feed conversion ratio associated TGF-β receptor type 3 gene in chicken

The recent advances in high throughput sequencing technology accelerate possible ways for the study of genome wide variation in several organisms and associated consequences. In the present study, mutations in TGFBR3 showing significant association with FCR trait in chicken during exome sequencing were further analyzed. Out of four SNPs, one nsSNP p.Val451Leu was found in the coding region of TGFBR3. In silico tools such as SnpSift and PANTHER predicted it as deleterious (0.04) and to be tolerated, respectively, while I-Mutant revealed that protein stability decreased. The TGFBR3 I-TASSER model has a C-score of 0.85, which was validated using PROCHECK. Based on MD simulation, mutant protein structure deviated from native with RMSD 0.08 Å due to change in the H-bonding distances of mutant residue. The docking of TGFBR3 with interacting TGFBR2 inferred that mutant required more global energy. Therefore, the present study will provide useful information about functional SNPs that have an impact on FCR traits.

Identification of novel transcripts deregulated in buccal cancer by RNA-seq

The differential transcriptome analysis provides better understanding of molecular pathways leading to cancer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and prediction of therapeutic outcome. This study describes the transcriptome analysis of buccal cancer and normal tissue by CLC Genomics Workbench from the data generated by Roches 454 sequencing platform, which identified total of 1797 and 2655 genes uniquely expressed in normal and cancer tissues, respectively with 2466 genes expressed in both tissues. Among the genes expressed in both tissues, 1842 were up-regulated whereas 624 were down-regulated in cancer tissue. Besides transcripts known to be involved in cancer, this study led to the identification of novel transcripts, with significantly altered expression in buccal cancer tissue, providing potential targets for diagnosis and cancer therapeutics. The functional categorization by the KEGG pathway and gene ontology analysis revealed enrichment of differentially expressed transcripts to various pathways leading to cancer, including the p53 signaling pathway. Moreover, the gene ontology analysis unfolded suppression of transcripts involved in actin mediated cell contraction process. The down-regulation of four of these transcripts MYL1, ACTA1, TCAP and DESMIN in buccal cancer were further supported by quantitative PCR signifying its possible implication in the cancer progression.

Metagenomic analysis of buffalo rumen microbiome: Effect of roughage diet on Dormancy and Sporulation genes.

Buffalo rumen microbiome experiences a variety of diet stress and represents reservoir of Dormancy and Sporulation genes. However, the information on genomic responses to such conditions is very limited. The Ion Torrent PGM next generation sequencing technology was used to characterize general microbial diversity and the repertoire of microbial genes present, including genes associated with Dormancy and Sporulation in Mehsani buffalo rumen metagenome. The research findings revealed the abundance of bacteria at the domain level and presence of Dormancy and Sporulation genes which were predominantly associated with the Clostridia and Bacilli taxa belonging to the phyla Firmicutes. Genes associated with Sporulation cluster and Sporulation orphans were increased from 50% to 100% roughage treatment, thereby promoting sporulation all along the treatments. The spore germination is observed to be the highest in the 75% roughage treatment both in the liquid and solid rumen fraction samples with respect to the decrease in the values of the genes associated with spore core dehydration, thereby facilitating spore core hydration which is necessary for spore germination.

RNA-Seq reveals differentially expressed isoforms and novel splice variants in buccal mucosal cancer.

Buccal mucosal cancer (BMC) is a multifactorial disease with poorly defined genetic profile and prognosis due to late detection stage and unavailability of reliable prognostic markers. To identify aberrant transcriptional events, we employed high throughput RNA-Seq analysis of BMC and normal tissue. Comparative transcriptome analysis with Cufflinks revealed 260 up and 328 down regulated genes whereas, 350 up and 397 down regulated isoforms by at least two folds over buccal normal in BMC. Study revealed 46 splice variants in normal and 106 in cancer, out of which 10 variants were validated with end point RT-PCR. Expression of two isoforms of CD74 was validated using RT-qPCR and found in accordance with RNA-Seq. Further extensive follow up analysis of modulator genes, isoforms and splice variants found in this study, might be useful in deep understanding of pathological changes in BMC and development of prospective intervention strategies.

Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq.

Muscle growth and development from the embryonic to the adult stage of an organism consists of a series of exquisitely regulated and orchestrated changes in expression of genes leading to muscle maturation. In this study, we performed whole transcriptome profiling of adult caprine skeletal muscle derived myoblast and fused myotubes. Using Ion Torrent PGM sequencing platform, a total of 948,776 and 799,976 reads were generated in myoblasts and fused myotubes, respectively. The sequence reads were analyzed on CLC Genomics Workbench using Bos taurus RNA database to study the gene expression in both stages to study different genes responsible for muscle development and regeneration. The up and down-regulated genes were analyzed for gene ontology (GO) and KEGG pathways by Database for Annotation, Visualization and Integrated Discovery (DAVID) database. We found many genes exclusive to multinuclear fused myotubes and contractile nature of skeletal muscle, whereas up-regulated genes in myoblast stage were related to cell division and transcriptional regulation. Out of 27 genes selected for expression validation by RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), 19 genes showed the expression pattern comparable with CLC Genomics Workbench findings. Further, mRNA originated muscle specific microRNAs (miRNA-1 and miRNA-133b) were also observed in the fused myotubes along with other miRNAs with possible importance in muscle development. This study highlights important genes responsible for muscle development and differentiation in adult skeletal muscle system.

Evaluation of genetic diversity and population structure of west-central Indian cattle breeds

Evaluations of genetic diversity in domestic livestock populations are necessary to implement region-specific conservation measures. We determined the genetic diversity and evolutionary relationships among eight geographically and phenotypically diverse cattle breeds indigenous to west-central India by genotyping these animals for 22 microsatellite loci. A total of 326 alleles were detected, and the expected heterozygosity ranged from 0.614 (Kenkatha) to 0.701 (Dangi). The mean number of alleles among the cattle breeds ranged from 7.182 (Khillar) to 9.409 (Gaolao). There were abundant genetic variations displayed within breeds, and the genetic differentiation was also high between the Indian cattle breeds, which displayed 15.9% of the total genetic differentiation among the different breeds. The genetic differentiation (pairwise FST ) among the eight Indian breeds varied from 0.0126 for the Kankrej-Malvi pair to 0.2667 for Khillar-Kenkatha pair. The phylogeny, principal components analysis, and structure analysis further supported close grouping of Kankrej, Malvi, Nimari and Gir; Gaolao and Kenkatha, whereas Dangi and Khillar remained at distance from other breeds.

Ion torrent next-generation sequencing reveals the complete mitochondrial genome of endangered mahseer Tor khudree (Sykes, 1839)

Abstract The complete mitochondrial genome of an endangered mahseer (Deccan mahseer), Tor khudree was sequenced using Ion torrent platform for the first time. The genome sequence was 16 573 bp in size, and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and 1 control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A: 31.9%, G: 15.6%, C: 27.68%, T: 24.76%, A + T content 56.6% and the G + C content 43.32%. The phylogenetic tree constructed using a maximum likelihood model showed sister relationship between T. khudree and Tor tambroides.

Evaluation of a topical herbal drug for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis

Background: Antibiotics have been in use in the treatment of bovine mastitis since decades; however, their use is associated with cost issues and human health concern. Use of herbal drugs does not generally carry these disadvantages. Many plants/herbs have been evaluated in the treatment of bovine mastitis with additional property of immunomodulation in affected mammary gland. Aim: To evaluate a topical herbal drug in two breeds of cattle for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis. Materials and Methods: The response to treatment was evaluated by enumerating somatic cell count (SCC), determining total bacterial load, and studying the expression of different cytokines (interleukin [IL]-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon (IFN)-γ and tumor necrosis factor [TNF]-α). Results: The pre- and post-treatment SCC in mastitic quarters statistically did not differ significantly, however, total bacterial load declined significantly from day 0 onwards in both the breeds. Highly significant differences (P < 0.01) were observed in all the cytokines on day 0, 5, and 21 postlast treatment in both the breeds. The expression level of all the cytokines showed a significant increase on day 5, while a decrease was noticed on day 21 in both the breeds of cattle. The comparison of cytokine expression profiles between crossbred and Gir cattle revealed a significant difference in expression of IL-6 and TNF-α. However, other cytokines exhibited a similar pattern of expression in both breeds, which was non-significant. Conclusion: The topical herbal drug exhibited antibacterial and immunomodulatory activities in subclinical mastitis and thus the work supports its use as alternative herbal therapy against subclinical udder infection in bovines.