Comparative Analysis of Molecular Structure, Function and Expression of Buffalo (Bubalus bubalis) Toll-Like Receptor 9

Toll-like receptor 9 (TLR9) has been characterized as a receptor that recognizes unmethylated CpG motif and triggers a pro-inflammatory cytokine response that influences both innate and adaptive immunity. Buffalo is an economically important livestock species in many Asian and Mediterranean countries, but there is little information available on its TLR9 structure and response to stimulation with its agonist CpG-ODNs. Hence in this study, we report the analysis of newly sequenced buffalo TLR9 gene fragment. In this study, buffalo TLR9 amino acid sequence revealed close association of TLR9 proteins within other bovines and small ruminants; but high divergence from other species. Multiple alignment of deduced amino acid sequence of Bubalus bubalis TLR9 with other species showed that 156/201 (74.28%) amino acids were conserved in all species. Leucine rich repeat (LRR) motifs in the ectodomain of TLR9 are responsible for molecular recognition of its agonist. The LRR pattern of Bubalus bubalis TLR9 protein was predicted towards N-terminal sequence and was found to be conserved among all species except Rattus norvegicus and Equus caballus. Blast analysis of buffalo TLR9 sequence with single nucleotide polymorphisms (SNPs) database revealed 13 SNPs out of which 7 were cds-synonymous and 6 were of the functional significance. Furthermore, kinetics of TLR9 and proinflammatory IL-beta and TNF-alpha cytokine expression by buffalo PBMCs influenced by CpG-ODN is also discussed

Evaluation of Physiological Parameters in Response to Endurance Exercise of Zanskar Ponies Adapted to High Altitude of Ladakh Region

Zanskar pony, a native horse breed of Ladakh mainly used for transportation in Trans-Himalayan region of India, is well adapted to high altitude hypobaric hypoxia environment. Due to extreme conditions of the Ladakh region, better endurance of these ponies under hypoxic and extreme cold conditions is of utmost concern for their recruitment in Indian Army. In the present study, 12 young trained Zanskar ponies were evaluated during endurance exercise at an altitude of 3292 meter above mean sea level. The animals were subjected to carriage transport with 65-70Kg load or riding on a track of 5-6 Km. Physiological parameters viz., pulse rate (PR), heart rate (HR), rectal temperature (RT), respiratory rate (RR) and oxygen saturation (SaO2) were recorded in Zanskar ponies during pre-exercise (T0), post- exercise (T1) and post recovery (T2, 2 hours post resting) stages. Results showed marked increase in PR, HR, RR and RT post exercise time points. The mean values of PR increased from 49.83±4.62 to 73.67±21.54 per minute, HR from 48±13.60 to 75±15.82 beats/min, RR from 37.83±9.70 to 57.67±13.48 per min and RT from 99.62±0.34 101.04±0.53 °F from pre stress to post endurance stress. The mean SaO2 level reduced significantly (88.58±6.75 at T0 versus 64.00±18.70 at T1 and 54.42±14.79 at T2) post exercise. This indicated limited availability of arterial oxygen for tissues which could be vital factor for adverse change in some of physio-biochemical parameters. Though the trend of physiological response was similar for all the 12 animals, still variation at individual animal level was observed during endurance stress. In future, some of these physiological parameters along with biochemical and molecular parameters could be evaluated as potential biomarkers in selecting ponies with superior endurance trait specifically under hypoxic conditions

Identification of stably expressed Internal Control Genes (ICGs) for normalization of expression data in liver of C57BL/6 mice injected with beta casomorphins.

In recent years, beta-casomorphin peptides (BCM7/BCM9) derived from the digestion of cow milk have drawn a lot of attention world over because of their proposed impact on human health. In order to evaluate the transcriptional modulation of target genes through RT-qPCR in response to these peptides, availability of appropriate reference or internal control genes (ICGs) will be the key. The present study was planned to identify a panel of stable ICGs in the liver tissue of C57BL/6 mice injected with BCM7/BCM9 cow milk peptides for 3 weeks. A total of ten candidate genes were evaluated as potential ICGs by assessing their expression stability using software suites; geNorm, NormFinder and BestKeeper. The suitability of the identified ICGs was validated by assessing the relative expression levels of target genes, HP and Cu/Zn SOD. Based on geNorm, PPIA and SDHA gene pair was identified to be most stably expressed in liver tissue during the animal trials. Similarly, NormFinder analysis also identified PPIA as the most stable gene. BestKeeper analysis showed crossing point SD value for all the genes in the acceptable range that is closer to 1. Overall, the study identified a panel of stable ICGs for reliable normalization of target genes expression data in mice liver tissues during BCM7/9 peptides trial

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Not AvailableBackground: India has a vast riverine and swamp buffalo diversity adapted to various agro-ecological conditions. In the present study, genetic diversity data for 10 different buffalo populations of India, using 20 highly polymorphic microsatellite markers has been generated for the genetic diversity analysis. The buffalo populations of Eastern Odisha state, were the primary focus. Methods and results: The minimal spanning network based on Bruvo's distance, PCA (Principal Component Analysis) based on the Fst (Fixation Index) values, and genetic admixture analysis using both the STRUCTURE and 'snapclust' were performed. The analysis could identify the Manda population as distinct from other Odisha buffalo breeds as well as adjoining Chhattisgarhi buffalo breeds. The total observed number of alleles ranged between 143 (Manda) and 301 (Paralakhemundi) with an average of 204 alleles per breed. The Sambhalpuri buffalo population also clustered into two separate subpopulations, half of the unique sub-population located geographically south-wards, displayed no admixture with any of the adjacent buffalo populations. The Manda buffalo population has shown sufficient allelic richness and heterozygosity under random mating being practiced in the field conditions. Conclusions: The study has led to the identification of the Manda as a distinct buffalo population, and the germplasm has been registered as a new Indian buffalo breed. Whereas, the Sambhalpuri population requires elaborate analysis to confirm the existence of two distinct sub-populations.Not Availabl

A nonsense mutation in cGMP-dependent type II protein kinase (PRKG2) causes dwarfism in American Angus cattle

Historically, dwarfism was the major genetic defect in U.S. beef cattle. Aggressive culling and sire testing were used to minimize its prevalence; however, neither of these practices can eliminate a recessive genetic defect. We assembled a 4-generation pedigree to identify the mutation underlying dwarfism in American Angus cattle. An adaptation of the Elston-Steward algorithm was used to overcome small pedigree size and missing genotypes. The dwarfism locus was fine-mapped to BTA6 between markers AFR227 and BM4311 . Four candidate genes were sequenced, revealing a nonsense mutation in exon 15 of cGMP-dependant type II protein kinase ( PRKG2 ). This C/T transition introduced a stop codon (R678X) that truncated 85 C-terminal amino acids, including a large portion of the kinase domain. Of the 75 mutations discovered in this region, only this mutation was 100% concordant with the recessive pattern of inheritance in affected and carrier individuals (log of odds score = 6.63). Previous research has shown that PRKG2 regulates SRY (sex-determining region Y) box 9 (SOX9)-mediated transcription of collagen 2 ( COL2 ). We evaluated the ability of wild-type (WT) or R678X PRKG2 to regulate COL2 expression in cell culture. Real-time PCR results confirmed that COL2 is overexpressed in cells that overexpressed R678X PRKG2 as compared with WT PRKG2 . Furthermore, COL2 and COL10 mRNA expression was increased in dwarf cattle compared with unaffected cattle. These experiments indicate that the R678X mutation is functional, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. Therefore, we present PRKG2 R678X as a causative mutation for dwarfism cattle

Cytokine levels in imiquimod, poly I:C or PPRV treated PBMCs.

<p>a) <b>TNFα,</b> b) IFNα, and c) IFNγ from supernatants of PPRV infected PBMC of different goat breeds. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. PPRV infected PBMC from Kanni and Salem black breeds show higher production of TNFα, IFNα and IFNγ than Barbari. Poly I:C and imiquimod treated PBMC, similarly, show higher production of TNFα and IFNα in Kanni and Salem black breeds than in Barbari. TNFα and IFNγ levels are expressed as the corrected mean ± SD of optical density [OD] of treatment groups from which the OD of mock infected supernatants is subtracted. IFNα concentrations in the experimental samples are expressed as pg/ml.</p

Virus replication in PBMCs stimulated with TLR3 and TLR7 agonists.

<p>a) Reduction in TCID₅₀ values of PPRV in goat PBMC on imiquimod treatment. Reduction in PPRV H gene expression levels in goat PBMC on treatment with b) poly I:C and c) imiquimod. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. Values represent mean ± SD of 40-corrected CT in 5 individual animals per goat breed. Significantly higher PPRV viral loads observed in PBMC of Barbari and Tellicherry breeds as compared to Kanni and Salem Black. Significant reduction in PPRV levels on poly I:C and imiquimod treatment in all breeds.</p