Sequence based structural characterization and genetic diversity analysis across coding and promoter regions of goat Toll-like receptor 5 gene.

The exploration of candidate immune response genes in goat may be vital in improving further our understanding about the species specific response to pathogens specifically among the ruminants. In this study, approximately 3.7 kb long genomic sequence of Toll-like receptor 5 (TLR5) covering the entire coding and 5'upstream regions of the gene, was characterized in the Indian goat breeds. Sequence analysis revealed a 2577-nucleotide long open reading frame (ORF) of goat TLR5, encoding 858 amino acids from single exon, similar to other ruminants. The domain structure analysis of goat TLR5 showed the presence of 13 leucine rich repeats (LRRs) in extracellular domain (amino acid position 1-634), single transmembrane domain (position 644-666), and a Toll/interleukin-1 receptor (position 692-837) in cytoplasmic domain, similar to other species. A total of 87 putative transcription factor binding sites were observed within the 5' upstream region of TLR5 gene in goat, 106 in cattle, and 103 in buffalo. Sixteen polymorphic sites were observed in goat TLR5 gene, out of which 10 non-synonymous SNPs were in the functionally important regions. However, none of the amino acid substitutions was found to be potentially damaging to the structure and function of the receptor protein. Further, one of the SNPs in the transmembrane region was genotyped by a TETRA-ARMS PCR in 444 goats of nine breeds from different geographical regions and having different utilities. A significant variation in allelic frequencies was observed across the milch and other types of goat breeds. The comparative modeling of goat TLR5 followed by molecular dynamics simulation gave an insight into its 3D structural arrangements. The molecular docking of Salmonella flagellin and TLR5 dimer elucidated LRRNT (N-terminal) to LRR4 as the key flagellin binding domains region in goat TLR5. The study shows that, although being highly conserved among the ruminants, comparatively high variations in goat TLR5 might give an opportunity to host for recognizing the wider spectrum of pathogens.

Exploring novel KDR inhibitors based on pharmaco-informatics methodology.

Kinase-insert domain-containing receptor (KDR) is one of the important mediators of Vascular endothelial growth factor (VEGF) function in endothelial cells. Inhibition of KDR can be therapeutically advantageous for treatment of a number of diseases. The present study focuses on exploring novel KDR inhibitors by means of pharmaco-informatics methodologies. Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis by atom-based pharmacophore mapping over a set of 85 molecules provides a proposition regarding the molecular fingerprint that can be optimized for designing more active inhibitors. The model was statistically validated with Q(2) = 0.865 for training and r(2) = 0.789, Pearson-r = 0.903 for test set molecules; r(2)(0.925) by external validation suggests model robustness and indicates it as a strong query for screening any compound library. Virtual screening shows the importance of active site and hinge region residue for interaction with KDR inhibitors. Remarkably the retrieved hits contain a urea backbone, implicating urea derivatives as promising candidate for designing KDR inhibitors. The hydrophobicity of active site, which has until now been overlooked, has been raised into the picture by this study. This can impact on KDR drug development. The study thus quantifies crucial structural requirements necessary for a favourable interaction with the receptor binding site while the cooperative pattern provides important structural clues to chemists for framing potent medicinal agents in future.

Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita).

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.