Transcriptional Profiling of PRKG2-Null Growth Plate Identifies Putative Down-Stream Targets of PRKG2

Kinase activity of cGMP-dependent, type II, protein kinase (PRKG2) is required for the proliferative to hypertrophic transition of growth plate chondrocytes during endochondral ossification. Loss of PRKG2 function in rodent and bovine models results in dwarfism. The objective of this study was to identify pathways regulated or impacted by PRKG2 loss of function that may be responsible for disproportionate dwarfism at the molecular level. Microarray technology was used to compare growth plate cartilage gene expression in dwarf versus unaffected Angus cattle to identify putative downstream targets of PRGK2. Pathway enrichment of 1284 transcripts (nominal p \u3c 0.05) was used to identify candidate pathways consistent with the molecular phenotype of disproportionate dwarfism. Analysis with the DAVID pathway suite identified differentially expressed genes that clustered in the MHC, cytochrome B, WNT, and Muc1 pathways. A second analysis with pathway studio software identified differentially expressed genes in a host of pathways (e.g. CREB1, P21, CTNNB1, EGFR, EP300, JUN, P53, RHOA, and SRC). As a proof of concept, we validated the differential expression of five genes regulated by P53, including CEBPA, BRCA1, BUB1, CD58, and VDR by real-time PCR (p \u3c 0.05). Known and novel targets of PRKG2 were identified as enriched pathways in this study. This study indicates that loss of PRKG2 function results in differential expression of P53 regulated genes as well as additional pathways consistent with increased proliferation and apoptosis in the growth plate due to achondroplastic dwarfism

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

Structural and functional annotation of the porcine immunome

Background: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.[br/] Results: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.[br/] Conclusions: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response

Transcriptional Profiling of PRKG2-Null Growth Plate Identifies Putative Down-Stream Targets of PRKG2

Kinase activity of cGMP-dependent, type II, protein kinase (PRKG2) is required for the proliferative to hypertrophic transition of growth plate chondrocytes during endochondral ossification. Loss of PRKG2 function in rodent and bovine models results in dwarfism. The objective of this study was to identify pathways regulated or impacted by PRKG2 loss of function that may be responsible for disproportionate dwarfism at the molecular level. Microarray technology was used to compare growth plate cartilage gene expression in dwarf versus unaffected Angus cattle to identify putative downstream targets of PRGK2. Pathway enrichment of 1284 transcripts (nominal p CEBPA, BRCA1, BUB1, CD58, and VDR by real-time PCR (p This article is from BMC Research Notes 8 (2015): 177, doi:10.1186/s13104-015-1136-6. Posted with permission.</p

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

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