- Establishing the involvement of the novel gene AGBL5 in retinitis pigmentosa by whole genome sequencing. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00101.2016
- While more than 250 genes are known to cause inherited retinal degenerations (IRD), nearly 40-50% of families have the genetic basis for their disease unknown. In this study we sought to identify the...
While more than 250 genes are known to cause inherited retinal degenerations (IRD), nearly 40-50% of families have the genetic basis for their disease unknown. In this study we sought to identify the underlying cause of inherited retinal degeneration (IRD) in a family by whole genome sequence (WGS) analysis. Clinical characterization including standard ophthalmic examination, fundus photography, visual field testing, electroretinography, and review of medical and family history was performed. WGS was performed on affected and unaffected family members using Illumina HiSeq X10. Sequence reads were aligned to hg19 using BWA-MEM and variant calling was performed using GATK. The called variants were annotated with SnpEff v4.11, PolyPhen v2.2.2, and CADD v1.3. Copy number variations (CNVs) were called using Genome STRiP (svtoolkit 2.00.1611) and SpeedSeq software. Variants were filtered to detect rare potentially deleterious variants segregating with disease. Candidate variants were validated by dideoxy sequencing. Clinical evaluation revealed typical adolescent onset recessive retinitis pigmentosa (arRP) in affected members. WGS identified about 4 million variants in each individual. Two rare and potentially deleterious compound heterozygous variants p.Arg281Cys and p.ARg487* were identified in the gene ATP/GTP binding protein like 5 (AGBL5) as likely causal variants. No additional variants in IRD genes that segregated with disease were identified. Mutation analysis confirmed the segregation of these variants with the IRD in the pedigree. Homology models indicated destabilization of AGBL5 due to the p.Arg281Cys change. Our findings establish the involvement of mutations in AGBL5 in RP and validate the WGS variant filtering pipeline we designed.
- Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00100.2016
- NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypert...
NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its c-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted c-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one c-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription and vesicle trafficking.
- Muscle dysfunction in a zebrafish model of Duchenne muscular dystrophy. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00088.2016
- Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. Howev...
Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days post fertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were 3 to 5-fold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening.
- Reactive oxygen species and bacterial biofilms in diabetic wound healing. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00066.2016
- Chronic wounds are a common and debilitating complication for the diabetic population. It is challenging to study the development of chronic wounds in hu-man patients; by the time it is clear that a ...
Chronic wounds are a common and debilitating complication for the diabetic population. It is challenging to study the development of chronic wounds in hu-man patients; by the time it is clear that a wound is chronic, the early phases of wound healing have passed and can no longer be studied. Because of this limita-tion, mouse models have been employed to better understand the early phases of chronic wound formation. In the past few years, a series of reports have high-lighted the importance of reactive oxygen species and bacterial biofilms in the development of chronic wounds in diabetics. We review these recent findings and discuss mouse models that are being utilized to enhance our understanding of these potentially important contributors to chronic wound formation in diabetic patients.
- Strain survey and genetic analysis of vasoreactivity in mouse aorta. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00054.2016
- Understanding the genetic influence on vascular reactivity is important for identifying genes underlying impaired vascular function. The purpose of this study was to characterize the genetic contribu...
Understanding the genetic influence on vascular reactivity is important for identifying genes underlying impaired vascular function. The purpose of this study was to characterize the genetic contribution to intrinsic vascular function and to identify loci associated with phenotypic variation in vascular reactivity in mice. Concentration response curves to phenylephrine (PE), potassium chloride (KCl), acetylcholine (ACh), and sodium nitroprusside (SNP) were generated in aortic rings from male mice (12-wk old) from 27 inbred mouse strains. Significant strain-dependent differences were found for both maximal responses and sensitivity for each agent, except for SNP Max (%). Strain differences for maximal responses to ACh, PE, and KCl varied by 2-5 fold. Based on these large strain differences, genome-wide association mapping (GWAS) was performed to identify loci associated with variation in responses to these agents. GWAS for responses to ACh identified 4 significant and 19 suggestive loci. Several suggestive loci for responses to SNP, PE and KCl (including one significant locus for KCl EC50) were also identified. These results demonstrate that intrinsic endothelial function, and more generally vascular function, is genetically determined and associated with multiple genomic loci. Furthermore, these results are supported by the finding that several genes residing in significant and suggestive loci for responses to ACh were previously identified in rat and/or human QTL/GWAS for cardiovascular disease. This study represents the first step toward the unbiased comprehensive discovery of genetic determinants that regulate intrinsic vascular function, particularly endothelial function.
- Transcriptomic differences in intra-abdominal adipose tissue in extremely obese adolescents with different stages of NAFLD. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00020.2016
- CONCLUSIONS: Unique transcriptomic differences exist in IAT from severely obese adolescents with distinct stages of NAFLD, providing an important resource for identifying potential novel therapeutic targets for childhood NASH.
- Lifestyle and DNA base composition in polychaetes. [Journal Article]
- PGPhysiol Genomics 2016 Oct 7; :physiolgenomics.00018.2016
- A comparative analysis of polychaete species, classified as motile and low-motile forms, highlighted that the former were characterized not only by a higher metabolic rate (MR), but also by a higher ...
A comparative analysis of polychaete species, classified as motile and low-motile forms, highlighted that the former were characterized not only by a higher metabolic rate (MR), but also by a higher genomic GC content. The fluctuation of both variables was not affected by the phylogenetic relationship of the species. Thus, present results further support that a very active lifestyle affects at the same time MR and GC, showing an unexpected similarity between invertebrates and vertebrates. In teleost, indeed, a similar pattern has been also observed comparing migratory and non-migratory species. Till now a cause-effect link between MR and GC has not been proved yet, but the fact that the two variables are significantly linked in all the organisms so far analyzed is, most probably, of relevant biological and evolutionary meaning. Present results fit very well within the frame of the metabolic rate hypothesis proposed to explain the DNA base composition variability among organisms. On the contrary, the thermostability hypothesis was not supported. At present, no data about the recombination rate in polychaetes were available to test the biased gene conversion (BGC) hypothesis.
- Microbial short chain fatty acid metabolites lower blood pressure via endothelial G-protein coupled receptor 41. [Journal Article]
- PGPhysiol Genomics 2016 Sep 23; :physiolgenomics.00089.2016
- Short chain fatty acid (SCFA) metabolites are byproducts of gut microbial metabolism that are known to affect host physiology via GPCRs. We previously showed that an acute SCFA bolus decreases blood ...
Short chain fatty acid (SCFA) metabolites are byproducts of gut microbial metabolism that are known to affect host physiology via GPCRs. We previously showed that an acute SCFA bolus decreases blood pressure (BP) in anesthetized mice, an effect mediated via Gpr41. In this study, our aims were to identify the cellular localization of Gpr41 and to determine its role in BP regulation. We localized Gpr41 to the vascular endothelium using RT-PCR: Gpr41 is detected in intact vessels, but is absent from denuded vessels. Furthermore, using pressure myography we confirmed that SCFAs dilate resistance vessels in an endothelium-dependent manner. Since we previously found that Gpr41 mediates a hypotensive response to acute SCFA administration, we hypothesized that Gpr41 knockout (KO) mice would be hypertensive. Here, we report that Gpr41 knockout (KO) mice have isolated systolic hypertension compared to wild-type (WT) mice; diastolic BP was not different between WT and KO. Older Gpr41 KO mice also exhibited elevated pulse wave velocity, consistent with a phenotype of systolic hypertension - however, there was no increase in ex vivo aorta stiffness (measured by electromechanical tensile testing). Plasma renin concentration was also similar in KO and WT mice. The systolic hypertension in Gpr41 KO is not salt-sensitive, as it is not significantly altered on either a high or low-salt diet. In sum, these studies suggest that endothelial Gpr41 lowers baseline BP, likely by decreasing active vascular tone without altering passive characteristics of the blood vessels, and that Gpr41 KO mice have hypertension of a vascular origin.
- Effects of dietary forage and calf starter on ruminal pH and transcriptomic adaptation of the rumen epithelium in Holstein calves during the weaning transition. [Journal Article]
- PGPhysiol Genomics 2016 Sep 23; :physiolgenomics.00086.2016
- We investigated the relationship between ruminal pH and transcriptomic adaptation of the rumen epithelium (RE) of calves fed calf starter with and without forage during the weaning transition. Holste...
We investigated the relationship between ruminal pH and transcriptomic adaptation of the rumen epithelium (RE) of calves fed calf starter with and without forage during the weaning transition. Holstein calves were assigned to groups fed calf starter either with forage (HAY group, n = 3) or without forage (CON group, n = 4). Ruminal pH was measured continuously, and rumen fluid and epithelium were collected 3 weeks after weaning. mRNA expression profiles of the RE were examined by one-color microarray. Differentially expressed genes (DEGs) were investigated using the ingenuity pathway analysis (IPA). Mean and maximum ruminal pH were significantly (P < 0.05) higher, and the duration of pH < 5.8 during 1 day was significantly (P < 0.05) shorter, in the HAY group. The proportion of ruminal acetate and the acetate-to-propionate ratio were significantly (P < 0.05) lower in the CON group. DEGs encoding transcription regulators (SREBP1), insulin-like growth factor binding proteins (IGFBP7 and CTGF), ketogenic enzymes (HMGCL, BDH1, and BDH2), and a transporter (SLC16A3) were identified (P < 0.05) between the two groups. A growth factor (TGFB1) and signaling pathway (EGF and EGFR) were activated as upstream regulators. These results suggest that dietary forage alleviates ruminal acidosis, and the decrease in ruminal pH may damage the RE, leading to changes in gene expression to repair the damage. Furthermore, rumen development may be regulated by growth factor (TGFB1) and signaling pathways (EGF and IGFBP) for adaptation to feeding on calf starter with and without forage during the weaning transition.
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- A genome-wide association study of plasma resistin levels identified rs1423096 and rs10401670 as possible functional variants in the Japanese population. [Journal Article]
- PGPhysiol Genomics 2016 Sep 23; :physiolgenomics.00040.2016
- Resistin is a cytokine inducing insulin resistance in mice. We previously identified single nucleotide polymorphisms (SNPs) at -420 (rs1862513) and -358 (rs3219175) located in the human resistin gene...
Resistin is a cytokine inducing insulin resistance in mice. We previously identified single nucleotide polymorphisms (SNPs) at -420 (rs1862513) and -358 (rs3219175) located in the human resistin gene (RETN) promoter as strong determinants for circulating resistin in the Japanese population. The objective was to identify additional functional variants for circulating resistin. We conducted a genome-wide association study in 448 Japanese subjects. A peak association signal was found on chromosome 19 where RETN is located. The top-hit SNP was SNP-358 G>A, followed by rs1423096 C>T, SNP-420 C>G, and rs10401670 C>T (P=5.39×10(-47), 1.81×10(-22), 2.09×10(-16), and 9.25×10(-15), respectively). Meta-analysis including another two independent general Japanese populations showed that circulating resistin was most strongly associated with SNP-358, followed by SNP-420, rs1423096, and rs10401670. Rs1423096 and rs10401670 were located in the 3'-region of RETN and were in strong linkage disequilibrium. Although these SNPs were also in linkage disequilibrium with the promoter SNPs, conditional and haplotype association analyses identified rs1423096 and rs10401670 as independent determinants for circulating resistin. Functionally, nuclear proteins specifically recognized T but not C at rs10401670 as evidenced by an electrophoretic mobility shift assay. The promoter activity of a luciferase reporter with T at either rs1423096 or rs10401670 was lower than that with C in THP-1 human monocytes. Therefore, rs1423096 and rs10401670, in addition to SNP-420 and SNP-358, were identified as possible functional variants affecting circulating resistin by the genome-wide search in the Japanese population.