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Physiol Genomics [journal]
- Scuba diving induces nitric oxide synthesis and the expression of inflammatory and regulatory genes of the immune response in neutrophils. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jul 8.
Objective: Scuba diving, characterized by hyperoxia and hyperbaria, could increase reactive oxygen species production which acts as signalling molecules to induce adaptation against oxidative stress. The aim was to study the effects of scuba diving immersion on neutrophil inflammatory response, the induction of oxidative damage and the NO synthesis. Design: Nine male divers performed a dive at 50 m depth for a total time of 35 min. Blood samples were obtained before diving at rest, after diving, and 3 h after the diving session. Measurements: Markers of oxidative and nitrosative damage, nitrite and the gene expression of genes related with the synthesis of nitric oxide and lipid mediators, cytokine synthesis and inflammation were determined in neutrophils. Results: The mRNA levels of genes related with the inflammatory and immune response of neutrophils, except TNF-α, myeloperoxidase and toll-like receptor (TLR) 2, significantly increased after the recovery period respect to pre-dive and post dive levels. NFκβ, IL-6 and TLR4 gene expression reported significant differences immediately after diving respect to the pre-dive values. Protein-nitrotyrosine levels significantly rose after diving and remained high during recovery, whereas no significant differences were reported in malondialdehyde. Neutrophil nitrite levels as indicative of iNOS activity progressively increased after diving and recovery. The iNOS protein levels maintained the basal values in all situations. Conclusion: Scuba diving which combines hyperoxia, hyperbaria and acute exercise induces nitrosative damage with increased nitrotyrosine levels and an inflammatory response in neutrophils.
- Genome-wide analysis of gestational gene-environment interactions in the developing kidney. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jul 8.
The G-protein coupled bradykinin B2 receptor (Bdkrb2) plays an important role in regulation of blood pressure under conditions of excess salt intake. Our previous work has shown that Bdkrb2 also plays a developmental role since Bdkrb2(-/-) embryos, but not their wild type or heterozygous littermates, are prone to renal dysgenesis in response to gestational high salt intake. Although impaired terminal differentiation and apoptosis are consistent findings in the Bdkrb2(-/-) mutant kidneys, the developmental pathways downstream of gene-environment interactions leading to the renal phenotype remain unknown. Here, we performed genome-wide transcriptional profiling on embryonic kidneys from salt-stressed Bdkrb2(+/+) and Bdkrb2(-/-) embryos. The results revealed significant alterations in key pathways regulating Wnt signaling, apoptosis, embryonic development, and cell-matrix interactions. In silico analysis revealed that nearly 12% of differentially regulated genes harbor one or more Pax2 DNA-binding sites in their promoter region. Further analysis showed that metanephric kidneys of salt-stressed Bdkrb2(-/-) have a significant downregulation of Pax2 gene expression. This was corroborated in Bdkrb2(-/-);Pax2(GFP+/tg) mice, demonstrating that Pax2 transcriptional activity is significantly repressed by gestational salt-Bdkrb2 interactions. We conclude that gestational gene (Bdkrb2) and environment (salt) interactions cooperate to impact gene expression programs in the developing kidney. Suppression of Pax2 likely contributes to the defects in epithelial survival, growth, and differentiation in salt-stressed BdkrB2(-/-) mice.
- Intima Modifier Locus 2 Controls Endothelial Cell Activation and Vascular Permeability. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jul 1.
Carotid intima formation is a significant risk factor for cardiovascular disease. C3H/FeJ (C3H/F) and SJL/J (SJL) inbred mouse strains differ in susceptibility to immune and vascular traits. Using a congenic approach we demonstrated that the Intima modifier 2 (Im2) locus on chromosome 11 regulates leukocyte infiltration. We sought to determine whether inflammation was due to changes in circulating immune cells or activation of vascular wall cells in genetically pure Im2 (C3H/F.SJL.11.1) mice. Complete blood counts showed no differences in circulating monocytes between C3H/F and C3H/F.SJL.11.1 compared to SJL mice. Aortic vascular cell adhesion molecule-1 (VCAM-1) total protein levels were dramatically increased in SJL and C3H/F.SJL.11.1 compared to C3H/F mice. Immunostaining of aortic endothelial cells (EC) showed a significant increase in VCAM-1 expression in SJL and C3H/F.SJL.11.1 compared to C3H/F under steady flow conditions. Immunostaining of EC membranes revealed a significant decrease in EC size in SJL and C3H/F.SJL.11.1 versus C3H/F in regions of disturbed flow. Vascular permeability was significantly higher in C3H/F.SJL.11.1 compared to C3H/F. Our results indicate that Im2 regulation of leukocyte infiltration is mediated by EC inflammation and permeability. RNA sequencing and pathway analyses comparing genes in the Im2 locus to C3H/F provides insight into candidate genes that regulate vascular wall inflammation and permeability highlighting important genetic mechanisms that control vascular intima in response to injury.
- Discovery and refinement of muscle weight QTLs in B6 x D2 advanced intercross mice. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jun 24.
The genes underlying variation in skeletal muscle mass are poorly understood. Although many quantitative trait loci (QTLs) have been mapped in crosses of mouse strains, the limited resolution inherent in these conventional studies has made it difficult to reliably pinpoint the causal genetic variants. The accumulated recombination events in an advanced intercross line (AIL), in which mice from two inbred strains are mated at random for several generations, can improve mapping resolution. We demonstrate these advancements in mapping QTLs for hindlimb muscle weights in an AIL (n=832) of the C57BL/6J (B6) and DBA/2J (D2) strains (generations F8 and F9-F13). We mapped muscle weight QTLs using the high-density MegaMUGA SNP panel. The QTLs highlight the shared genetic architecture of four hindlimb muscles, and suggest that the genetic contributions to muscle variation are substantially different in males and females, at least in the B6D2 lineage. Out of the 15 muscle weight QTLs identified in the AIL, 9 overlapped the genomic regions discovered in an earlier B6D2 F2 intercross. Mapping resolution, however, was substantially improved to a median QTL interval of 12.5 Mb. Subsequent sequence analysis of the QTL regions revealed 20 genes with nonsense or potentially damaging missense mutations. Further refinement of the muscle weight QTLs using additional functional information, such as gene expression differences between alleles, will be required to discern the causal genes.
- Assessment of flow distribution in the mouse fetal circulation at late gestation using high frequency Doppler ultrasound. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jun 24.
This study was to evaluate the flow distribution in the mouse fetal circulation at late gestation using high frequency ultrasound. Twelve fetuses (E17.5) from twelve pregnant CD1 mice were studied using 40MHz ultrasound to assess the flow in eleven vessels based on Doppler measurements of blood velocity and M-mode measurements of diameter. Specifically, the intra-hepatic umbilical vein (UVIH), ductus venosus (DV), foramen ovale (FO), ascending aorta (AA), main pulmonary artery (MPA), ductus arteriosus (DA), descending thoracic aorta (DTA), common carotid artery (CCA), inferior vena cava (IVC), and right and left superior vena cavae (RSVC, LSVC) were examined, and anatomically confirmed by micro-CT. The mouse fetal circulatory system was found similar to that of the humans in terms of the major circuit and three shunts, but characterized by bilateral superior vena cavae and a single umbilical artery. The combined cardiac output (CCO) was 1.22±0.05 ml/min, with the left ventricle (flow in AA) contributing 47.8±2.3% and the right ventricle (flow in MPA) 52.2±2.3%. Relative to the CCO, the flow percentages were 13.6±1.0% for the UVIH, 10.4±1.1% for the DV, 35.6±2.4% for the DA, 41.9±2.6% for the DTA, 3.8±0.3% for the CCA, 29.5±2.2% for the IVC, 12.7±1.0% for the RSVC and 9.9±0.9% for the LSVC. The calculated flow percentage was 16.6±3.4% for the pulmonary circulation and 31.2±5.3% for the FO. In conclusion, the flow in mouse fetal circulation can be comprehensively evaluated with ultrasound. The baseline data of the flow distribution in normal mouse fetus serves as the reference range for future studies.
- Quantitative genomics of voluntary exercise in mice: transcriptional analysis and mapping of expression QTL in muscle. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jun 17.
Motivation and ability both underlie voluntary exercise, each with a potentially unique genetic architecture. Muscle structure and function are one of many morphological and physiological systems acting to simultaneously determine exercise ability. We generated a large (n = 815) advanced intercross line of mice (G4) derived from a line selectively bred for increased wheel running (high runner) and the C57BL/6J inbred strain. We previously mapped quantitative trait loci (QTL) contributing to voluntary exercise, body composition, and changes in body composition as a result of exercise. Using brain tissue in a subset of the G4 (n = 244), we have also previously reported expression QTL (eQTL) colocalizing with the QTL for the higher-level phenotypes. Here, we examined the transcriptional landscape of hind limb muscle tissue via global mRNA expression profiles. Correlations revealed an ~1,168% increase in significant relationships between muscle transcript expression levels and the same exercise and body composition phenotypes examined previously in the brain. The exercise trait most often significantly correlated with gene expression in the brain was running duration while in the muscle it was maximum running speed. This difference may indicate that time spent engaging in exercise behavior may be more influenced by central (neurobiological) mechanisms, while intensity of exercise may be largely controlled by peripheral mechanisms. Additionally, we used subsets of cis-acting eQTL, colocalizing with QTL, to identify candidate genes based on both positional and functional evidence. We discuss three plausible candidate genes (Insig2, Prcp, Sparc) and their potential regulatory role.
- Progesterone receptor (PGR) dependent regulation of genes in the oviducts of female mice. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jun 10.
Oviducts play a critical role in gamete and embryo transport, as well as supporting early embryo development. Progesterone receptor (PGR) is a transcription factor highly expressed in oviductal cells, while its activating ligand, progesterone, surges to peak levels as ovulation approaches. Progesterone is known to regulate oviduct cilia beating and muscular contractions in vitro, but how PGR may mediate this in vivo is poorly understood. We used PGR null mice to identify genes potentially regulated by PGR in the oviducts during the periovulatory period. Histologically, oviducts from PGR null mice showed no gross structural or morphological defects compared to normal, littermates. However, microarray analysis of oviducts at 8 h post-hCG revealed over 1000 PGR-dependent genes. Ten genes were selected for validation using reverse-transcription polymerase chain reaction (RT-PCR) based on their potential roles in oocyte/embryo transport and support. Eight genes were confirmed to be down-regulated (Adamts1, Itga8, Edn3, Prlr, Ptgfr, Des, Myocd and Actg2) and one up-regulated (Agtr2) in PGR null oviducts. Expression of these genes was also assessed in oviducts of naturally cycling mice during ovulation, day 1 and day 4 of pregnancy. Adamts1, Itga8, Edn3, Prlr and Ptgfr were significantly up-regulated in oviducts at ovulation/mating. However, most genes showed basal levels of expression at other times. The exceptions were Prlr and Ptgfr which showed pulsatile increases on day 1 and/or day 4 of pregnancy. This is the first, comprehensive study to elucidate putative PGR-regulated genes in the oviduct and reveals key downstream targets potentially mediating oocyte and embryo transport.
- Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jun 3.
Although, cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function and impaired lung anti-oxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells, with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24-h oxidative stress induced by 15 µM 2, 3-dimethoxy-1, 4-naphtoquinone (DMNQ). In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared to NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.
- Identification of reference genes for RT-qPCR in ovine mammary tissue during late-pregnancy, lactation and in response to maternal nutritional programming. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Jun 3.
The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRP3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analysing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.
- Mechanisms for the adverse effects of late gestational increases in maternal cortisol on the heart revealed by transcriptomic analyses of the fetal septum. [JOURNAL ARTICLE]
- Physiol Genomics 2014 May 27.
We have previously shown in sheep that 10 days of modest chronic increase in maternal cortisol resulting from maternal infusion of cortisol (1 mg/kg/d) caused fetal heart enlargement and Purkinje cell apoptosis. In subsequent studies we extended the cortisol infusion to term, finding a dramatic incidence of stillbirth in the pregnancies with chronically increased cortisol. To investigate effects of maternal cortisol on the heart, transcriptomic analyses were performed on the septa using ovine microarrays and Webgestalt and Cytoscape programs for pathway inference. Analyses of the transcriptomic effects of maternal cortisol infusion for 10days (130d-cortisol vs 130d-control), or ~25 days (140d-cortisol vs 140d-control) and of normal maturation (140d-control vs 130d- control) were performed. Gene ontology terms related to immune function and cytokine actions were significantly overrepresented as genes altered by both cortisol and maturation in the septa. After 10 days of cortisol, growth factor and muscle cell apoptosis pathways were significantly overrepresented, consistent with our previous histologic findings. In the term fetuses ( ~25 days of cortisol) nutrient pathways were significantly overrepresented, consistent with altered metabolism and reduced mitochondria. Analysis of mitochondrial number by mitochondrial DNA expression confirmed a significant decrease in mitochondria. The metabolic pathways modeled as altered by cortisol treatment to term were different from those modeled during maturation of the heart to term, and thus changes in gene expression in these metabolic pathways may be indicative of the fetal heart pathophysiologies seen in pregnancies complicated by stillbirth, including gestational diabetes, Cushing's disease and chronic stress.