Project description:Objective - The TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease (CAD) in humans. The lipid associated SNPs identified by genome-wide association studies (GWAS) are located ~ 30 kb downstream from TRIB1 suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits. Methods & Results - Characterization of the risk locus reveals that it encompasses a gene, TRIB1 associated locus (TRIBAL) comprised of a well conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and re-sequencing identified a single nucleotide polymorphism (SNP), rs2001844, within the promoter region that associates with increased plasma triglycerides, reduced HDL-C and CAD risk. Furthermore, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs (lncRNA), including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes. Conclusions - These studies demonstrate an interplay between a novel locus,TRIBAL, and TRIB1. TRIBAL is located in the GWAS identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression and associates with plasma triglyceride concentrations. HepG2 hepatoma cells were stably infected with TRIBAL1 or no insert carrying lentiviruses
Project description:Shigella is an intracellular bacterial pathogen known to activate numerous innate immunity and inflammatory signaling pathways. Many of these pathways are also involved in mTOR signaling. We used HEK293T cell cultures and sought to understand how Shigella infection and rapamycin treatment affect the transcriptome and in particular whether there were commonly affected pathways in the two experimental conditions. 8 samples were used - 2 control that were uninfected/untreated -- 1 sample lost due to RNA degradation, 3 infected with Shigella for 4 hours, and 3 treated with Rapamycin (50 ug/mL) for 4 hours. All samples were from independent experiments. We used the program AltAnalyze to perform pairwise comparisons between Uninfected with Infected, and Uninfected with Rapamycin using default parameters.
Project description:Genome-wide transcriptome analyses of transgenic mice, that express Cre recombinase flanked by mutated estrogen receptors (MerCreMer; mcm), and carry loxP-flanked sequences of p53 and Mdm2 was performed in the presence and absence of Tamoxifen.<br>The molecular mechanisms underlying heart failure remain poorly understood. As such, identifying the factors which effectively maintain cardiac tissue homeostasis is of great scientific and clinical importances. The tumor suppressor Trp53 (p53) inhibits cell growth after acute stress by regulating gene transcription. The mammalian genome contains hundreds of p53 binding sites. However, whether p53 participates in the regulation of cardiac tissue homeostasis under normal conditions is not known. To examine the physiologic consequences of p53 and Mdm2 ablation in adult cardiomyocytes in vivo, cardiac morphology and function were assessed in conditional mutant mice.
Project description:Measles virus infects serum activated airway epithelial cells and many adenocarcinoma cell lines. A microarray analysis was performed on virus permissive versus non-permissive cells. Membrane protein genes that were upregulated in permissive cells were tested as receptor/entry factors. Membrane protein genes that were upregulated in smooth airway epithelial cells (SAEC) following growth in 10% fetal calf serum that made the cell line permissive to measles virus were identified. Membrane protein genes that were upregulated in adenocarcinoma cells that were permissive to wild type measles virus infection were identified. [SAEC]: Airway cells (SAEC) grown in serum free media (SAGM) were purchaced from Lonza. Half the cells were cultured in SAGM, the other half were transferred into Dulbecco's 10% fetal calf serum for 24 hrs. RNA was harvested from the cells by the Qiagen RNAeasy [Adenocarcinoma cells]: MCF7, MDA-MB-468, T47D, NCI-H358, NCI-H125, MGH24 cells were permissive and A549 and MDA-MB-231 cells were non-permissive.
Project description:Methylation profiling of CpG Island Methylation of genomic DNA samples extracted from placentae of Intra-Uterine Growth Restricted newborns and gestational age matched controls. These data can be used for methylation comparisons between these two types of samples. The results of such comparisons can be used to generate hypotheses on the involvement of DNA methylation variation in placenta development and fetal growth. Two-condition Methyl DNA Immunoprecipitation (MeDIP) experiment, IUGR vs. Control placenta samples. Biological replicates: 8 control, 8 IUGR cases.
Project description:We used microarrays to detail the global changes in gene expression resulting from miR-95 overexpression PC3 prostate cells stably overexpressing control miR or miR-95 were processed for Affymetrix gene array
Project description:The hepatic endoplasmic reticulum contains a series of enzymes that oxidize and conjugate lipid and steroids. Together, these enzymes form a molecular machine that plays key roles in the metabolism of both endogenous and xenobiotic compounds. To characterize this molecular machine, we used quantitative proteomics to assess the frequency of occurrence of detected peptides within each primary sequence, leading to the assessment of the relative abundances of 137 of these proteins. These 137 proteins include over 38 different cytochrome P450s, 11 glucuronosyltransferases, and 9 carboxylesterases. Our sensitive approach was able to detect P450 allelic isoforms which differ by only a single amino acid and clearly resolved 4 splice variants of the glucuronosyltransferases. We identified a cytosolically-exposed DID motif for 3 cytochrome P450s that were located with high abundance in the Golgi apparatus as well the lack of a C-terminal HXEL motif for the sole carboxylesterase highly abundant in the Golgi. Using gene expression microarrays, we also characterized the hepatic transcriptome, and a comparison of proteomics and transcriptomics indicated a requirement for both technologies in order to provide insight into protein families of drug detoxification. In this way, a major hurdle of hepatotoxicity related to drug development may be overcome. Experiment Overall Design: Four replicate Sprague-Dawley rats were assessed, each on an individual microarray.
Project description:In many mammals, halogenated aromatic hydrocarbon (HAH) exposure causes wasting syndrome, defined as lethal weight loss as a result of severe and persistent hypophagia. The most potent HAH in causing wasting is 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), which exerts its toxic effects through the aryl hydrocarbon receptor (AHR) – a transcription factor. Because TCDD toxicity is thought to predominantly arise from dysregulation of AHR-transcribed genes, we hypothesized that wasting syndrome is due to TCDD-induced dysregulation of genes involved in regulation of food-intake. We therefore focused on the hypothalamus, as it is the regulatory center of food-intake and energy balance in the central nervous system. We profiled mRNA abundance in hypothalamic tissue from two rat strains with widely differing sensitivities to wasting syndrome: TCDD-sensitive Long-Evans rats and TCDD-resistant Han/Wistar rats, 23 hours after exposure to TCDD (100 μg/kg) or corn oil vehicle. We found that TCDD exposure caused minimal transcriptional dysregulation effects in the hypothalamus, with only 6 genes changed in Long-Evans rats and 15 genes in Han/Wistar rats. Two of the most dysregulated genes were Cyp1a1 and Nqo1, which are induced by TCDD across a wide range of tissues and are considered sensitive markers of TCDD exposure. The minimal response of the hypothalamic transcriptome to a lethal dose of TCDD at an early time-point suggests that the hypothalamus is not the predominant site of initial events leading to hypophagia and associated wasting. TCDD may affect feeding behaviour via events upstream or downstream of the hypothalamus, and further work is required to evaluate this at the level of individual hypothalamic nuclei and subregions. Two strains, each with drug-treated vs vehicle-control
Project description:2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities across various experimental models. In rodents alone, there is huge divergence in the toxicological response across species, between strains within a species and even between sexes within a strain. This difference in sensitivities between sexes has been characterized in multiple rodent models, however with contrasting results: the severity of toxicity is greater in female rats than in males while male mice and guinea pigs are more sensitive to the effects of TCDD than females. While the presence of estrogens or androgens has a known impact on response, the specific transcriptional events that cause this difference remain unclear. We sought to characterize the transcriptional environment of male and female C57BL/6 mice treated with 500 μg/kg TCDD and followed across a time-course. This dose was chosen as it is greater than the LD50 for male mice yet produces minimal effects in females. Additionally, evaluation along a time-course allows for the detection of specific changes that occur throughout the development of phenotypic toxicities. The transcriptional profile across the time-course was highly divergent between the sexes. Female TCDD-treated mice demonstrated a large number of altered transcripts as early as 6 hours following treatment, suggesting a large primary response, possibly indicative of the activation of numerous defense mechanisms. Conversely, male animals showed the greatest TCDD-mediated response 144 hours following exposure, potentially implicating significant secondary responses for the increased appearance of TCDD toxicities. Nr1i3 is statistically significantly altered at all time-points in the sensitive male animals. This mRNA encodes a transcription factor (constitutive androstane receptor; CAR) involved in the regulation of xenobiotic metabolism, as well as lipid metabolism, cell cycle and apoptosis. In particular, increased levels of CAR may result in induction of the anti-apoptotic gene Gadd45b and carboxylesterase Ces1d, both of which we identify as transcriptionally altered and may be responsible for phenotypic hepatic abnormalities, such as steatohepatitis and tumor formation. Further evaluation across studies of mice and rats into the role of Nr1i3 and associated genes are crucial to enhance our understanding of various TCDD-induced toxicities. Adult male and female C57BL/6 mice were treated by gavage with either 500 ug/kg TCDD in corn oil or corn oil vehicle alone. Animals were euthanized at either 6, 24, 72 or 144 hours after treatment and tissues were harvested. RNA was isolated from hepatic tissue and the transcriptome for each animal assayed on an individual microarray. Please note that 7 samples (out of total 65 samples) were identified as outliers and therefore the data were processed without the outliers as well. The normalized data without outliers were provided in the 'normalized_data_without_outliers.txt' file.