Project description:Hippo effectors Yap and Taz, Vgll1-4 and Tead 1-4 transcription factors have been linked to satellite cell/ myoblast proliferation and differentiation, muscle function and disease. The molecular function of Vgll3 in skeletal muscle stem cells was investigated using analysis of gene expression by microarrays to determine Vgll3 mediated gene expression changes and compare those to previously determined Taz and Yap mediated changes. Satellite cell-derived myoblasts were transduced with Vgll3 retrovirus for 24h or 48h, with empty retrovirus vector as control. Triplicate microarray analysis of empty vector controls and Vgll3 transgenic primary myoblasts were conducted.
Project description:Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. Here we describe a novel Caenorhabditis elegans nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors.
Project description:Plutella xylostella is the major cosmopolitan pest of brassica and other crucifer crops, the larval midgut of which is a dynamic organ that interfaces with a divers earray of physiological and toxicological processes. The draft sequence of the P.xylostella genome was recently released, but its annotation remains challenging because of the low coverage of this branch of life.Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirm or refute protein prediction, which has been termed proteogenomics. In this study we carried out an in-depth proteogenomic analysis using shotgun HPLC-ESI-MS/MS approach with a multi-algorithme pipeline to complement genome annotation in the P.xylostella larval midgut.
Project description:In total 3 mice brain tissues were used for this experiment. Tissue from each mouse brain was divided for immunoprecipitation with 5ug of either rabbit anti-TDP-43 antibody (Abcam) or normal rabbit IgG (Sigma) .The antibodies were first incubated with the lysate overnight at 4 degrees C, after which 50 uL of protein G Dynabeads(tm) (Invitrogen(tm)) added and the solution incubated for 1 hour at 4 degrees C with rotation. Following several washing with washing buffer (Invitrogen(tm)), the protein-RNA complex was eluted from 20 uL of bead-protein complex using 10 uL of elution buffer (Invitrogen(tm)) and seperated on a 10% NuPage(tm) Bis-Tris gel (Invitrogen(tm)). RNA was isolated from the remaining 30 uL of protein-bead complex using TRIzol reagent (Invitrogen) followed by DNaseI treatment (Ambion). The TDP-43 immunoprecipitated RNA was converted to cDNA, fragmented and biotin labelled using WT cDNA synthesis & amplification kit and Terminal Labeling Kit (Affymetrix(tm)) for Affymetrix(tm) GeneChip(tm) Mouse Gene 1.0 ST and 3'-IVT expression analysis kit (Affymetrix(tm)) for GeneChip(tm) Mouse 430_2 arrays according to the standard protocol. Labelled RNA was hybridised to arrays in a hybridization oven (Affymetrix(tm)) at 45 degrees C rotating at 60 rpm for 17 hours and scanned using the Affymetrix(tm) GeneChip Scanner. Successful hybridisation to the microarray was checked using Expression Console software (Affymetrix(tm)) and the data (.CEL files) transferred to Partek Genomics Suite for statistical analysis. TDP-43 and IgG immunoprecipitated RNA samples were each hybridised to 3 GeneChip Mouse Gene 1.0 ST and 3 GeneChip Mouse 430 (n = 6 for each group).
Project description:EOS1 is required for tolerance to oxidative stress in Saccharomyces cerevisiae; mutants are defective in the gene sensitive to hydrogen peroxide and tolerant to tunicamycin. To clarify the function of Eos1, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress the hydrogen peroxide-sensitive eos1 mutation. We identified such a gene as IZH2, which has previously been reported to be a Zap1-regulated gene. However, the EOS1 and IZH2 genes do not themselves appear to be functionally interchangeable. Double disruption of the EOS1 and IZH2 genes yielded a slow-growth phenotype, suggesting that the two proteins are involved in related cellular processes. DNA microarray analysis revealed decreased expression of Zap1-regulated genes in the eos1-deletion mutant (delta eos1). Thus, it is likely that Eos1 is involved in zinc homeostasis. Yeast cells were cultivated in YPD at 30 oC with shaking at 150 rpm, and then the cells were harvested at the log phase (OD600=1.0). Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were used as the DNA microarrays. Statistical analysis of the expression data were performed using GeneSpring ver. 7.3.1 (Agilent Technologies, Palo Alto, CA, USA) based on the gene expression data from two independent experiments.
Project description:Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.
Project description:Background Pannexin1 (Panx1), an ATP release channel, is present in most mammalian tissues, but the role of Panx1 in health and disease is not fully understood. Panx1 may serve to modulate AKI; ATP is a precursor to adenosine and may function to block inflammation, or ATP may act as a danger-associated molecular pattern and initiate inflammation.Methods We used pharmacologic and genetic approaches to evaluate the effect of Panx1 on kidney ischemia-reperfusion injury (IRI), a mouse model of AKI.Results Pharmacologic inhibition of gap junctions, including Panx1, by administration of carbenoxolone protected mice from IRI. Furthermore, global deletion of Panx1 preserved kidney function and morphology and diminished the expression of proinflammatory molecules after IRI. Analysis of bone marrow chimeric mice revealed that Panx1 expressed on parenchymal cells is necessary for ischemic injury, and both proximal tubule and vascular endothelial Panx1 tissue-specific knockout mice were protected from IRI. In vitro, Panx1-deficient proximal tubule cells released less and retained more ATP under hypoxic stress.Conclusions Panx1 is involved in regulating ATP release from hypoxic cells, and reducing this ATP release may protect kidneys from AKI.
Project description:OE21, OE33 and AGS cell lines were selected for progressive resistance to oxaliplatin, cisplatin and docetaxel. Selection began at a drug dose that was 20 fold less than the IC50 concentration. Cells were grown at the same drug concentration over 4 passages and then cell viability tests performed.Drug concentrations were increased 2 to 4 - fold until the IC50 daughter/ IC50 parental ≥ 3.The panel of drug resistant cell lines generated in this way were AGSCIS5, AGSOX8, AGSDOC6, OE33CIS4, OE33OX4 and OE21OX4 with the subscript denoting the drug and final concentration of drug(μM) that cells were exposed to. Gene expression profiling was performed using Affymetrix Exon 1.0 ST Arrays ) with 3 independent replicates per cell line from three different passages