Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:The goal of this study is to investigate the cell type-specific targets of miR-126-3p in human lung microvascular endothelial cells (HLMVEC). Following the transfections of HLMVEC with non-targeting negative controls, miR-126 mimics, or miR-126 antisense inhibitors, we calculated the copy number concentration of miR-126 in each sample and performed genome-wide RNA sequencing. Plotting the gene expression data for each transfection condition (Scramble, 126-OE and 126-KD) against their respective miR-126 concentrations, we performed a linear regression analysis to discover genes that were the most sensitive to changes in miR-126 levels. We identified 1258 genes that were upregulated and 1436 genes that were downregulated by miR-126-3p. Further comparison between the downregulated genes in HLMVEC and targets predicted by online databases including TargetScan and miRDB revealed 6 genes as potential direct targets of miR-126-3p. Our study is the first to report targets of miR-126-3p in HLMVEC and demonstrate the effect of miR-126 level alteration on the HLMVEC global transcriptome. These results add to the diverse functions of miR-126-3p in different endothelial cell types and provide basis for the development of cell type-specific treatment for lung diseases.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:"The analysis was designed to search for putative seed sequences for miR-126 in the 3’UTR of up-regulated genes. ECs were co-transfected with anti-miR-126 and a plasmid vector bearing the GFP-coding sequence and three complementary sites for miR-126 downstream. As a consequence of the presence of miR-126 binding sites the GFP expression was under the control of miR-126. Fluorescente cells were the cells transfected with anti-miR-126 oligo. MiR-126 target enrichment analysis was performed on the basis of the Miranda database, as provided by Diana miRGen. Significance was estimated on the basis of hyper geometric distribution p-values comparing the occurrence of miR targets in the signature with respect to the universe, defined as the genes expressed in HUVEC. Frequency of log 2 ratio between the subset of mir-126 targets and the genes which are not targets of miR-126 were plotted, highlighting enrichment of the target of miR in differentially regulated genes. "