We hypothesized that altered extracellular osmolality per se could affect the transcriptome of the kidney inner medullary collecting duct (IMCD) cells, and hence it might change renal tubular function. The data sets of transcriptomics were incorporated into the "omic" data sets of metabolomics. Primary cultured IMCD cells of rat kidney were grown in hyperosmolar culture medium (640 mOsm/KgH2O) for 4 d, and then the cells were cultured in the medium with either reduced (300 mOsm/KgH2O) or the same osmolality for 1 or 2 d more.
Patterns of gene and metabolite define the effects of extracellular osmolality on kidney collecting duct.
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Analysis of DNA methylation and gene expression changes during regulated endothelial progenitor cells (EPCs) to outgrowth endothelial cells (OECs). Results provide information of DNA methylation and gene expression pattern during cord-blood derived EPCs differentiation. Taken together, we discovered specific set of genes regulated by hyper- and hypo-methylation during differentiation. Overall design: mRNA and MeDIP seq using total RNA and genomic DNA isolated from cord blood-derived EPCs and OECs.
Integrative analysis of DNA methylation and mRNA expression during differentiation of umbilical cord blood derived mononuclear cells to endothelial cells.
Specimen part, SubjectView Samples
Chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. HK-2 cells were cultured to confluence in 100mm dishes. Total RNA was extracted (QIAGEN, Valencia, CA, USA), and the concentration in the samples was measured using a Micro UV-Vis fluorescence spectrophotometer (Malcom, Tokyo, JAPAN). Sample of 10g of Total RNA from HK-2 cells were labeled with biotin (3'IVT Labeling Kit, Affymetrix, USA) and hybridized (GeneAtlas Hybridization, Wash, and Stain Kit for 3' IVT Arrays, Affymetrix).
Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation.
Specimen part, Cell line, TreatmentView Samples
N-glycolylneuraminic acid (Neu5Gc) is generated by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH). However, humans lack this common mammalian cell surface molecule, Neu5Gc, due to inactivation of the CMAH gene during evolution. CMAH is one of several human-specific genes whose function has been lost by disruption or deletion of the coding frame. It has been suggested that CMAH inactivation has resulted in biochemical or physiological characteristics that have resulted in human-specific diseases. To identify differential gene expression profiles associated with the loss of Neu5Gc expression, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip, using the main tissues (liver, lung, kidney, and heart) from a control mouse and a Cmah-null mouse.
Gene expression and pathway analysis of effects of the CMAH deactivation on mouse lung, kidney and heart.
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Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into GABA and succinate before entering the TCA cycle in support of cell growth as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were identified to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown indispensable for polyamine utilization. The newly identified dadRAX locus, encoding the regulator, alanine transaminase and racemase respectively, coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintain alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, the alternative gamma-glutamylation pathway for the conversion of putrescine into GABA was also discussed. Subsequently, GabD, GabT and PA5313 were identified for GABA utilization. Growth defect of PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA was also demonstrated in vitro. Polyamine utilization in general was proven independent of the PhoPQ two-component system even the expression of which was induced by polyamines. Multiple potent catabolic pathways as depicted in this study could serve pivotal roles in control of intracellular polyamine levels.
Transcriptome analysis of agmatine and putrescine catabolism in Pseudomonas aeruginosa PAO1.
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Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues, such as adult mammalian brains, is challenging. Here, we integrate sucrose-gradient assisted nuclei purification with droplet microfluidics to develop a highly scalable single-nucleus RNA-Seq approach (sNucDrop-Seq), which is free of enzymatic dissociation and nuclei sorting. By profiling ~18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-Seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity, but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo. Overall design: Single-nucleus RNA sequencing analysis of adult mouse cerebral cortex
Dissecting Cell-Type Composition and Activity-Dependent Transcriptional State in Mammalian Brains by Massively Parallel Single-Nucleus RNA-Seq.
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Gene-profiling of Tregs across inbred strains. There is a wide inter-individual range in the frequency of FoxP3+ Treg cells, but little is known about the underlying genetic or epigenetic mechanisms. We explored this issue accross inbred strains of mice. During this study, we established the gene expression profiles of Treg cells from the various inbred strains of mice.
Unstable FoxP3+ T regulatory cells in NZW mice.
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Microarray profiles of M176, M354 and WT FoxP3 -transdued (in vitro) and -Treg from mice
Different molecular complexes that mediate transcriptional induction and repression by FoxP3.
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