Chip-SIP analysis of Monterey Bay surface waters incubated with organic carbon substrates
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ABSTRACT: October 2013 surface seawater collected from Monterey Bay was incubated with 1 micromolar 13C labeled glucose, starch, acetate, lipids, protein, or amino acids for 12 hours. Community RNA was extracted and hybridized to a Roche Nimblegen microarray and analyzed by NanoSIMS to obtain isotope ratio data for all probe spots. Two Chips for fluorescence, and 15 Chips for different substrates from samples incubated for 12 or 36 hours.
Project description:Comparison of the DNA methylation profiles of CD14+ monocytes from human peripheral blood with derived dendritic cells (DCs) and macrophages (MACs) obtained by exposure with GM-CSF/IL-4 and GM-CSF, respectively. Effects on the methylation profiles of DCs and MACs of JAK3 inhibitor PF-956980 The methylation profiles of bisulfite-modified DNA of human CD14+ monocytes were compared with derived dendritic cells (DCs), macrophages (MACs) following GM-CSF/IL-4 and GM-CSF incubation, and DC and MAC samples incubated with JAK3 inhibitor PF-956980 using the Infinium HumanMethylation450 BeadChips (Illumina, Inc., San Diego, CA,). This platform allows the interrogation of >485,000 methylation sites per sample at single-nucleotide resolution, and comprises an average of 17 CpG sites per gene in the 99% of RefSeq genes. 96% of CpG islands are covered, with additional coverage in CpG island shores and the regions flanking them. The samples were hybridized in the array following the manufacturerâÂÂs instructions. Total DNA isolated by standard procedures from CD14+ cells (total monocytes, MOs) corresponding to three sets of samples of monocytes (MOs), derived DCs and MACs (DCs and iMACs; DMSO as these samples were differentiated in the absence of JAK3 inhibitors) and DCs and MACs differentiated in the presence of JAK3 inhibitor PF-956980.
Project description:October 2013 surface seawater collected from Monterey Bay was incubated with 1 micromolar 13C labeled glucose, starch, acetate, lipids, protein, or amino acids for 12 hours. Community RNA was extracted and hybridized to a Roche Nimblegen microarray and analyzed by NanoSIMS to obtain isotope ratio data for all probe spots.
Project description:A long-standing question in developmental and reproductive biology is when the mammalian embryo becomes sufficiently distinct from its oocyte precursor. Myriads of studies examined the messenger RNAs that change during the oocyte-to-embryo transition, whereas proteins have been much less studied, in spite of their greater vicinity to phenotype. In the present study we modified the widely used embryo culture medium KSOM (PMID 12470333, PMID 10859270) to make it apt for our application. We replaced the serum albumin with polyvinylpyrrolidone and also replaced the natural Arginine and Lysine with their “heavy” isotopic variants Arginine 13C 15N and Lysine 13C 15N. Fertilized oocytes were retrieved from oviducts of gonadotropin-primed B6C3F1 females mated to CD1 males, and cultured at 37 degrees Celsius under 5% CO2 in KSOM containing 0.3 mM Arginine 13C 15N and 0.2 mM Lysine 13C 15N, which are the regular concentrations of these two amino acids in KSOM medium (PMID 12470333; PMID 10859270). After 4 days of culture, the embryos of the isotopic group had undergone blastocyst formation just like the control embryos cultured in normal medium. Samples of approx. 500 “heavy”-labeled blastocysts were collected zona-free and subjected to mass spectrometric analysis. The median labeling rate was 83%, ranging from 0% in proteins that did not incorporate any Arginine 13C 15N and Lysine 13C 15N, to 100% in proteins that were completely labeled. Our study demonstrates that a commonly used, chemically defined medium can be adapted for Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and combined with high-resolution mass spectrometry, in a preimplantation embryo setting. This allows to tackle long-standing questions in developmental and reproductive biology, such as the identification of putative maternal (0% labeled), putative embryonic (100% labeled) or shared proteins in live mammalian embryos.
Project description:To assess the pattern of TGF-beta and TLR4-induced gene expression changes at the genome-wide level, confluent healthy skin fibroblasts were incubated with the classic TLR4 ligand, LPS, TGF-beta or transfected with CD4TLR4. At the end of incubation, total RNA was harvested and processed for hybridizations to Illumina Human HT-12V4 microarray chips.
Project description:Expression profiles at different time points during dendritic cell differentiation (induced by specific culture conditions) including monocytes as well as expression profiles between monocytes and completely differentiated cells (macrophages at day7 and dendritic cells at day7, respectively) were compared. Monocyte-derived dendritic cells (DC) were obtained by culturing elutriated monocytes with 20U/ml IL-4, 280U/ml GM-CSF and 10% FCS; monocyte-derived macrophages (MAC) were obtained by culturing elutriated monocytes with 2% AB serum. Three to seven biological replicates that are derived from independent healthy donors were included. One-color based gene expression. 2 datasets: dendritic cell kinetic study and comparison of monocyte, macrophage, and dendritic cells
Project description:Retinoblastoma (Rb) is the most common intraocular tumor in childhood and represent a very robust model about hereditary develop for cancer. We believe that miRNome expression may give clues to understanding Rb heterogeneity and to comprehend biological features in order to find new biomarkers for better treatments. We explore 12 Rb tumors from enucleated patients with no chemotherapy history from Children Hospital of Mexico Federico Gomez and Pediatric Hospital Medical Center XXI century by informed parental consent to collaborate with retrospective research. 5 bilateral and 7 unilateral, 4 female and 8 male samples were tailed and labeled with biotin to hybridize in GeneChip miRNA 4.0 Affymetrix array. Chips were washed and scanned 18hrs after hybridization.
Project description:[1] Lactic acidosis time course: MCF7 cells were exposed to lactic acidosis for different length of time. We used microarrays to examine the genomic programs of cells incubated under lactic acidosis for different length of time [2] Metabolic profiling: MCF7 cells were exposed to control condition, 25mM lactic acidosis, glucose deprivation (zero glucose) and hypoxia (1% oxygen level). [3] Mouse study: Lactic acidosis triggers starvation response with paradoxical induction of TXNIP through MondoA. Wild-type mouse embryo fibroblasts (MEFs) and TXNIP-null MEFs were exposed to Ctrl versus lactic acidosis conditions for 24hrs and the RNAs from cells were extracted with MiRVana kit (Ambion) and applied to Affymetrix 430A mouse chips We used microarrays to examine the genomic programs of cells incubated under different microenvironmental stresses.