Project description:Biogenic methane formation, methanogenesis, a key process in the global carbon cycle is the only energy metabolism known to sustain growth of the microorganisms employing it, the methanogenic archaea. All known methanogenic pathways converge at the methane-liberating step where also the terminal electron acceptor of methanogenic respiration, the heterodisulfide of coenzyme M and coenzyme B is formed. Carbon monoxide (CO) utilization of Methanosarcina acetivorans is unique in that the organism can shift from methanogenesis towards acetogenesis. Here, we show that M. acetivorans can dispense of methanogenesis for energy conservation completely. By disrupting the methanogenic pathway through targeted mutagenesis, followed by adaptive evolution, a strain capable of sustained growth by CO-dependent acetogenesis was created. Still, a minute flux through the methane-liberating reaction remained essential, which was attributed to the involvement of the heterodisulfide in at least one essential anabolic reaction. Genomic and proteomic analysis showed that substantial metabolic rewiring had occurred in the strain. Most notably, heterodisulfide reductase, the terminal respiratory oxidoreductase was eliminated to funnel the heterodisulfide towards anabolism. These results suggest that the metabolic flexibility of “methanogenic” archaea is much greater than anticipated and open avenues for probing the mechanism of energetic coupling and the crosstalk between catabolism and anabolism.
Project description:To understand the the effect of microparticles conjugated with vascular endothelial growth factor (VEGF) on human endothelial cells derived from human umbilical cord blood (UCB) CD34+ hematopoietic stem cells, we have employed microRNA microarray profiling. The microparticles used in this study were purchased from Invitrogen (Dynal magnetic microparticles coated with streptavidin, 4.5 micron in size) and modified in our lab. Cell suspensions were mixed with blank microparticles (without VEGF), soluble VEGF or VEGF-conjugated microparticles and seeded as hanging drops to prepare endothelial cell aggregates. Cell aggregates without any treatments were used as control. After 2 h or 8 h, the cell aggregates were collected and miRNA expression profiles were detected.
Project description:Four stable and robust TCE-dechlorinating microbial communities were enriched from TCE-contaminated groundwater under four different conditions exploring two parameters, high and low methanogenic activity (Meth and NoMeth), with and without vitamin B12 supplement (MethB12 and NoMethB12, Meth and NoMeth, respectively). Identical amounts of lactate (2.7 mmol) and TCE (20 μl) were supplied as electron donor and electron acceptor. All four cultures were capable of reductively dechlorinating TCE to VC and ethene. Genomic DNA of the four enrichments was applied on a quad-Dhc-genome microarray in order to characterize the gene content of Dehalococcoides species present in the four enrichments
Project description:One of strategies to regenerate cartilage defect is transplantation of mesenchymal stem cells (MSCs). Improvements of therapeutic potential of MSCs are needed to achieve successful cartilage regeneration by transplantation of a limited number of cells. Aggregated culture is a popular method in ES and iPS cells to maintain or enhance their potentials. Here we investigated gene expression profile of aggregated MSCs. 621 genes were up-regulated and 409 genes were down-regulated more than 5-fold in MSC-aggregates compared with the number in MSCs in a monolayer culture. The most up-regulated gene was BMP2, which is one of the genes involved in chondrogenesis. Anti-inflammatory genes were also up-regulated in MSC-aggregates. The microarray data for selected genes were confirmed by real-time PCR. Human synovial MSCs was isolated from synovium of 3 distinct donors. The gene expression profile of MSC-aggregates cultured in hanging drop for 3days was compared with that of MSCs in a monolayer culture.
Project description:We report the isolation and sequencing of tau aggregates from [1] HEK293 cells expressing Tau-RD-P301S-CFP/YFP that have been seeded with preformed fibrils from the brain of P301S mice (B6-Tg(Thy1-MAPT*P301S)2541; referred to as Tg2541 mice). Tau aggregates were isolated by differential centrifugation followed by fluorescence automated particle sorting using a BD FACSaraia. We found that these tau aggregates were enriched for particular small non-coding RNAs, including snoRNAs and snRNAs. [2] the following mice: FvBB6F1-Tg(Camk2a-tTa),(tetO-MAPT*wt)21221 (referred to as rTg21221 or WT tau mice in the paper) and FvBB6F1-Tg(Camk2a-tTA)1Mmay, (tet)-tdTomato-Syp/EGFP)1.1Luo/J,(tetO-MAPT*P301L)4510 (referred to as rTg4510 or P301L mice in the paper). Briefly, tau aggregates were isolated by 1% sarkosyl extraction (to enrich for insoluble proteins) followed by immunoprecipitation of tau using the tau-12 antibody (see Methods section of associated paper for further details). We found that these tau aggregates were enriched for particular small non-coding RNAs, including snRNAs and some snoRNAs. [3] Sequencing of HEK293 tau biosensor cells with and without tau aggregates reveals evidence of significant splicing alterations. Specifically we observed an increase in intron retention events in cells that contain tau aggregates relative to cells without tau aggregates.