Project description:Gram negative bacteria are surrounded by the cell envelope, a structure comprised of an outer membrane (OM) and an inner membrane (IM). These two membranes delimit the periplasmic space, a compartment containing the peptidoglycan (PG), a giant polymer surrounding the cell and functioning as an extracytoplasmic skeleton. In the model organism Escherichia coli, covalently anchoring the OM to the peptidoglycan is crucial for envelope integrity. When attachment is prevented, the OM forms blebs and detaches from the cell body. Intriguingly, the only bacterial protein known to covalently attach the OM to the PG (the Braun lipoprotein or Lpp) is present in a small number of γ-proteobacteria, raising the question of OM-PG attachment in other species. Here, we show that in -proteobacteria Brucella abortus and Agrobacterium tumefaciens, the OM is attached to the peptidoglycan via the formation of covalent crosslinks between the N-terminus of integral OM proteins (OMPs; including porins) and peptide stems of peptidoglycan.
Project description:The deoxyguanosine kinase (DGUOK) deficiency causes mtDNA depletion and mitochondrial dysfunction. We reported prolonged survival of Dguok knockout (Dguok-/-) mice despite low (<5%) mtDNA content in the liver. However, the molecular mechanisms, enabling the extended survival, remain unknown. Using transcriptomics, proteomics and metabolomics followed by in vitro assays, we aimed to identify the molecular pathways involved in the extended survival of Dguok-/- mice. At the early stage, the serine synthesis and folate cycle were activated but declined later. Increased activity of the mitochondrial citric acid cycle (TCA cycle) and the urea cycle and degradation of branched amino acids were hallmarks of the extended lifespan in DGUOK-deficiency. Furthermore, the increased synthesis of TCA cycle intermediates was supported by coordination of two pyruvate kinase genes, Pklr and Pkm, indicating a central coordinating role of pyruvate kinases to support the long-term survival in mitochondrial dysfunction.
Project description:Importance: Pectobacterium species cause soft rot in potato and other host plants primarily by secreting a battery of plant cell wall degrading enzymes. In addition, several different secretion systems are mobilized during infection. Previous studies of gene expression and regulation thereof primarily focused on the onset of infection. This work investigated transcriptome changes in Pectobacterium during the infection of potato tubers up to 72 hours post inoculation to elucidate biological processes during a longer infection period. Methods: The transcriptomes of aggressive strains of the two species P. carotovorum subsp. carotovorum and P. polaris were investigated during infection of potato minitubers (cv. 'Asterix') at 24, 48 and 72 hours after inoculation by RNA sequencing. The transcriptomes were compared to that of bacteria grown on minimal M9 medium, and transcriptomes from later infection time points (48 and 72 hours after inoculation) were compared to early infection (24 hours after inoculation). Results: Plant cell wall degrading enzymes and secretion system associated genes were largely upregulated during infection compared to in vitro growth, but downregulated in the later phases of infection compared to the early infection phase. The downregulation was not sufficiently explained by the expression of known virulence regulators such as the RsmA/B or the ExpA/S systems.
Project description:The purpose of this experiment of high-coverage DNA sequencing of 58 frequently mutated genes in hepatocellular carcinoma (HCC) is to confirm clonal distribution of the known HCC drivers in samples corresponding to multiple regions of a tumor.
Project description:The microbiota generates structurally diverse small molecules that can regulate host physiology and disease. Of these microbiota metabolites, bile acids have emerged as important modulators of host immunity and microbial pathogenesis. While the modes of action for different bile acids on host pathways are becoming more apparent, the mechanisms by which these prominent microbiota metabolites suppress microbial virulence pathways are less clear. To identify the direct protein targets of bile acids in Salmonella, we have generated three photoaffinity bile acid reporters (alk-X-CDCA, alk-X-UDCA, alk-X-LCA) and performed chemical proteomics. Using a combination of photocrosslinking with bile acid chemical reporters and label-free proteomics, we performed a quantitative analysis of bile acid interacting proteins in Salmonella with or without UV-mediated photocrosslinking. These studies revealed bile acid can interact with many Salmonella proteins, such as extracellular or secreted proteins, T3SS components and motility-related proteins, as predicted by Gene Ontology analysis. In addition, cytoplasmic and membrane proteins including metabolic enzymes are also identified. Notably, HilD, an important transcriptional regulator of S. Typhimurium virulence was also identified by the alk-X-CDCA reporter. This study highlights the utility of chemical proteomics to identify the direct protein targets and mechanisms of action for microbiota metabolites in bacterial pathogens.
Project description:The aim of this sequencing experiment was to make available tissue expression panels for selected fish species for comparative expression studies between the species. Tissue samples were collected for zebrafish (Danio rerio), medaka (Oryzias latipes), and rainbow trout (Oncorhynchus mykiss). Tissue types included liver, skin, muscle, heart, gut, gill, eye, brain for all three species, with additionally pyloric caeca, kidney, head kidney, and spleen for rainbow trout. Only liver samples were taken in replicate of four or three for rainbow trout. All fish were raised under standard rearing conditions for the species. Total RNA was extracted from the tissue samples and paired‐end sequencing of sample libraries was completed on an Illumina HiSeq 2500 with 125‐bp reads. Processed count tables per species as raw counts, FPKM, or TPM, were generated from read alignment to the Ensembl genomes of the respective species using STAR and gene level counting using RSEM and Ensembl gene annotation.
Project description:We performed ChIP-Seq analysis of SOX10, histone H3 lysine 27 acetylation (H3K27ac) and H3K27 trimethylation (H3K27me3) in melanocytes to profile the genomic binding sites of SOX10 and the chromatin landscape. In parallel, we generated Sox10 haploinsufficient cell lines using gene knockout approaches and conducted microarray gene expression analysis to identify functional gene targets of SOX10 transcriptional regulation in melanocytes. We demonstrate that SOX10 predominantly engages “open” chromatin, binds to melanocyte enhancer elements and plays a central role in transcriptional activation and repression of functionally distinct classes of genes. Furthermore, we identified cis-regulatory sequence motifs of putative co-regulatory transcription factors that define SOX10-activated and SOX10-repressed target genes. Our results uncover novel mechanisms and roles of SOX10 in global transcriptional regulation of diverse regulatory pathways in the melanocyte lineage. ChIP-seq profiling of SOX10, H3K27ac, and H3K27me3 in the mouse melanocyte cell line melan-Ink4a-Arf-1 (melan-a).
Project description:This experiment involves RNAseq data on 3 parental lines and 2 hybrid crosses. The parents: inbred lines B104 and Mo17, and the GA20OX transgenic line in B104 background. The hybrid crosses: B104 x Mo17, GA20OX x Mo17.
Project description:Plants were grown for 4 weeks under short day (10 h light/14 h dark and 20 °C/16 °C, respectively). The relative humidity during the day and night was 50 %. Then, plants were transferred at 11 am (5 h after turning on the light) for 4 h either to dark (0 µE, 22 °C) or low light (100-130 µE, 22 °C) conditions and subsequently harvested.