Project description:Excessive degradation of the colonic mucin layer by Bacteroides within the human gut microbiota drives inflammatory bowel disease in mice. Bacterial carbohydrate sulfatases are key enzymes in gut colonization, as they are elevated in human inflammatory bowel disease and correlate with disease severity. Selective inhibitors of carbohydrate sulfatases could function as sulfatase-selective drugs, allowing precise control of sulfatase activity while preserving these otherwise beneficial bacteria. Arylsulfamates are covalent inhibitors that target a catalytic formylglycine residue of steroid sulfatases, which is also conserved in carbohydrate sulfatases. Here, using a library of aryl- and carbohydrate sulfamates, we find that they are ineffective against Bacteroides carbohydrate sulfatases yet can inhibit human gut microbiota species grown on sulfated glycans. Leveraging thermal proteome profiling, we identify a lipid kinase as the target responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and unveils the adverse effect that arylsulfamates have on Bacteroides species of the human gut microbiota.
Project description:2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide utagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc) – the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.
Project description:This study aimed to evaluate the cost-effective and genome-wide cell-free reduced representation bisulfite sequencing (cfRRBS) method combined with computational deconvolution for effective disease monitoring in patients with esophageal adenocarcinoma (EAC). cfDNA methylation profiling with cfRRBS was performed on 162 blood plasma samples from 33 EAC cancer patients and 28 blood plasma samples from 20 healthy donors. In addition, for reproducibility testing purposes of the method, 9 plasma samples were re-prepped (library was re-made) and re-sequenced once (n=9) or twice (n=1). As a reference for the data deconvolution cfRRBS was performed on 7 EAC tumor tissue (FFPE) samples.
Project description:The large-scale application of genomic and metagenomic sequencing technologies has yielded a number of insights about the metabolic potential of symbiotic human gut microbes. Bacteria that colonize the mucosal layer that overlies the gut epithelium have access to highly-sulfated polysaccharides (i.e., mucin oligosaccharides and glycosaminoglycans), which they could potentially forage as nutrient sources. To be active, sulfatases must undergo a critical post-translational modification catalyzed in anaerobic bacteria by the AdoMet enzyme anSME (anaerobic Sulfatase-Maturating Enzyme). In the present study, we have tested the role of this pathway in the prominent gut symbiont Bacteroides thetaiotaomicron, which possesses more predicted sulfatases (28) than in the human genome and a single predicted anSME. In vitro studies revealed that deletion of its anSME (BT0238) results in loss of sulfatase activity and impaired ability to use sulfated polysaccharides as carbon sources. Co-colonization of germ-free animals with both isogenic strains, or invasion experiments involving the introduction of one then the other strain, established that anSME activity and the sulfatases that are activated via this pathway, are important fitness factors for B. thetaiotaomicron, especially when mice are fed a simple sugar diet that requires this saccharolytic bacterium to adaptively forage on host glycans as nutrients. Whole genome transcriptional profiling of wild-type and the anSME mutant in vivo revealed that loss of this enzyme alters expression of genes involved in mucin utilization and that this disrupted ability to access mucosal glycans likely underlies the observed dramatic colonization defect. Comparative genomic analysis reveals that 100% of 46 fully sequenced human gut Bacteroidetes contain homologs of BT0238 and genes encoding sulfatases, suggesting that this is an important and evolutionarily conserved feature.
Project description:We performed ChIP-seq targeting the glucocorticoid receptor (GR) in the U2OS-GR cell line and the androgen receptor (AR) in the U2OS-AR cell line. The cell lines are derived from U2OS ATTC:HTB-96 and stably transfected with an expression construct for either rat GR or human AR, respectively. The U2OS-GR cells were treated with dexamethasone (1 µM) for 90 minutes. The U2OS-AR cells were treated with R1881 (5 nM) for 4 hours.
Project description:Evaluation of the transcriptomic profile of the rabbit embryo along the preimplantation period during in vivo development. Three embryonic stages were used: four cell embryos (H32 post-coïtum); morula (H58 pc) and blastocyst (H90 pc). Keywords: time course rabbit embryo 18 samples
Project description:ChIP-Seq analysis revealed that suberoylanilidehydroxamic acid (SAHA) increases genome-wide H4 acetylation in differentially regulated genes, except for the 500 bp upstream of transcription start sites (TSS). Chromatin immunoprecipitation (ChIP) with massively parallel high throughput sequencing (Seq) was used to map genome-wide histone H4 acetylation (K4/7/11/15) in the presence or absence of SAHA in differentiating MC3T3 sc4 osteoblasts.
Project description:We analyze the effect of magnetic 3D bioprinting using NanoShuttle-PL on the proteome of primary human skin fibroblasts and epidermal squamous cell carcinoma cells. NanoStuttle-PL employs magnetic nanoparticles that interact electrostatically with cells rendering them magnetic and allowing manipulation of their 3D assembly using external magnetic forces.
Project description:Tumor suppressor p53 regulates various role in the cell including cell cycle arrest, DNA repair and apoptosis. Current research achieved to investigate p53 target genes in human osteosarcoma cell line-SaOS2 cell. Examination of p53 binding protein by transfecting flag-tagged wild type p53 into SaOS2 cells.