Project description:A common genomic feature of most EAEC strains is the presence of a virulence plasmid termed pAA. Plasmid-encoded virulence determinants are, among others, a transcriptional activator termed AggR, a member of the AraC-XylS family of transcription factors. We have previously determined the direct correlation between (p)ppGpp, expression of AggR and biofilm development in strain EAEC 042 (https://doi.org/10.3389/fmicb.2018.00717). In this work we characterize a novel variant of the aggR gene. We modified its 3´UTR by insertion of a FRT sequence, which have generated a series of different phenotypes. We used RNA-seq to compare the transcriptome of the wt strain and its aggR3UTRFRT variant grown at 37ºC in LB medium.
Project description:The cholestasis frequently occurs in patients with inflammatory processes. However, the mechanisms of inflammation in inducing cholestasis are still unclear. Here, by using lipopolysaccharide (LPS)-induced cholestasis mouse models, we found that most genes encoding enzymes responsible for bile acid biosynthesis were downregulated in the hepatic tissues. Notably, among these genes, only Cyp7b1 was directly reduced by LPS in hepatocytes. The content of UDCA was decreased in the liver of inflammation-model mice which might be caused by the downregulation of Cyp7b1, and UDCA supplementation could alleviate the cholestasis and liver injury caused by LPS. Mechanistically, we observed that the level of m6A methylation in the 3’-UTR of Cyp7b1 mRNA was decreased, and inhibition of m6A methylation could reduce the expression of Cyp7b1 in Hep1-6 cells. Finally, a novel short variant FTO was identified to be upregulated in the liver of mice with LPS, and knockdown of the short variant FTO could rescue the expression of Cyp7b1 by improving the m6A modification in its mRNA, and then mitigate the cholestasis and liver injury in mice with LPS treatment. Collectively, our study uncovers that LPS-induced inflammation results in cholestasis by upregulating the expression of a short variant FTO, which subsequently leads to the reduction of Cyp7b1 mRNA by removing its m6A modification at the 3’-UTR region.
Project description:Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem: c.-27C>A located near the transcription initiation site and c.85G>T. The c.-27C>A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation. Five members of a three-generation Caucasian Lynch syndrome family with an autosomal dominant MLH1 epimutation linked to a single nucleotide variant (c.-27C>A) within the MLH1 5'UTR were examined for copy number variations and retention of heterozygosity on chromosome 3. These five carriers of constitutional MLH1 methylation and the c.-27C>A variant were compared with 300 healthy Caucasian controls from the Wellcome Trust Case Control Consortium using three algorithms (QuantiSNP, PennCNV, COKGEN) to detect any copy number variants. The five family members studied were female (the proband II5, her affected mother I1, and three asymptomatic relatives II2, II4 and III2) are labeled according to the pedigree in Figure 3 of the associated publication (Hitchins et al., Cancer Cell, 2011). The supplementary file 'GSE30348_gw6.lrr_baf.txt' contains log R ratio and B-allele frequency values in a tab-delimited format with one marker per row.
Project description:Cancer immunotherapies targeting the adaptive immune system have revolutionized the treatment for many cancer patients. However, a considerable fraction of patients does not benefit from targeting only the adaptive arm of the immune system. Here, we demonstrate that priming the innate immune system via systemic delivery of a novel synthetic TLR2/6 agonist, AXA-042, represents a rational strategy to boost anti-tumor immunity. TLR2 and TLR6 expression in mouse and human was predominantly confined to tumor-infiltrating myeloid immune cells. Activation of TLR2-signalling induced a pro-inflammatory phenotype in myeloid immune cells with increased cytokine production which resulted in strong T cell dependent anti-tumor immune responses. Systemic delivery of AXA-042 was well tolerated in mouse and non-human primates, demonstrated innate response engagement and anti-tumor efficacy as monotherapy and in combination with checkpoint inhibitors across a wide range of preclinical mouse models. Preclinical efficacy and safety studies provide evidence that priming and re-shaping the innate immune system with the systemic administration of a TLR2 agonist may represent a promising approach for cancer patients. AXA-042 has completed GLP toxicology studies and is currently in clinical development (ACTRN12622000993796) in advanced solid tumors.
Project description:NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP) and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs e.g. Crohn disease, asthma and atopic eczema. It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2wt or NOD2L1007fsinsC to stimulation with MDP. Importantly, a clear loss-of-function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, while the NOD2wt cells showed differential regulation of growth factors, chemokines and several antagonists of NF-κB, e.g. TNFAIP3 (A20) and IER3. To elucidate the MDP-induced activation program we generated isogenic HEK293 cells stably expressing wildtype NOD2 or NOD2L1007fsinsC using a FRT-recombinase based approach. Cells carrying the inserted vector cassette were used as controls (mock-transfectant). To comprehensively analyze NOD2-mediated innate immune responses we analyzed transcriptomal signature patterns using genome-wide cDNA microarrays. Samples were harvested from cell cultures under normal growth conditions 0 h, 2 h and 6 h after MDP‑ stimulation of the cells.
Project description:FUS is one of the pathogenic RNA-binding proteins for amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilized SynGAP mRNA at its 3’UTR and maintained spine maturation and cognitive function in mice. To elucidate whether this mechanism could be pathogenic for ALS, we identified SynGAP 3’UTR variant at the binding site of FUS, different from that in mice, from a multicenter cohort in Japan. Human induced pluripotent stem cells (hiPSC)-derived motor neurons with SynGAP variant showed spine abnormality with aberrant SynGAP splicing. To evaluate how SynGAP variant altered the access of RNA binding proteins to SynGAP 3'UTR, we performed pull down assay by using biotinylated RNA probes with or without the variant.