Phyllosphere QS receptors C14:1 and C14:2 labeling 1
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ABSTRACT: Extracted cultures of phyllosphere bacteria's QS receptors (heterologously expressed in E. Coli) the have been incubated with an a,b-unsaturated QS signal
Project description:Arthrobacter chlorophenolicus A6 is a 4-chlorophenol degrading soil bacterium with high phyllosphere colonization capacity. Till now the genetic basis for the phyllosphere competency of Arthrobacter or other pollutant-degrading bacteria is uncertain. We investigated global gene expression profile of A. chlorophenolicus grown in the phyllosphere of common bean (Phaseolus vulgaris) compared to growth on agar surfaces.
Project description:We perform differential RNA-seq comparing a treatment with 1.4% saccharin to a mock treatment in E. coli K12 in order to describe the alterations in global transcription produced by this artificial sweetener. Three biological replicates of saturated E. coli K12 cultures were diluted 1/100 in 25 mL LB medium (in duplicate, to obtain 3 treated cultures and 3 mock controls). The cultures were grown (37 ºC, 180 rpm) to approximately OD600 0.3 and they were treated with a final concentration of 1.4% saccharin or a water control. The cultures were further incubated until reacing OD600 0.8. At this point, cells were harvested, treated with RNAlate for preservation of total RNA and stored at -80 C. After that, total RNA was extracted from each sample. As a result, 305 genes appeared upregulated by the saccharin treatment, whereas 419 were downregulated.
Project description:Biofilm formation by Escherichia coli was significantly inhibited when co-cultured with Stenotrophomonas maltophilia in static systems. Genes of E. coli involved in species interactions with S. maltophilia were identified in order to allow the study of the mechanisms of inhibited E. coli biofilm formation in co-culture. A total of 89 and 108 genes were identified as differentially expressed in mixed species cultures when growing as biofilm and as planktonic cultures, respectively, compared to the counterpart of pure cultured E. coli. Differential expression of certain identified genes was confirmed using E. coli reporter strains combined with single-cell based flow cytometry analysis. Co-culture with S. maltophilia affected genes involved in metabolism, signal transduction, cell wall composition, and biofilm formation of E. coli. Several selected genes were further confirmed as affecting E. coli biofilm formation in mixed species cultures with S. maltophilia. The data suggest that these genes were involved in species interactions between E. coli and S. maltophilia. This SuperSeries is composed of the SubSeries listed below.
Project description:Arthrobacter chlorophenolicus A6 is a 4-chlorophenol degrading soil bacterium with high phyllosphere colonization capacity. Till now the genetic basis for the phyllosphere competency of Arthrobacter or other pollutant-degrading bacteria is uncertain. We investigated global gene expression profile of A. chlorophenolicus grown in the phyllosphere of common bean (Phaseolus vulgaris) compared to growth on agar surfaces. We designed transcriptome arrays and investigated which genes had different transcript levels in the phyllosphere of common bean (Phaseolus vulgaris) as compared to agar surfaces. Since water availability is considered an important factor in phyllosphere survival and activity, we included both high and low relative humidity treatments for the phyllosphere-grown cells. In addition, we determined the expression profile under pollutant exposure by the inclusion of two agar surface treatments, i.e. with and without 4-chlorophenol.
Project description:LC3-associated phagocytosis (LAP) represents a non-canonical function of autophagy proteins in which ATG8 family proteins (LC3 and GABARAP proteins) are lipidated onto single-membrane phagosomes as particles are engulfed by phagocytic cells. LAP plays roles in innate immunity, inflammation and anti-cancer responses and is initiated upon phagocytosis of particles that stimulate Toll-like receptors (TLR), Fc-receptors, and upon engulfment of dying cells. However, how this molecular process is initiated remains elusive. Here we report that receptors that engage LAP enrich phosphatidylserine (PS) in the phagosome membrane via membrane-proximal domains that are necessary and sufficient for LAP to proceed. Subsequently, PS recruits the Rubicon-containing PI3-kinase complex to initiate the enzymatic cascade leading to LAP. Manipulation of plasma membrane PS content, PS-binding by Rubicon, or the PS-clustering domains of receptors prevents LAP and phagosome maturation. Therefore, the initiation of LAP represents a novel mechanism of PS-mediated signal transduction upon ligation of surface receptors.
Project description:KrÜppel-like factor 10 (KLF10), deficient in two thirds of 105 resected pancreatic adenocarcinoma (PDAC) patients, was associated with rapid loco-regional recurrence and large tumor size. Additional KLF10 depletion in KC (LSL: KrasG12D; Pdx1-CRE) mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. In Panc-1 cells with KLF10 deficiency (Panc-1-pLKO-shKLF10), increased sphere formation, stem cell markers expression, and tumor growth were noted compared with those of control. Over-expressing KLF10 genetically or pharmacologically using metformin, reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed Notch signal molecules including Notch receptors 3 and 4 were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch 3 and 4 receptors by competing with ELF3, a positive regulator, for promoter binding. Downregulating Notch signal genetically or pharmacologically ameliorated stem cell phenotypes of Panc-1-pLKO-shKLF10. Elevating KLF10 expression by metformin with concomitant evodiamine, a non-toxic Notch 3 methylation stimulator, delayed murine tumor growth of PDAC with KLF10 deficiency without prominent toxicity. These results demonstrated a novel signal pathway of KLF10 in modulating stem cell phenotypes of PDAC via transcriptional regulating Notch signal pathway. Elevating KLF10 and suppressing Notch signal may cooperatively reduce PDAC tumorigenesis and malignant progression.
Project description:A major challenge in the field of mechanobiology relates to the lack of methods that enable identification of mechanically active cells and force transmitting receptors for subsequent biochemical analysis. For example, potent T cell activation requires the transmission of biophysical forces between the T cell receptor (TCR) and its peptide-loaded major histocompatibility complex (pMHC) antigens. Therefore, methods that can chemically tag mechanically active T cells and TCRs are highly desirable, as these techniques will enable a deeper understanding of the mechanisms governing immune responses and may also have broad applications in immunotherapy. Herein, we report a technique dubbed mechano-ID, which allows for mechanically selective proximity tagging by leveraging DNA-based molecular force probes that recruit proximity tagging enzymes. We demonstrate mechano-ID tagging of T cells using microscopy and flow cytometry and this is further confirmed using western blotting of mechanically active T cell receptors.
Project description:This project uses an MCF10A clone (B2B1) expressing chimeric synthetic receptors for ErbB1 (EGFR) and ErbB2 (HER2) along with BirA*-fused inducible alleles of CSE1L or NUP37. Cells were cultured with reconstituted basement membrane, BirA* alleles induced with doxycycline, ErbB receptors dimerized with a synthetic small molecule, and biotinylation was performed for 24 hours. Lysates underwent a two-step biotin affinity purification before gel electrophoresis and mass spectrometry on selected gel fragments.