Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression of triclosan susceptible and tolerant E. coli O157:H19 in response to triclosan exposure

ABSTRACT: Triclosan is a biocidal active agent commonly found in domestic cleaning products, hand sanitizers, cosmetics and personal care products. It is used to control microbial contamination and has a broad-spectrum of activity against many Gram-positive and Gram-negative bacteria. The development of triclosan tolerance with potential cross resistance to clinically relevant antibiotics in zoonotic pathogens is of concern given the widespread use of this active agent in clinical, food processing and domestic environments. Some studies have proposed that an over-dependence on triclosan-containing products could lead to the emergence of clinically important pathogens that are highly tolerant to both biocides and antibiotics. Currently, there is limited understanding of the mechanisms contributing to the emergence of triclosan tolerance in foodborne pathogens at a genetic level. We used microarray analysis to compare gene expression between a wildtype E. coli O157:H19 isolate (WT) with a minimum inhibitory concentration (MIC) to triclosan of 6.25 ug/ml and its laboratory generated triclosan tolerant mutant (M) with a MIC of >8000 ug/ml. Gene expression profiling was performed on untreated E. coli O157:H19 wildtype (WTu) and mutant (Mu), and on the wildtype and mutant treated with 6 ug/ml triclosan for 30 minutes (WTt and Mt respectively). RNA was extracted from three independent biological replicates for WTu, Mu, WTt & Mt for hybridization on Affymetrix GeneChip E. coli Genome 2.0 Arrays. Micorarray analysis including pre-processing, normalisation and statistical analysis were performed using R (R, 2007) version 2.6 and Bioconductor (Gentleman et al. 2004, Genome Biol. 5:R80) version 2.1 as previously described by Morris et al.(2009, Physiol. Genomics 39:28-37).

ORGANISM(S): Escherichia coli O157:H7

SUBMITTER: Mary Lenahan

PROVIDER: E-GEOD-39343 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Epigenetic modifications, such as aberrant DNA promoter methylation is frequently observed in cervical cancer. Identification of hypermethylated regions maybe useful for discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3) or worse may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions were characterised using genome-wide methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methyl-DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium resulting in the identification of hypermethylated differentially methylated regions (DMRs). Validation of 9 selected DMRs by MSP or BSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was applied exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples (p<0.001). Clinical validation of both markers in cervical scrapings from patients referred with an abnormal cervical smear, confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion (p<0.001) and the ROC analysis was discriminative (p<0.005). These possible methylation markers represent COL25A1 and KATNAL2 promoters and their observed increased methylation upon progression is in agreement with their biological function (cytoskeleton regulation). In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and could be potential biomarkers for early detection. Epigenetic modifications, such as aberrant DNA promoter methylation is frequently observed in cervical cancer. Identification of hypermethylated regions maybe useful for discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3) or worse may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions were characterised using genome-wide methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methyl-DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium resulting in the identification of hypermethylated differentially methylated regions (DMRs). Validation of 9 selected DMRs by MSP or BSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was applied exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples (p<0.001). Clinical validation of both markers in cervical scrapings from patients referred with an abnormal cervical smear, confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion (p<0.001) and the ROC analysis was discriminative (p<0.005). These possible methylation markers represent COL25A1 and KATNAL2 promoters and their observed increased methylation upon progression is in agreement with their biological function (cytoskeleton regulation). In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and could be potential biomarkers for early detection. MeDIP with subsequent microarray analysis was performed on DNA isolated from frozen macrodissected epithelial tissue of CIN3 lesions (n=15) and normal cervices (n=10).

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Gene expression of triclosan susceptible and tolerant E. coli O157:H19 in response to triclosan exposure

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