Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of Mycobacterium tuberculosis high-persister (hip) mutants.

ABSTRACT: Bacterial persister cells are phenotypic variants of regular cells that are tolerant to antibiotics. High-persister (hip) mutants of Mycobacterium tuberculosis produce 10- to 1,000-fold more persister cells than the wild type strain when challenged with various antibiotics. Comparison of gene expression pattern of the hip mutants may provide clues as to the genetic mechanisms underlying persister formation. Transcriptome analysis will provide information on what differentiates M. tuberculosis hip strains from regular strains, which will be useful in the development of anti-persister therapy for persister cell eradication. Twelve independent M. tuberculosis hip mutants and the wild type strain were grown to stationary phase in duplicate. To avoid analyzing the drug effect, total RNA was extracted from the cultures prior to the addition of antibiotic for hybridization to Affymetrix microarrays. Data analysis was performed by comparing the hip mutants to the wild type control.

ORGANISM(S): Mycobacterium tuberculosis

SUBMITTER: Kim Lewis

PROVIDER: E-GEOD-55647 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:With aging, significant changes in circadian rhythms occur, including a shift in phase toward a âmorningâ chronotype and a loss of rhythmicity in circulating hormones. However, the effects of aging on molecular rhythms in the human brain have remained elusive. Here we employed a previously-described time-of-death analyses to identify transcripts throughout the genome that have a significant circadian rhythm in expression in the human prefrontal cortex (Brodmannâs areas (BA) 11 and 47). Expression levels were determined by microarray analysis in 146 individuals. Rhythmicity in expression was found in ~10% of detected transcripts (p<0.05). Using a meta-analysis across the two brain areas, we identified a core set of 235 genes (q<0.05) with significant circadian rhythms of expression. These 235 genes showed 92% concordance in the phase of expression between the two areas. In addition to the canonical core circadian genes, a number of other genes were found to exhibit rhythmic expression in the brain. Notably, we identified more than one thousand genes (1186 in BA11; 1591 in BA47) that exhibited age-dependent rhythmicity or alterations in rhythmicity patterns with aging. Interestingly, a set of transcripts gained rhythmicity in older individuals, which may represent a compensatory mechanism due to a loss of canonical clock function. Thus, we confirm that rhythmic gene expression can be reliably measured in human brain and identified for the first time significant changes in molecular rhythms with aging that may contribute to altered cognition, sleep and mood in later life. Using the resources of the University of Pittsburghâs Brain Tissue Donation Program, 210 subjects were identified. Samples were obtained after consent from next-of-kin during autopsies conducted at the Allegheny County Medical Examinerâs Office (Pittsburgh, USA). The absence of lifetime psychiatric disorders was determined by an independent committee of experienced clinical research scientists using information from clinical records, toxicology results and a standardized psychological autopsy. Subjects of unwitnessed death were removed from the study. A total of 146 individuals with the following characteristics were analyzed in this study: mean (range) age of 50.7 (16-96) years, 78% male, 85% Caucasian, mean postmortem interval (PMI) for brain collection 17.3 (4.8-28) hours, mean pH 6.7 (5.8-7.6) and RNA integrity number (RIN) 8.0 (5.9-9.6). For each subject, the right hemisphere was blocked coronally, frozen and stored at -80C. Blocks containing Brodmann area 11 (BA11) or Brodmann area 47 (BA47) of the orbital PFC were cut on a cryostat, and cortical gray matter was collected for RNA extraction as previously described. All procedures were approved by the University of Pittsburghâs Institutional Review Board for Biomedical Research and Committee for Research Involving the Dead.

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Gene expression analysis of Mycobacterium tuberculosis high-persister (hip) mutants.

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