Summary To characterize somatic alterations in colorectal carcinoma (CRC), we conducted genome-scale analysis of 276 samples, analyzing exome sequence, DNA copy number, promoter methylation, mRNA and microRNA expression. A subset (97) underwent low-depth-of-coverage whole-genome sequencing. 16% of CRC have hypermutation, three quarters of which have the expected high microsatellite instability (MSI), usually with hypermethylation and MLH1 silencing, but one quarter has somatic mismatch repair gene mutations. Excluding hypermutated cancers, colon and rectum cancers have remarkably similar patterns of genomic alteration. Twenty-four genes are significantly mutated. In addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9, and FAM123B/WTX. Recurrent copy number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive CRC and important role for MYC-directed transcriptional activation and repression.
High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules.
The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes ( ANME-2 ؍ 0.167 · week ؊1 ), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes ( ANME-1 ؍ 0.218 · week ؊1 ). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 · week ؊1 , and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.Geochemical studies have shown that the anaerobic oxidation of methane (AOM) in marine sediments is a microbially mediated phenomenon and represents a significant component of carbon cycling in marine environments (4,25,32). Based on field and laboratory experiments and observations using rRNA-targeted fluorescent in situ hybridization, AOM is believed to be mediated by a consortium of archaea and sulfatereducing bacteria (2,5,7,20). Phylogenetic and isotopic analyses have shown that two groups of anaerobic methanotrophic archaea (ANME-1 and ANME-2) cooccur in sediments with sulfate-reducing bacteria and incorporate methane-derived carbon into cellular biomass (19, 30). Genomic and biochemical analyses suggest that microbial AOM may be achieved in part by reversal of the canonical methanogenic pathway (15, 23), and ongoing studies have shown that ANME-1 and ANME-2 methane-oxidizing archaea (or MOA) are distributed throughout the world in anoxic marine sediments where methane and sulfate are available (22,26,31,35,37,39,41).Our understanding of MOA physiology has been advanced by employing both in situ isotopic tracers and serum vial incubations to examine the relation between AOM and sulfate reduction and the effect of physical factors,...
Background-Human atherosclerotic lesions contain elevated levels of urokinase plasminogen activator (uPA), expressed predominantly by macrophages. Methods and Results-To test the hypothesis that macrophage-expressed uPA contributes to the progression and complications of atherosclerosis, we generated transgenic mice with macrophage-targeted overexpression of uPA. The uPA transgene was bred into the apolipoprotein E-null background, and transgenic mice and nontransgenic littermate controls were fed an atherogenic diet. uPA-transgenic mice had significantly elevated uPA activity in the atherosclerotic artery wall, of a magnitude similar to elevations reported in atherosclerotic human arteries. Compared with littermate controls, uPA-transgenic mice had accelerated atherosclerosis, dilated aortic roots, occlusive proximal coronary artery disease, myocardial infarcts, and early mortality. Conclusions-These data support the hypothesis that overexpression of uPA by artery wall macrophages is atherogenic and suggest that uPA inhibitors might be therapeutically useful.
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