Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution.

RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous 'synthetic nucleic acid substitution' strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-'substituted' NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the β,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation

Resveratrol has antiinflammatory and antifibrotic effects in the peptidoglycan‐polysaccharide rat model of Crohn's disease

Background: Resveratrol has antiinflammatory and antifibrotic effects. Resveratrol decreases proliferation and collagen synthesis by intestinal smooth muscle cells. We hypothesized that resveratrol would decrease inflammation and fibrosis in an animal model of Crohn's disease. Methods: Peptidoglycan‐polysaccharide (PG‐PS) or human serum albumin (HSA) was injected into the bowel wall of Lewis rats at laparotomy. Resveratrol or vehicle was administered daily by gavage 1–27 days postinjection. On day 28, gross abdominal and histologic findings were scored. Cecal collagen content was measured by colorimetric analysis of digital images of trichrome‐stained sections. Cecal levels of procollagen, cytokine, and growth factor mRNAs were determined. Results: PG‐PS‐injected rats (vehicle‐treated) developed more fibrosis than HSA‐injected rats by all measurements: gross abdominal score ( P < 0.001), cecal collagen content ( P = 0.04), and procollagen I and III mRNAs ( P ≤ 0.0007). PG‐PS‐injected rats treated with 40 mg/kg resveratrol showed a trend toward decreased gross abdominal score, inflammatory cytokine mRNAs, and procollagen mRNAs. PG‐PS‐injected rats treated with 100 mg/kg resveratrol had lower inflammatory cytokine mRNAs (IL‐1β [3.50 ± 1.08 vs. 10.79 ± 1.88, P = 0.005], IL‐6 [17.11 ± 9.22 vs. 45.64 ± 8.83, P = 0.03], tumor necrosis factor alpha (TNF‐α) [0.80 ± 0.14 vs. 1.89 ± 0.22, P = 0.002]), transforming growth factor beta 1 (TGF‐β1) mRNA (2.24 ± 0.37 vs. 4.06 ± 0.58, P = 0.01), and histologic fibrosis score (6.4 ± 1.1 vs. 9.8 ± 1.0; P = 0.035) than those treated with vehicle. There were trends toward decreased gross abdominal score and decreased cecal collagen content. Procollagen I, procollagen III, and IGF‐I mRNAs also trended downward. Conclusions: Resveratrol decreases inflammatory cytokines and TGF‐β1 in the PG‐PS model of Crohn's disease and demonstrates a promising trend in decreasing tissue fibrosis. These findings may have therapeutic applications in inflammatory bowel disease. (Inflamm Bowel Dis 2011;)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90530/1/21843_ftp.pd

Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis

A cross-sample statistical model for SNP detection in short-read sequencing data

Highly multiplex DNA sequencers have greatly expanded our ability to survey human genomes for previously unknown single nucleotide polymorphisms (SNPs). However, sequencing and mapping errors, though rare, contribute substantially to the number of false discoveries in current SNP callers. We demonstrate that we can significantly reduce the number of false positive SNP calls by pooling information across samples. Although many studies prepare and sequence multiple samples with the same protocol, most existing SNP callers ignore cross-sample information. In contrast, we propose an empirical Bayes method that uses cross-sample information to learn the error properties of the data. This error information lets us call SNPs with a lower false discovery rate than existing methods

Ultrasensitive detection of rare mutations using next-generation targeted resequencing

With next-generation DNA sequencing technologies, one can interrogate a specific genomic region of interest at very high depth of coverage and identify less prevalent, rare mutations in heterogeneous clinical samples. However, the mutation detection levels are limited by the error rate of the sequencing technology as well as by the availability of variant-calling algorithms with high statistical power and low false positive rates. We demonstrate that we can robustly detect mutations at 0.1% fractional representation. This represents accurate detection of one mutant per every 1000 wild-type alleles. To achieve this sensitive level of mutation detection, we integrate a high accuracy indexing strategy and reference replication for estimating sequencing error variance. We employ a statistical model to estimate the error rate at each position of the reference and to quantify the fraction of variant base in the sample. Our method is highly specific (99%) and sensitive (100%) when applied to a known 0.1% sample fraction admixture of two synthetic DNA samples to validate our method. As a clinical application of this method, we analyzed nine clinical samples of H1N1 influenza A and detected an oseltamivir (antiviral therapy) resistance mutation in the H1N1 neuraminidase gene at a sample fraction of 0.18%

Rare germline variants in DNA repair genes and the angiogenesis pathway predispose prostate cancer patients to develop metastatic disease

Background Prostate cancer (PrCa) demonstrates a heterogeneous clinical presentation ranging from largely indolent to lethal. We sought to identify a signature of rare inherited variants that distinguishes between these two extreme phenotypes. Methods We sequenced germline whole exomes from 139 aggressive (metastatic, age of diagnosis < 60) and 141 non-aggressive (low clinical grade, age of diagnosis ≥60) PrCa cases. We conducted rare variant association analyses at gene and gene set levels using SKAT and Bayesian risk index techniques. GO term enrichment analysis was performed for genes with the highest differential burden of rare disruptive variants. Results Protein truncating variants (PTVs) in specific DNA repair genes were significantly overrepresented among patients with the aggressive phenotype, with BRCA2, ATM and NBN the most frequently mutated genes. Differential burden of rare variants was identified between metastatic and non-aggressive cases for several genes implicated in angiogenesis, conferring both deleterious and protective effects. Conclusions Inherited PTVs in several DNA repair genes distinguish aggressive from non-aggressive PrCa cases. Furthermore, inherited variants in genes with roles in angiogenesis may be potential predictors for risk of metastases. If validated in a larger dataset, these findings have potential for future clinical application

Integrative eQTL-Based Analyses Reveal the Biology of Breast Cancer Risk Loci

该论文是在本文通讯作者美国哈佛大学医学院代纳法伯癌症中心马修.弗里德曼教授实验室完成的。Germline determinants of gene expression in tumors are infrequently studied due to the complexity of transcript regulation caused by somatically acquired alterations. We performed expression quantitative trait locus (eQTL)-based analyses using the multi-level information provided in The Cancer Genome Atlas (TCGA). Of the factors we measured, cis-acting eQTLs accounted for 1.2% of the total variation of tumor gene expression, while somatic copy-number alteration and CpG methylation accounted for 7.3% and 3.3%, respectively. eQTL analyses of 15 previously reported breast cancer risk loci resulted in the discovery of three variants that are significantly associated with transcript levels (false discovery rate [FDR] < 0.1). Our trans-based analysis identified an additional three risk loci to act through ESR1, MYC, and KLF4. These findings provide a more comprehensive picture of gene expression determinants in breast cancer as well as insights into the underlying biology of breast cancer risk loci

Cancer risk in hospitalised psoriasis patients: a follow-up study in Sweden

We examined overall and specific cancer risks among Swedish subjects who had been hospitalised one or more times for psoriasis. A database was created by identifying such patients from the Swedish Hospital Discharge Register and linking them with the Cancer Registry. Follow-up of patients was carried out from the last hospitalisation through 2004. A total of 15 858 patients were hospitalised for psoriasis during 1965–2004, of whom 1408 developed cancer, giving an overall standardised incidence ratios (SIRs) of 1.33. A significant excess was noted for squamous cell skin cancer, and for cancers of the upper aerodigestive tract, oesophagus, stomach, liver, pancreas, lung, kidney and bladder as well as non-Hodgkin lymphoma. Many of these may reflect the effects of alcohol drinking and tobacco smoking. Patients with multiple hospitalisations showed high risk, particularly for oesophageal (SIR 6.97) and skin (SIR 4.76) cancers

Identifying strategies to improve access to credible and relevant information for public health professionals: a qualitative study

BACKGROUND: Movement towards evidence-based practices in many fields suggests that public health (PH) challenges may be better addressed if credible information about health risks and effective PH practices is readily available. However, research has shown that many PH information needs are unmet. In addition to reviewing relevant literature, this study performed a comprehensive review of existing information resources and collected data from two representative PH groups, focusing on identifying current practices, expressed information needs, and ideal systems for information access. METHODS: Nineteen individual interviews were conducted among employees of two domains in a state health department – communicable disease control and community health promotion. Subsequent focus groups gathered additional data on preferences for methods of information access and delivery as well as information format and content. Qualitative methods were used to identify themes in the interview and focus group transcripts. RESULTS: Informants expressed similar needs for improved information access including single portal access with a good search engine; automatic notification regarding newly available information; access to best practice information in many areas of interest that extend beyond biomedical subject matter; improved access to grey literature as well as to more systematic reviews, summaries, and full-text articles; better methods for indexing, filtering, and searching for information; and effective ways to archive information accessed. Informants expressed a preference for improving systems with which they were already familiar such as PubMed and listservs rather than introducing new systems of information organization and delivery. A hypothetical ideal model for information organization and delivery was developed based on informants' stated information needs and preferred means of delivery. Features of the model were endorsed by the subjects who reviewed it. CONCLUSION: Many critical information needs of PH practitioners are not being met efficiently or at all. We propose a dual strategy of: 1) promoting incremental improvements in existing information delivery systems based on the expressed preferences of the PH users of the systems and 2) the concurrent development and rigorous evaluation of new models of information organization and delivery that draw on successful resources already operating to deliver information to clinical medical practitioners

A powerful test for multiple rare variants association studies that incorporates sequencing qualities

Next-generation sequencing data will soon become routinely available for association studies between complex traits and rare variants. Sequencing data, however, are characterized by the presence of sequencing errors at each individual genotype. This makes it especially challenging to perform association studies of rare variants, which, due to their low minor allele frequencies, can be easily perturbed by genotype errors. In this article, we develop the quality-weighted multivariate score association test (qMSAT), a new procedure that allows powerful association tests between complex traits and multiple rare variants under the presence of sequencing errors. Simulation results based on quality scores from real data show that the qMSAT often dominates over current methods, that do not utilize quality information. In particular, the qMSAT can dramatically increase power over existing methods under moderate sample sizes and relatively low coverage. Moreover, in an obesity data study, we identified using the qMSAT two functional regions (MGLL promoter and MGLL 3′-untranslated region) where rare variants are associated with extreme obesity. Due to the high cost of sequencing data, the qMSAT is especially valuable for large-scale studies involving rare variants, as it can potentially increase power without additional experimental cost. qMSAT is freely available at http://qmsat.sourceforge.net/