Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Safety and Efficacy of Nivolumab Monotherapy in Recurrent or Metastatic Cervical, Vaginal, or Vulvar Carcinoma: Results From the Phase I/II CheckMate 358 Trial

Purpose: Nivolumab was assessed in patients with virus-associated tumors in the phase I/II CheckMate 358 trial (ClinicalTrials.gov identifier: NCT02488759). We report on patients with recurrent/metastatic cervical, vaginal, or vulvar cancers. Patients and methods: Patients received nivolumab 240 mg every 2 weeks. Although patients with unknown human papillomavirus status were enrolled, patients known to have human papillomavirus-negative tumors were ineligible. The primary end point was objective response rate. Duration of response (DOR), progression-free survival, and overall survival were secondary end points. Safety and patient-reported outcomes were exploratory end points. Results: Twenty-four patients (cervical, n = 19; vaginal/vulvar, n = 5) were enrolled. Most patients had received prior systemic therapy for metastatic disease (cervical, 78.9%; vaginal/vulvar, 80.0%). Objective response rates were 26.3% (95% CI, 9.1 to 51.2) for cervical cancer and 20.0% (95% CI, 0.5 to 71.6) for vaginal/vulvar cancers. At a median follow-up of 19.2 months, median DOR was not reached (range, 23.3 to 29.5+ months; + indicates a censored observation) in the five responding patients in the cervical cohort; the DOR was 5.0 months in the single responding patient in the vaginal/vulvar cohort. Median overall survival was 21.9 months (95% CI, 15.1 months to not reached) among patients with cervical cancer. Any-grade treatment-related adverse events were reported in 12 of 19 patients (63.2%) in the cervical cohort and all five patients in the vaginal/vulvar cohort; there were no treatment-related deaths. In the cervical cohort, nivolumab treatment generally resulted in stabilization of patient-reported outcomes associated with health status and health-related quality of life. Conclusion: The efficacy of nivolumab in patients with recurrent/metastatic cervical and vaginal or vulvar cancers is promising and warrants additional investigation. No new safety signals were identified with nivolumab treatment in this population

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Variation and Plasticity of DNA Methylation in Genome Regulation

DNA methylation is an important and well-studied epigenetic mark. It plays a pivotal role in imprinting, X-chromosome inactivation and genome stability and regulation in many normal development processes. DNA methylation, along with histone modifications, orchestrates the cell type- and developmental stage-specific chromatin landscapes and influences gene expression in vertebrates. In this thesis, I utilized the latest next-generation sequencing based methods to map DNA methylation levels at a genome-wide scale to investigate two areas of interest: the variation of DNA methylation across a large number of sample types and its functional potentials; the plasticity of DNA methylation in response to environmental stimuli including both the commensal gut microbiota and pathogenic Helicobactor pylori. In the first section of the thesis, I analyzed the largest collection of complete human DNA methylomes at single CpG resolution with 54 normal human samples representing 21 cell types to better understand the variation of DNA methylation at a genome-wide scale. I uncovered the general pattern of DNA methylation for normal somatic cells with a near-constant ratio of methylated and unmethylated CpGs, but the specific CpGs that are methylated or unmethylated can be dynamic and cell type-specific. I segmented the genome into regions with distinct DNA methylation signatures and focused the analysis on 22.6% of autosomal CpGs that can be variably methylated across cell types. These variably methylated regions (VMRs) are associated with enhancer chromatin states, and some have been validated as enhancers. They are also associated with transcription factor binding sites and GWAS variants enrichment sites. The evidence suggests a regulatory role of variable DNA methylation in modulating cell type specificity. In the second section of the thesis, I examined the plasticity of DNA methylation in response to environmental stimuli in two projects where I utilized H. pylori infection of a gastric cell line and the gnotobiotic mouse as two model systems. In both projects, commensal gut microbiota and pathogenic H, pylori were considered as microbial environmental factors, and I conducted experiments to collect samples with and without the influence of the environmental factor. I then applied two sequencing based assays, MeDIP-seq and MRE-seq, to profile the genome-wide DNA methylation and compared their patterns to identify genomic regions that show significant differences in DNA methylation. The overall DNA methylation patterns remain similar in both cases upon the impact of microbes. Focusing on local DNA methylation, for the mouse project, I identified hundreds of differentially methylated regions in the tissues examined and the colon is the site with the biggest difference. Some of these regions are associated with enhancer histone modification signatures, and genes near these regions are enriched for functions relevant for the tissue type. In the H. pylori project, I did not observe dramatic differences in DNA methylation between untreated and treated gastric cells, which might be due to insufficient infection time and conditions. In summary, I applied the latest high-throughput sequencing technologies for DNA methylation profiling to the study of the variation and plasticity of DNA methylation in cell type specificity and in response to environmental stimuli. These studies demonstrated the power of high-throughput epigenomic data integration in uncovering novel insights into the role of DNA methylation at unprecedented scales, and provided a foundation for additional studies on the role of DNA methylation in multicellular organism development and in genome-environment interaction

The Types Division and Severity Threshold of Traffic Conflict on Freeway Interchange

This paper selects time to collision (TTC) as the traffic conflict index. Shooting high-definition video from high altitude above freeway interchange by unmanned aircraft, the TTC was calculated by processing software. The mechanism of traffic conflict on Freeway Interchange area was researched by combining subjective and objective method, and then defined severity. Firstly, conflict observers were trained. Twelve observers were trained repeatedly on the judgment of traffic conflict type and severity cognitive until a consensus was reached. Secondly, the subjective judgment on traffic conflict types and severity was made. The video clips containing traffic conflict were selected, and then all observers together determined the type of each conflict and severity. Thirdly, TTC samples were analyzed. The obviously unsuitable TTC samples were excluded, and the rest would be tested whether meet the sample size requirement of each type of traffic conflict. Finally, the TTC threshold value was defined. According to the cumulative frequency distribution of each type TTC, 85% value was selected as severity of traffic conflict threshold

The Types Division and Severity Threshold of Traffic Conflict on Freeway Interchange

This paper selects time to collision (TTC) as the traffic conflict index. Shooting high-definition video from high altitude above freeway interchange by unmanned aircraft, the TTC was calculated by processing software. The mechanism of traffic conflict on Freeway Interchange area was researched by combining subjective and objective method, and then defined severity. Firstly, conflict observers were trained. Twelve observers were trained repeatedly on the judgment of traffic conflict type and severity cognitive until a consensus was reached. Secondly, the subjective judgment on traffic conflict types and severity was made. The video clips containing traffic conflict were selected, and then all observers together determined the type of each conflict and severity. Thirdly, TTC samples were analyzed. The obviously unsuitable TTC samples were excluded, and the rest would be tested whether meet the sample size requirement of each type of traffic conflict. Finally, the TTC threshold value was defined. According to the cumulative frequency distribution of each type TTC, 85% value was selected as severity of traffic conflict threshold

Uncovering the transcriptomic and epigenomic landscape of nicotinic receptor genes in non-neuronal tissues

Abstract Background Nicotinic acetylcholine receptors (nAChRs) play an important role in cellular physiology and human nicotine dependence, and are closely associated with many human diseases including cancer. For example, previous studies suggest that nAChRs can re-wire gene regulatory networks in lung cancer cell lines. However, the tissue specificity of nAChRs genes and their regulation remain unexplored. Result In this study, we integrated data from multiple large genomic consortiums, including ENCODE, Roadmap Epigenomics, GTEx, and FANTOM, to define the transcriptomic and epigenomic landscape of all nicotinic receptor genes across many different human tissues and cell types. We found that many important nAChRs, including CHRNA3, CHRNA4, CHRNA5, and CHRNB4, exhibited strong non-neuronal tissue-specific expression patterns. CHRNA3, CHRNA5, and CHRNB4 were highly expressed in human colon and small intestine, and CHRNA4 was highly expressed in human liver. By comparing the epigenetic marks of CHRNA4 in human liver and hippocampus, we identified a novel liver-specific transcription start site (TSS) of CHRNA4. We further demonstrated that CHRNA4 was specifically transcribed in hepatocytes but not transcribed in hepatic sinusoids and stellate cells, and that transcription factors HNF4A and RXRA were likely upstream regulators of CHRNA4. Our findings suggest that CHRNA4 has distinct transcriptional regulatory mechanisms in human liver and brain, and that this tissue-specific expression pattern is evolutionarily conserved in mouse. Finally, we found that liver-specific CHRNA4 transcription was highly correlated with genes involved in the nicotine metabolism, including CYP2A6, UGT2B7, and FMO3. These genes were significantly down-regulated in liver cancer patients, whereas CHRNA4 is also significantly down-regulated in cancer-matched normal livers. Conclusions Our results suggest important non-neuronally expressed nicotinic acetylcholine receptors in the human body. These non-neuronal expression patterns are highly tissue-specific, and are epigenetically conserved during evolution in the context of non-conserved DNA sequence

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The epigenetic landscape around CHRNB4, CHRNA5, CHRNA3, CHRNB2, and CHRNA4 in human liver, CD34-HSC, brain, colon, and lung tissues. (PDF 288 kb