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Methods for profiling DNA methylation differ in the physical principles used to detect modified cytosines. Harris et al . compare the performances of four sequencing-based technologies for genome-wide analysis of DNA methylation and combine two methods to enable detection of allelic differences in epigenetic marks. 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.

Epidermal growth factor receptor (EGFR) exon 20 insertion mutations (ex20ins) account for ≤ 12% of all EGFR‐mutant nonsmall cell lung cancers. We analysed real‐world datasets to determine the frequency of ex20ins variants, and the ability of polymerase chain reaction (PCR) and next‐generation sequencing (NGS) to identify them. Three real‐world United States NGS databases were used: GENIE, FoundationInsights, and GuardantINFORM. Mutation profiles consistent with in‐frame EGFR ex20ins were summarized. GENIE, FoundationInsights, and GuardantINFORM datasets identified 180, 627, and 627 patients with EGFR ex20ins respectively. The most frequent insertion region of exon 20 was the near loop (~ 70%), followed by the far loop (~ 30%) and the helical (~ 3–6%) regions. GENIE, FoundationInsights, and GuardantINFORM datasets identified 41, 102, and 96 unique variants respectively. An analysis of variants projected that ~ 50% of EGFR ex20ins identified by NGS would have been missed by PCR‐based assays. Given the breadth of EGFR ex20ins identified in the real‐world US datasets, the ability of PCR to identify these mutations is limited. NGS platforms are more appropriate to identify patients likely to benefit from EGFR ex20ins‐targeted therapies. Three next‐generation sequencing (NGS) databases (GENIE, FoundationInsights and GuardantINFORM) were analyzed to estimate the frequency of exon 20 insertion (ex20ins) variants in epidermal growth factor receptor (EGFR)–mutant non‐small cell lung cancer (NSCLC). Results indicate ~ 50% of ex20ins identified by NGS would have been missed by PCR and the near loop is the most frequent insertion site.

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.

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types.

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. 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. 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.

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.

New-generation human body motion sensors for wearable electronics and intelligent medicine are required to comply with stringent requirements in terms of ultralight weight, flexibility, stability, biocompatibility, and extreme precision. However, conventional sensors are hard to fulfill all these criteria due to their rigid structure, high-density sensing materials used as the constituents, as well as hermetical and compact assembly strategy. Here, we report an ultralight sensing material based on radial anisotropic porous silver fiber (RAPSF), which has been manufactured by phase separation and temperature-controlled grain growth strategy on a modified blow-spinning system. The resistance of RAPSF could be dynamically adjusted depending on the deflected shape. Furthermore, an all-fiber motion sensor (AFMS) with an ultra-low density of 68.70 mg cm −3 and an overall weigh of 7.95 mg was fabricated via layer-by-layer assembly. The sensor exhibited outstanding flexibility, breathability, biocompatibility, and remarkable body motion recognition ability. Moreover, the AFMS was shown to have great potential as an artificial intelligence throat sensor for throat state identification at the accuracy above 85%, allowing one to spot the early onset of the viral throat illness.

The fatigue behaviors of high-strength bearing steel were investigated with rotating bending fatigue loading with a frequency of 52.5 Hz. It was revealed that the high-strength steel tended to initiate at interior non-metallic inclusions in a very high-cycle fatigue regime. During fractography observation, it was also seen that the inclusion acting as a failure-originating site was seldom smaller than 10 μm. Moreover, prior austenite grains could also act as the originating source of failure when inclusion was absent. The crystal plasticity finite element method (CPFEM) was adopted to simulate the residual stress distribution around non-metallic inclusions of different sizes under different loading amplitudes. The accumulated plastic strain around the inclusion suggested that the existence of inclusion may reduce material strength and lead to more fatigue damage. The value of accumulated plastic strain around different inclusion sizes also resembled the crack nucleation or propagation of the materials. The simulation results also indicated that inclusions smaller than 5 μm had little influence on fatigue lifetimes, while inclusions larger than 10 μm had a significant influence on fatigue lifetimes.