BackgroundReduced representation bisulfite sequencing (RRBS) was developed to measure DNA methylation of high-CG regions at single base-pair resolution, and has been widely used because of its minimal DNA requirements and cost efficacy; however, the CpG coverage of genomic regions is restricted and important regions with low-CG will be ignored in DNA methylation profiling. This method could be improved to generate a more comprehensive representation.ResultsBased on in silico simulation of enzyme digestion of human and mouse genomes, we have optimized the current single-enzyme RRBS by applying double enzyme digestion in the library construction to interrogate more representative regions. CpG coverage of genomic regions was considerably increased in both high-CG and low-CG regions using the double-enzyme RRBS method, leading to more accurate detection of their average methylation levels and identification of differential methylation regions between samples. We also applied this double-enzyme RRBS method to comprehensively analyze the CpG methylation profiles of two colorectal cancer cell lines.ConclusionThe double-enzyme RRBS increases the CpG coverage of genomic regions considerably over the previous single-enzyme RRBS method, leading to more accurate detection of their average methylation levels. It will facilitate genome-wide DNA methylation studies in multiple and complex clinical samples.

BackgroundDifferences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis. The simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine is likely to stimulate the discovery of aberrantly methylated genes with increased accuracy in human hepatocellular carcinoma.ResultsHere, we performed ultra-performance liquid chromatography/tandem mass spectrometry and single-base high-throughput sequencing, Hydroxymethylation and Methylation Sensitive Tag sequencing, HMST-seq, to synchronously measure these two modifications in human hepatocellular carcinoma samples. After identification of differentially methylated and hydroxymethylated genes in human hepatocellular carcinoma, we integrate DNA copy-number alterations, as determined using array-based comparative genomic hybridization data, with gene expression to identify genes that are potentially silenced by promoter hypermethylation.ConclusionsWe report a high enrichment of genes with epigenetic aberrations in cancer signaling pathways. Six genes were selected as tumor suppressor gene candidates, among which, ECM1, ATF5 and EOMES are confirmed via siRNA experiments to have potential anti-cancer functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0533-9) contains supplementary material, which is available to authorized users.

Objective: Huangqin decoction (HQD), a classical traditional Chinese medicinal formula, has been commonly used to treat gastrointestinal diseases for thousands of years. We investigated the anti-inflammatory effects and underlying mechanisms of HQD on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC).Methods: Experimental mice were given 3% DSS, and HQD (2.275, 4.55, and 9.1 g/kg), or mesalazine (ME, 200 mg/kg) orally for 7 days. Body weight loss, disease activity index (DAI), colon length, histology, and levels of inflammatory cytokines were measured to evaluate the effects of HQD on colitis. The effects of HQD on the Ras-phosphoinositide-3kinase (PI3K)-Akt-hypoxia inducible factor 1 alpha (HIF-1a) and nuclear factor-kappa B (NF-kB) pathways were evaluated by Western blot analysis. In addition, the gut microbiota was characterized using high-throughput Illumina MiSeq sequencing. Results:The results showed that HQD significantly reduced the body weight loss, ameliorated DAI, restored colon length, and improved the intestinal epithelial cell barrier in mice with DSS-induced colitis. The messenger RNA (mRNA) expression levels of inflammatory mediators were decreased following HQD treatment. Furthermore, the Ras-PI3K-Akt-HIF-1a and NF-kB pathways were significantly inhibited by HQD. Finally, treatment with HQD resulted in recovery of gut microbiota diversity.Conclusions: HQD ameliorates DSS-induced colitis through regulation of the gut microbiota, and suppression of Ras-PI3K-Akt-HIF-1a and NF-kB pathways. Our results suggested that HQD may be a potential candidate for treatment of UC.GRAPHICAL ABSTRACT | This study demonstrated that supplementation of Huangqin decoction (HQD) attenuated dextran sulfate sodium (DSS)-induced ulcerative colitis by regulating the gut microbiota and suppressing Ras-PI3K-Akt-HIF-1a and NF-kB pathways in mice.

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BackgroundDNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported.Methodology/Principal FindingsThe DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to “neurogenesis” and “cell differentiation” by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples.Conclusions/SignificanceWe characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research.

The consumption of probiotics and fermented foods has been very popular in recent decades. The primary aim of our study was to evaluate the effect of probiotics on the gut microbiota and the changes in inflammatory cytokines after an average of 6.7 weeks of probiotic administration among normal pregnant women. Thirty-two healthy pregnant women at 32 weeks of gestation were recruited and divided into two groups. The probiotic group ingested combined probiotics until after birth. The base characteristics of the probiotics and control groups showed no significant differences. The structure of the fecal microbiota at the genus level varied during the third trimester, and administration of probiotics had no influence on the composition of the fecal microbiota however, many highly abundant taxa and core microbiota at the genus level changed in the probiotic group when compared to the control group. The analysis of cytokines showed that IL-5, IL-6, TNF-α, and GM-CSF had equal levels between the baseline and control groups but were significantly increased after probiotic administration (baseline = control < probiotics). Additionally, levels of IL-1β, IL-2, IL-12, and IFN-γ significantly increased among the three groups (baseline < control < probiotics). This result demonstrated that probiotics helped to shift the anti-inflammatory state to a pro-inflammatory state. The correlation analysis outcome suggested that the relationship between the microbiota and the cytokines was not strain-dependent. The gut microbiota varied during the third trimester. The probiotics demonstrated immunomodulation effects that helped to switch over to a pro-inflammatory immune state in the third trimester, which was important for labor.

BackgroundDNA methylation plays important roles in gene regulation during both normal developmental and disease states. In the past decade, a number of methods have been developed and applied to characterize the genome-wide distribution of DNA methylation. Most of these methods endeavored to screen whole genome and turned to be enormously costly and time consuming for studies of the complex mammalian genome. Thus, they are not practical for researchers to study multiple clinical samples in biomarker research.ResultsHere, we display a novel strategy that relies on the selective capture of target regions by liquid hybridization followed by bisulfite conversion and deep sequencing, which is referred to as liquid hybridization capture-based bisulfite sequencing (LHC-BS). To estimate this method, we utilized about 2 μg of native genomic DNA from YanHuang (YH) whole blood samples and a mature dendritic cell (mDC) line, respectively, to evaluate their methylation statuses of target regions of exome. The results indicated that the LHC-BS system was able to cover more than 97% of the exome regions and detect their methylation statuses with acceptable allele dropouts. Most of the regions that couldn't provide accurate methylation information were distributed in chromosomes 6 and Y because of multiple mapping to those regions. The accuracy of this strategy was evaluated by pair-wise comparisons using the results from whole genome bisulfite sequencing and validated by bisulfite specific PCR sequencing.ConclusionsIn the present study, we employed a liquid hybridisation capture system to enrich for exon regions and then combined with bisulfite sequencing to examine the methylation statuses for the first time. This technique is highly sensitive and flexible and can be applied to identify differentially methylated regions (DMRs) at specific genomic locations of interest, such as regulatory elements or promoters.

5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicates 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. Here we describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in a subset of cytosines in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-Sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occurs in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. Future application of this technology would enable us to uncover the status of methylation and hydroxymethylation in dynamic biological processes and disease development in multiple biological samples.