LiBis: an ultrasensitive alignment augmentation for low-input bisulfite sequencing.

The cell-free DNA (cfDNA) methylation profile in liquid biopsy has been utilized to diagnose early-stage disease and estimate therapy response. However, typical clinical procedures are capable of purifying only very small amounts of cfDNA. Whole-genome bisulfite sequencing (WGBS) is the gold standard for measuring DNA methylation; however, WGBS using small amounts of fragmented DNA introduces a critical challenge for data analysis, namely a low-mapping ratio. The resulting low sequencing depth and low coverage of CpG sites genome-wide is a bottleneck for the clinical application of cfDNA-based WGBS assays. We developed LiBis (Low-input Bisulfite Sequencing), a novel method for low-input WGBS data alignment. By dynamically clipping initially unmapped reads and remapping clipped fragments, we judiciously rescued those reads and uniquely aligned them to the genome. By substantially increasing the mapping ratio by up to 88%, LiBis dramatically improved the number of informative CpGs and the precision in quantifying the methylation status of individual CpG sites. LiBis significantly improved the cost efficiency of low-input WGBS experiments by dynamically removing contamination introduced by random priming. The high sensitivity and cost effectiveness afforded by LiBis for low-input samples will allow the discovery of genetic and epigenetic features suitable for downstream analysis and biomarker identification using liquid biopsy.

Increased Pathogenicity and Virulence of Middle East Respiratory Syndrome Coronavirus Clade B In Vitro and In Vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-ß) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.

Human pluripotent stem cell-derived chondroprogenitors for cartilage tissue engineering.

The cartilage of joints, such as meniscus and articular cartilage, is normally long lasting (i.e., permanent). However, once damaged, especially in large animals and humans, joint cartilage is not spontaneously repaired. Compensating the lack of repair activity by supplying cartilage-(re)forming cells, such as chondrocytes or mesenchymal stromal cells, or by transplanting a piece of normal cartilage, has been the basis of therapy for biological restoration of damaged joint cartilage. Unfortunately, current biological therapies face problems on a number of fronts. The joint cartilage is generated de novo from a specialized cell type, termed a 'joint progenitor' or 'interzone cell' during embryogenesis. Therefore, embryonic chondroprogenitors that mimic the property of joint progenitors might be the best type of cell for regenerating joint cartilage in the adult. Pluripotent stem cells (PSCs) are expected to differentiate in culture into any somatic cell type through processes that mimic embryogenesis, making human (h)PSCs a promising source of embryonic chondroprogenitors. The major research goals toward the clinical application of PSCs in joint cartilage regeneration are to (1) efficiently generate lineage-specific chondroprogenitors from hPSCs, (2) expand the chondroprogenitors to the number needed for therapy without loss of their chondrogenic activity, and (3) direct the in vivo or in vitro differentiation of the chondroprogenitors to articular or meniscal (i.e., permanent) chondrocytes rather than growth plate (i.e., transient) chondrocytes. This review is aimed at providing the current state of research toward meeting these goals. We also include our recent achievement of successful generation of "permanent-like" cartilage from long-term expandable, hPSC-derived ectomesenchymal chondroprogenitors.

Structural basis for effects of CpA modifications on C/EBPß binding of DNA.

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPß binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPß DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPß. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

GsmPlot: a web server to visualize epigenome data in NCBI.

BACKGROUND: Epigenetic regulation is essential in regulating gene expression across a variety of biological processes. Many high-throughput sequencing technologies have been widely used to generate epigenetic data, such as histone modification, transcription factor binding sites, DNA modifications, chromatin accessibility, and etc. A large scale of epigenetic data is stored in NCBI Gene Expression Omnibus (GEO). However, it is a great challenge to reanalyze these large scale and complex data, especially for researchers who do not specialize in bioinformatics skills or do not have access to expensive computational infrastructure. RESULTS: GsmPlot can simply accept GSM IDs to automatically download NCBI data or can accept user's private bigwig files as input to plot the concerned data on promoters, exons or any other user-defined genome locations and generate UCSC visualization tracks. By linking public data repository and private data, GsmPlot can spark data-driven ideas and hence promote the epigenetic research. CONCLUSIONS: GsmPlot web server allows convenient visualization and efficient exploration of any NCBI epigenetic data in any genomic region without need of any bioinformatics skills or special computing resources. GsmPlot is freely available at https://gsmplot.deqiangsun.org/.

Decoding the dynamic DNA methylation and hydroxymethylation landscapes in endodermal lineage intermediates during pancreatic differentiation of hESC.

Dynamic changes in DNA methylation and demethylation reprogram transcriptional outputs to instruct lineage specification during development. Here, we applied an integrative epigenomic approach to unveil DNA (hydroxy)methylation dynamics representing major endodermal lineage intermediates during pancreatic differentiation of human embryonic stem cells (hESCs). We found that 5-hydroxymethylcytosine (5hmC) marks genomic regions to be demethylated in the descendent lineage, thus reshaping the DNA methylation landscapes during pancreatic lineage progression. DNA hydroxymethylation is positively correlated with enhancer activities and chromatin accessibility, as well as the selective binding of lineage-specific pioneer transcription factors, during pancreatic differentiation. We further discovered enrichment of hydroxymethylated regions (termed '5hmC-rim') at the boundaries of large hypomethylated functional genomic regions, including super-enhancer, DNA methylation canyon and broad-H3K4me3 peaks. We speculate that '5hmC-rim' might safeguard low levels of cytosine methylation at these regions. Our comprehensive analysis highlights the importance of dynamic changes of epigenetic landscapes in driving pancreatic differentiation of hESC.

Smurf2, an E3 ubiquitin ligase, interacts with PDE4B and attenuates liver fibrosis through miR-132 mediated CTGF inhibition.

We previously reported that Smad ubiquitin regulatory factor 2 (Smurf2) activity was decreased in human fibrotic livers. Here, we overexpressed Smurf2 in livers of transgenic mice and observed inhibited collagen deposition and hepatic stellate cell activation in fibrotic model induced by carbon tetrachloride treatment or bile duct ligation. Hepatic Smurf2 overexpression also inhibited the production of connective tissue growth factor (CTGF), a central mediator of liver fibrosis. Using miRNA array and bioinformatics analyses, we identified miR-132 as a mediator of this inhibitory effect. miR-132 directly targets the 3'-untranslated region of CTGF and was transcriptionally upregulated by cAMP-PKA-CREB signaling. In addition, Smurf2 activated cAMP-PKA-CREB pathway by interacting with phosphodiesterase 4B (PDE4B) and facilitating its degradation. Thus, we have demonstrated a previously unrecognized anti-fibrotic pathway controlled by Smurf2.

Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling.

The Ten-eleven translocation (TET) family of 5-methylcytosine (5mC) dioxygenases catalyze the conversion of 5mC into 5-hydroxymethylcytosine (5hmC) and further oxidized species to promote active DNA demethylation. Here we engineered a split-TET2 enzyme to enable temporal control of 5mC oxidation and subsequent remodeling of epigenetic states in mammalian cells. We further demonstrate the use of this chemically inducible system to dissect the correlation between DNA hydroxymethylation and chromatin accessibility in the mammalian genome. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype-phenotype relations in various biological systems.

Oncogenic targets Mmp7, S100a9, Nppb and Aldh1a3 from transcriptome profiling of FAP and Pirc adenomas are downregulated in response to tumor suppression by Clotam.

Intervention strategies in familial adenomatous polyposis (FAP) patients and other high-risk colorectal cancer (CRC) populations have highlighted a critical need for endoscopy combined with safe and effective preventive agents. We performed transcriptome profiling of colorectal adenomas from FAP patients and the polyposis in rat colon (Pirc) preclinical model, and prioritized molecular targets for prevention studies in vivo. At clinically relevant doses in the Pirc model, the drug Clotam (tolfenamic acid, TA) was highly effective at suppressing tumorigenesis both in the colon and in the small intestine, when administered alone or in combination with Sulindac. Cell proliferation in the colonic crypts was reduced significantly by TA, coincident with increased cleaved caspase-3 and decreased Survivin, ß-catenin, cyclin D1 and matrix metalloproteinase 7. From the list of differentially expressed genes prioritized by transcriptome profiling, Mmp7, S100a9, Nppb and Aldh1a3 were defined as key oncogene candidates downregulated in colon tumors after TA treatment. Monthly colonoscopies revealed the rapid onset of tumor suppression by TA in the Pirc model, and the temporal changes in Mmp7, S100a9, Nppb and Aldh1a3, highlighting their value as potential early biomarkers for prevention in the clinical setting. We conclude that TA, an "old drug" repurposed from migraine, offers an exciting new therapeutic avenue in FAP and other high-risk CRC patient populations.

BSeQC: quality control of bisulfite sequencing experiments.

MOTIVATION: Bisulfite sequencing (BS-seq) has emerged as the gold standard to study genome-wide DNA methylation at single-nucleotide resolution. Quality control (QC) is a critical step in the analysis pipeline to ensure that BS-seq data are of high quality and suitable for subsequent analysis. Although several QC tools are available for next-generation sequencing data, most of them were not designed to handle QC issues specific to BS-seq protocols. Therefore, there is a strong need for a dedicated QC tool to evaluate and remove potential technical biases in BS-seq experiments. RESULTS: We developed a package named BSeQC to comprehensively evaluate the quality of BS-seq experiments and automatically trim nucleotides with potential technical biases that may result in inaccurate methylation estimation. BSeQC takes standard SAM/BAM files as input and generates bias-free SAM/BAM files for downstream analysis. Evaluation based on real BS-seq data indicates that the use of the bias-free SAM/BAM file substantially improves the quantification of methylation level. AVAILABILITY AND IMPLEMENTATION: BSeQC is freely available at: http://code.google.com/p/bseqc/.

RRBSMAP: a fast, accurate and user-friendly alignment tool for reduced representation bisulfite sequencing.

SUMMARY: Reduced representation bisulfite sequencing (RRBS) is a powerful yet cost-efficient method for studying DNA methylation on a genomic scale. RRBS involves restriction-enzyme digestion, bisulfite conversion and size selection, resulting in DNA sequencing data that require special bioinformatic handling. Here, we describe RRBSMAP, a short-read alignment tool that is designed for handling RRBS data in a user-friendly and scalable way. RRBSMAP uses wildcard alignment, and avoids the need for any preprocessing or post-processing steps. We benchmarked RRBSMAP against a well-validated MAQ-based pipeline for RRBS read alignment and observed similar accuracy but much improved runtime performance, easier handling and better scaling to large sample sets. In summary, RRBSMAP removes bioinformatic hurdles and reduces the computational burden of large-scale epigenome association studies performed with RRBS. AVAILABILITY: http://rrbsmap.computational-epigenetics.org/ http://code.google.com/p/bsmap/ CONTACT: wl1@bcm.tmc.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of the Dynamic Cushioning Property of Expanded Polyethylene Based on the Stress-Energy Method.

This paper aimed to experimentally clarify the dynamic crushing performance of expanded polyethylene (EPE) and analyze the influence of thickness and dropping height on its mechanical behavior based on the stress-energy method. Hence, a series of impact tests are carried out on EPE foams with different thicknesses and dropping heights. The maximum acceleration, static stress, dynamic stress and dynamic energy of EPE specimens are obtained through a dynamic impact test. Then, according to the principle of the stress-energy method, the functional relationship between dynamic stress and dynamic energy is obtained through exponential fitting and polynomial fitting, and the cushion material constants a, b and c are determined. The maximum acceleration-static stress curves of any thickness and dropping height can be further fitted. By the equipartition energy domain method, the range of static stress can be expanded, which is very fast and convenient. When analyzing the influence of thickness and dropping height on the dynamic cushioning performance curves of EPE, it is found that at the same drop height, with the increase of thickness, the opening of the curve gradually becomes larger. The minimum point on the maximum acceleration-static stress curve also decreases with the increase of the thickness. When the dropping height is 400 mm, compared to foam with a thickness of 60 mm, the tested maximum acceleration value of the lowest point of the specimen with a thickness of 40 mm increased by 45.3%, and the static stress is both 5.5 kPa. When the thickness of the specimen is 50 mm, compared to the dropping height of 300 mm, the tested maximum acceleration value of the lowest point of the specimen with a dropping height of 600 mm increased by 93.3%. Therefore, the dynamic cushioning performance curve of EPE foams can be quickly obtained by the stress-energy method when the precision requirement is not high, which provides a theoretical basis for the design of cushion packaging.

An Analysis of the Vibration Transmission Properties of Assemblies Using Honeycomb Paperboard and Expanded Polyethylene.

In the process of logistics, shock and vibration are the most important factors contributing to product damage. Assembling honeycomb paperboard and EPE is commonly used to provide cushioning and anti-vibration effects to materials. Therefore, it is necessary to study the vibration transmission properties of this kind of assembly in the anti-vibration process. The aim of this paper was to experimentally determine the vibration transmission properties of assemblies with honeycomb paperboard and expanded polyethylene (EPE). Through a sinusoidal sweep vibration test of this assembly, the vibration transmission characteristic curves of assemblies with honeycomb paperboard and EPE of different thicknesses were obtained and compared. Assuming the assembly and mass block as a single degree of freedom with a small damping linear system, the damping energy dissipation of the assembly and the resonance frequency were obtained. The vibration transmission property curves of the assembly can be divided into four regions. With an excitation acceleration of 0.5 g and a honeycomb paperboard with a thickness of 60 mm (F60), the vibration transmission rate and the resonance frequency-of the material dampened with EPE at a thickness of 60 mm (E60), and the assembly (F30/E30) with a 30 mm thick honeycomb paperboard and 30 mm thick EPE-increased by -2.5% and -17.5%, -86.9% and 79.3%, and -95.9% and -85.7%, respectively. Compared to the assembly with 20 mm thick honeycomb and 20 mm thick EPE (F20/E20), the vibration transmission rate, the resonance frequency, and the material damping and damping energy dissipation of F40/E20, F30/E30, and F20/E40 increased by 75.6%, 48.3%, and 66.1%; 1.2%, -21.5%, and -38.9%; 241.5%, 82.8%, and 13.3%; and 12.5%, 98.9%, and 106.8%, respectively. Compared to F60 and E60, the damping energy dissipation of F30/E30 increased by 2816.7% and 133.3%, respectively. The assembly of F30/E30 has the smallest vibration transmission rate and the most vibration energy dissipation among these assemblies. This means that the assembly of F30/E30 absorbs the most external vibration energy, while the acceleration that is transmitted to the internal product is minimal. Therefore, in the design of cushioning packaging, according to the characteristics and natural frequency of the internal products, an appropriate assembly can be selected, which should have a lower vibration transmission rate and more vibration energy dissipation, and should not resonate with the internal product. This will provide a theoretical basis for the design of cushioning packaging.

Crushing Responses of Expanded Polypropylene Foam.

This paper aimed to experimentally clarify the crushing mechanism and performance of expanded polypropylene foam (EPP) and analyze the influence of density and thickness on its mechanical behavior and energy absorption properties under static crushing loadings. Hence, a series of compression tests were carried out on EPP foams with different densities and thicknesses. For foam with a density of 60 kg/m3, the mean crushing strength, energy absorption (Ea), energy absorption efficiency (Ef), specific energy absorption (SEA), and energy absorption per unit volume (w) increased by 245.3%, 187.2%, 42.3%, 54.3%, and 242.8%, respectively, compared to foam with a density of 20 kg/m3. Meanwhile, compared to foam with a thickness of 30 mm, the mean crushing strength, energy absorption (Ea), energy absorption efficiency (Ef), SEA, and energy absorption per unit volume (w) for foam with a thickness of 75 mm increased by 53.3%, 25.2%, -10.8%, -4.7%, and -10.6%, respectively. The results show that foam density has a significantly greater influence on static compressive performance than foam thickness. The microstructures of the EPP foam before and after static compression were compared by observing with a scanning electron microscope (SEM), and the failure mechanism was analyzed. Results showed that the load and energy as well as the deformation and instability processes of its cells were transferred layer by layer. The influence of density on the degree of destruction of the internal cells was obvious. Due to its larger mass and larger internal damping, thicker foams were less damaged, and less deformation was produced. Additionally, the EPP foam exhibited a considerable ability to recover after compression.

Pan-cancer Analysis Reveals Cancer-dependent Expression of SOX17 and Associated Clinical Outcomes.

BACKGROUND/AIM: SRY-box containing gene 17 (SOX17) plays a pivotal role in cancer onset and progression and is considered a potential target for cancer diagnosis and treatment. However, the expression pattern of SOX17 in cancer and its clinical relevance remains unknown. Here, we explored the relationship between the expression of SOX17 and drug response by examining SOX17 expression patterns across multiple cancer types. MATERIALS AND METHODS: Single-cell and bulk RNA-seq analyses were used to explore the expression profile of SOX17. Analysis results were verified with qPCR and immunohistochemistry. Survival, drug response, and co-expression analyses were performed to illustrate its correlation with clinical outcomes. RESULTS: The results revealed that abnormal expression of SOX17 is highly heterogenous across multiple cancer types, indicating that SOX17 manifests as a cancer type-dependent feature. Furthermore, the expression pattern of SOX17 is also associated with cancer prognosis in certain cancer types. Strong SOX17 expression correlates with the potency of small molecule drugs that affect PI3K/mTOR signaling. FGF18, a gene highly relevant to SOX17, is involved in the PI3K-AKT signaling pathway. Single-cell RNA-seq analysis demonstrated that SOX17 is mainly expressed in endothelial cells and barely expressed in other cells but spreads to other cell types during the development of ovarian cancer. CONCLUSION: Our study revealed the expression pattern of SOX17 in pan-cancer through bulk and single-cell RNA-seq analyses and determined that SOX17 is related to the diagnosis, staging, and prognosis of some tumors. These findings have clinical implications and may help identify mechanistic pathways amenable to pharmacological interventions.

Mechanical Behavior of Closed-Cell Ethylene-Vinyl Acetate Foam under Compression.

The static and dynamic compressions of closed-cell ethylene-vinyl acetate (EVA) foams with different densities were conducted under various strain rates. The stress-strain curves were processed to determine the corresponding curves of energy absorption per unit volume and energy absorption efficiency, and energy absorption diagrams were produced. The influences of density and strain rate on the elastic modulus, yield strength, energy absorption per unit volume, optimal strain, densification strain, and energy absorption diagrams were analyzed and discussed. The whole stress-strain curve can be fitted with the Rusch formula. The strain rate does not change the shape of stress-strain curve, and has little influence on the elastic modulus. There exists the optimal density of EVA foam corresponding to its maximum energy absorption efficiency. Under a fixed strain rate, the optical energy absorption per unit volume is proportional to the optical stress on the envelope line in the energy absorption diagrams of EVA foams with different densities. The change in strain rate leads to the envelope line in the energy absorption diagrams of EVA foams with a given density having the larger slope and a negative intercept where the optical energy absorption per unit volume relies linearly on the optical stress. The empirical formulas of elastic modulus, yield strength, optimal strain, and envelope lines and their slopes are derived from the tested results.

Epigenome-wide development and validation of a prognostic methylation score in intrahepatic cholangiocarcinoma based on machine learning strategies.

Background: Clinical parameter-based nomograms and staging systems provide limited information for the prediction of survival in intrahepatic cholangiocarcinoma (ICC) patients. In this study, we developed a methylation signature that precisely predicts overall survival (OS) after surgery. Methods: An epigenome-wide study of DNA methylation based on whole-genome bisulfite sequencing (WGBS) was conducted for two independent cohorts (discovery cohort, n=164; validation cohort, n=170) from three hepatobiliary centers in China. By referring to differentially methylated regions (DMRs), we proposed the concept of prognostically methylated regions (PMRs), which were composed of consecutive prognostically methylated CpGs (PMCs). Using machine learning strategies (Random Forest and the least absolute shrinkage and selector regression), a prognostic methylation score (PMS) was constructed based on 14 PMRs in the discovery cohort and confirmed in the validation cohort. Results: The C-indices of the PMS for predicting OS in the discovery and validation cohorts were 0.79 and 0.74, respectively. In the whole cohort, the PMS was an independent predictor of OS [hazard ratio (HR) =8.12; 95% confidence interval (CI): 5.48-12.04; P<0.001], and the C-index (0.78) of the PMS was significantly higher than that of the Johns Hopkins University School of Medicine (JHUSM) nomogram (0.69, P<0.001), the Eastern Hepatobiliary Surgery Hospital (EHBSH) nomogram (0.67, P<0.001), American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system (0.61, P<0.001), and MEGNA prognostic score (0.60, P<0.001). The patients in quartile 4 of PMS could benefit from adjuvant therapy (AT) (HR =0.54; 95% CI: 0.32-0.91; log-rank P=0.043), whereas those in the quartiles 1-3 could not. However, other nomograms and staging system failed to do so. Further analyses of potential mechanisms showed that the PMS was associated with tumor biological behaviors, pathway activation, and immune microenvironment. Conclusions: The PMS could improve the prognostic accuracy and identify patients who would benefit from AT for ICC patients, and might facilitate decisions in treatment of ICC patients.

TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation.

Existing knowledge of the role of epigenetic modifiers in pancreas development has exponentially increased. However, the function of TET dioxygenases in pancreatic endocrine specification remains obscure. We set out to tackle this issue using a human embryonic stem cell (hESC) differentiation system, in which TET1/TET2/TET3 triple knockout cells display severe defects in pancreatic ß-cell specification. The integrative whole-genome analysis identifies unique cell-type-specific hypermethylated regions (hyper-DMRs) displaying reduced chromatin activity and remarkable enrichment of FOXA2, a pioneer transcription factor essential for pancreatic endoderm specification. Intriguingly, TET depletion leads to significant changes in FOXA2 binding at the pancreatic progenitor stage, in which gene loci with decreased FOXA2 binding feature low levels of active chromatin modifications and enriches for bHLH motifs. Transduction of full-length TET1 but not the TET1-catalytic-domain in TET-deficient cells effectively rescues ß-cell differentiation accompanied by restoring PAX4 hypomethylation. Taking these findings together with the defective generation of functional ß-cells upon TET1-inactivation, our study unveils an essential role of TET1-dependent demethylation in establishing ß-cell identity. Moreover, we discover a physical interaction between TET1 and FOXA2 in endodermal lineage intermediates, which provides a mechanistic clue regarding the complex crosstalk between TET dioxygenases and pioneer transcription factors in epigenetic regulation during pancreas specification.

Deficiency of a novel lncRNA-HRAT protects against myocardial ischemia reperfusion injury by targeting miR-370-3p/RNF41 pathway.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) contribute to myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms by which lncRNAs modulate myocardial I/R injury have not been thoroughly examined and require further investigation. A novel lncRNA named lncRNA-hypoxia/reoxygenation (H/R)-associated transcript (lncRNA-HRAT) was identified by RNA sequencing analysis. The expression of lncRNA-HRAT exhibited a significant increase in the I/R mice hearts and cardiomyocytes treated with H/R. LncRNA-HRAT overexpression facilitates H/R-induced cardiomyocyte apoptosis. Furthermore, cardiomyocyte-specific deficiency of lncRNA-HRAT in vivo after I/R decreased creatine kinase (CK) release in the serum, reduced myocardial infarct area, and improved cardiac dysfunction. Molecular mechanistic investigations revealed that lncRNA-HRAT serves as a competing endogenous RNA (ceRNA) of miR-370-3p, thus upregulating the expression of ring finger protein 41 (RNF41), thereby aggravating apoptosis in cardiomyocytes induced by H/R. This study revealed that the lncRNA-HRAT/miR-370-3p/RNF41 pathway regulates cardiomyocyte apoptosis and myocardial injury. These findings suggest that targeted inhibition of lncRNA-HRAT may offer a novel therapeutic method to prevent myocardial I/R injury.

Single-Cell RNA-Sequencing Atlas Reveals the Tumor Microenvironment of Metastatic High-Grade Serous Ovarian Carcinoma.

Ovarian cancer is the most common and lethal gynecological tumor in women worldwide. High-grade serous ovarian carcinoma (HGSOC) is one of the histological subtypes of epithelial ovarian cancer, accounting for 70%. It often occurs at later stages associated with a more fatal prognosis than endometrioid carcinomas (EC), another subtype of epithelial ovarian cancer. However, the molecular mechanism and biology underlying the metastatic HGSOC (HG_M) immunophenotype remain poorly elusive. Here, we performed single-cell RNA sequencing analyses of primary HGSOC (HG_P) samples, metastatic HGSOC (HG_M) samples, and endometrioid carcinomas (EC) samples. We found that ERBB2 and HOXB-AS3 genes were more amplified in metastasis tumors than in primary tumors. Notably, high-grade serous ovarian cancer metastases are accompanied by dysregulation of multiple pathways. Malignant cells with features of epithelial-mesenchymal transition (EMT) affiliated with poor overall survival were identified. In addition, cancer-associated fibroblasts with EMT-program were enriched in HG_M, participating in angiogenesis and immune regulation, such as IL6/STAT3 pathway activity. Compared with ECs, HGSOCs exhibited higher T cell infiltration. PRDM1 regulators may be involved in T cell exhaustion in ovarian cancer. The CX3CR1_macro subpopulation may play a role in promoting tumor progression in ovarian cancer with high expression of BAG3, IL1B, and VEGFA. The new targets we discovered in this study will be useful in the future, providing guidance on the treatment of ovarian cancer.