Integrative analysis of transcriptome, DNA methylome and chromatin accessibility reveals candidate therapeutic targets in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM. DNA methylation and chromatin accessibility, as well as gene expression, were assessed by nucleosome occupancy and methylome sequencing (NOMe-seq) and RNA-seq, respectively, using the cardiac tissues of HCM patients. Compared with those of the controls, the transcriptome, DNA methylome and chromatin accessibility of the HCM myocardium showed multifaceted differences. At the transcriptome level, HCM hearts returned to the fetal gene program through decreased sarcomeric and metabolic gene expression and increased extracellular matrix gene expression. In the DNA methylome, hypermethylated and hypomethylated differentially methylated regions (DMRs) were identified in HCM. At the chromatin accessibility level, HCM hearts showed changes in different genome elements. Several transcription factors (TFs), including SP1 and EGR1, exhibited a fetal-like pattern of binding motifs in nucleosome-depleted regions (NDRs) in HCM. In particular, the inhibition of SP1 or EGR1 in an HCM mouse model harboring sarcomere mutations markedly alleviated the HCM phenotype of the mutant mice and reversed fetal gene reprogramming. Overall, this study not only provides a high precision multi-omics map of HCM heart tissue but also sheds light on the therapeutic strategy by intervening the fetal gene reprogramming in HCM.

DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure.

DEAD-box helicase 17 (DDX17) is a typical member of the DEAD-box family with transcriptional cofactor activity. Although DDX17 is abundantly expressed in the myocardium, its role in heart is not fully understood. We generated cardiomyocyte-specific Ddx17-knockout mice (Ddx17-cKO), cardiomyocyte-specific Ddx17 transgenic mice (Ddx17-Tg), and various models of cardiomyocyte injury and heart failure (HF). DDX17 is downregulated in the myocardium of mouse models of heart failure and cardiomyocyte injury. Cardiomyocyte-specific knockout of Ddx17 promotes autophagic flux blockage and cardiomyocyte apoptosis, leading to progressive cardiac dysfunction, maladaptive remodeling and progression to heart failure. Restoration of DDX17 expression in cardiomyocytes protects cardiac function under pathological conditions. Further studies showed that DDX17 can bind to the transcriptional repressor B-cell lymphoma 6 (BCL6) and inhibit the expression of dynamin-related protein 1 (DRP1). When DDX17 expression is reduced, transcriptional repression of BCL6 is attenuated, leading to increased DRP1 expression and mitochondrial fission, which in turn leads to impaired mitochondrial homeostasis and heart failure. We also investigated the correlation of DDX17 expression with cardiac function and DRP1 expression in myocardial biopsy samples from patients with heart failure. These findings suggest that DDX17 protects cardiac function by promoting mitochondrial homeostasis through the BCL6-DRP1 pathway in heart failure.

Transcriptome-Wide 5-Methylcytosine Profiling of lncRNAs in the Mouse Cerebral Ischemia Model.

An increasing body of research has demonstrated the significant role of long non-coding RNAs (lncRNAs) in the pathogenesis of stroke. They can actively contribute to the disease's progression either by directly participating in its pathogenesis or by acting as mediators through competing endogenous RNA (ceRNA) mechanisms. Concurrently, epigenetics plays a pivotal role in the pathological mechanisms underlying stroke. Epigenetic factors serve as valuable markers for disease progression, diagnostic biomarkers, and novel therapeutic targets. One of the most prevalent epigenetic modifications is 5-methylcytosine (m5C). However, the specific profiles of 5-methylcytosine in lncRNAs associated with stroke remain to be solved. Within the scope of this research, we performed a thorough transcriptome-wide analysis of m5C methylation within lncRNAs by methylated RNA immunoprecipitation sequencing (MeRIP-Seq), within a mouse stroke model induced by middle cerebral artery occlusion. Our findings reveal substantial disparities in both the quantity and distribution of m5C within the mouse stroke model compared to normal mice. This suggests a potential linkage between stroke and lncRNA m5C modifications, offering valuable insights into the mechanisms of stroke pathogenesis and the development of new drug targets.

Mapping crossover events of mouse meiotic recombination by restriction fragment ligation-based Refresh-seq.

Single-cell whole-genome sequencing methods have undergone great improvements over the past decade. However, allele dropout, which means the inability to detect both alleles simultaneously in an individual diploid cell, largely restricts the application of these methods particularly for medical applications. Here, we develop a new single-cell whole-genome sequencing method based on third-generation sequencing (TGS) platform named Refresh-seq (restriction fragment ligation-based genome amplification and TGS). It is based on restriction endonuclease cutting and ligation strategy in which two alleles in an individual cell can be cut into equal fragments and tend to be amplified simultaneously. As a new single-cell long-read genome sequencing method, Refresh-seq features much lower allele dropout rate compared with SMOOTH-seq. Furthermore, we apply Refresh-seq to 688 sperm cells and 272 female haploid cells (secondary polar bodies and parthenogenetic oocytes) from F1 hybrid mice. We acquire high-resolution genetic map of mouse meiosis recombination at low sequencing depth and reveal the sexual dimorphism in meiotic crossovers. We also phase the structure variations (deletions and insertions) in sperm cells and female haploid cells with high precision. Refresh-seq shows great performance in screening aneuploid sperm cells and oocytes due to the low allele dropout rate and has great potential for medical applications such as preimplantation genetic diagnosis.

Drug repurposing screening and mechanism analysis based on human colorectal cancer organoids.

Colorectal cancer (CRC) is a highly heterogeneous cancer and exploring novel therapeutic options is a pressing issue that needs to be addressed. Here, we established human CRC tumor-derived organoids that well represent both morphological and molecular heterogeneities of original tumors. To efficiently identify repurposed drugs for CRC, we developed a robust organoid-based drug screening system. By combining the repurposed drug library and computation-based drug prediction, 335 drugs were tested and 34 drugs with anti-CRC effects were identified. More importantly, we conducted a detailed transcriptome analysis of drug responses and divided the drug response signatures into five representative patterns: differentiation induction, growth inhibition, metabolism inhibition, immune response promotion, and cell cycle inhibition. The anticancer activities of drug candidates were further validated in the established patient-derived organoids-based xenograft (PDOX) system in vivo. We found that fedratinib, trametinib, and bortezomib exhibited effective anticancer effects. Furthermore, the concordance and discordance of drug response signatures between organoids in vitro and pairwise PDOX in vivo were evaluated. Our study offers an innovative approach for drug discovery, and the representative transcriptome features of drug responses provide valuable resources for developing novel clinical treatments for CRC.

Development and external validation of a nomogram predicting overall survival for Gastric adenocarcinoma patients with radical gastrectomy.

PURPOSE: The aim of this study was to develop and externally validate a nomogram to accurately predict the overall survival (OS) of patients with gastric adenocarcinoma who underwent radical gastrectomy. MATERIALS AND METHODS: A total of 3492 patients with gastric adenocarcinoma who underwent radical gastrectomy from 2012 to 2017 were included as the training cohort. Survival analysis was performed via Kaplan Meier method and log-rank test. Independent postoperative prognostic factors in patients with gastric adenocarcinoma were analyzed using univariate and multifactorial COX analysis methods. The prognosis nomogram was established in the training cohort and verified externally in the Surveillance, Epidemiology and End Results (SEER) database. RESULTS: According to the univariate and multifactorial COX analyses, metastatic lymph node ratio (MLNR) and five other independent prognostic factors (age at surgery, type of gastrectomy, tumor size, T stage, and pathological grade) were included in the prognostic nomogram. The nomogram had better prognostic predictive ability than the American Joint Committee on Cancer (AJCC) TNM staging in both the training (C-index: 0.736 VS. 0.668) and external validation cohort (C-index: 0.712 VS. 0.627). The calibration plots showed that the predicted survival rate was in good agreement with the actual survival rate. And the decision curve analysis (DCA) curves revealed that nomogram showed stronger ability in predicting 1-year, 3-year, and 5-year OS. CONCLUSION: This study estimated the excellent prognostic predictive power and clinical application potential of the MLNR-based nomogram, which may be used to facilitate postoperative clinical treatment decisions and potentially improve patient survival outcomes.

Comparison of surgical and endovascular left subclavian artery revascularization during thoracic aortic endovascular repair: a systematic review and meta-analysis.

Objective: The purpose of this systematic review and meta-analysis was to incorporate data from the latest clinical studies and compare the safety and efficacy of surgical left subclavian artery (LSA) revascularization and endovascular LSA revascularization during thoracic endovascular aortic repair (TEVAR). Methods: This study was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was registered with the PROSPERO database on 16 April 2023 (CRD42023414579). The Embase, MEDLINE (PubMed), and the Cochrane Library databases were searched from January 2000 to May 2023. Results: A total of 14 retrospective cohort studies with a total of 1,695 patients, were included for review. The peri-operative stroke rates of the surgical and endovascular LSA revascularization groups were 3.8% and 2.6%, respectively (P = 0.97). The peri-operative technical success rates for the surgical and endovascular LSA revascularization groups were 95.6% and 93.0%, respectively (P = 0.24). The peri-operative spinal cord ischemia rates were 1.6% (n = 18) and 1.9% (n = 7) in the surgical and endovascular LSA revascularization groups, respectively (P = 0.90). The peri-operative type Ⅰ endoleak rates for the surgical and endovascular LSA revascularization groups were 6.6% and 23.2%, respectively (P = 0.25). The subgroup analysis showed that the incidence of peri-operative type I endoleak in the parallel stent group was significantly higher than that in the surgical LSA revascularization group (P < 0.0001). The peri-operative left upper limb ischemia rates for the surgical and endovascular LSA revascularization groups were 1.2% and 0.6%, respectively (P = 0.96). The peri-operative mortality rates of the surgical and endovascular LSA revascularization groups were 2.0% and 2.0%, respectively (P = 0.88). Conclusion: There was no significant difference in the terms of short-term outcomes when comparing the two revascularization techniques. The quality of evidence assessed by GRADE scale was low to very-low. Surgical and endovascular LSA revascularization during TEVAR were both safe and effective. Compared with surgical LSA revascularization techniques, parallel stent revascularization of LSA significantly increased the rate of type I endoleak.

Relationships of tumor differentiation and immune infiltration in gastric cancers revealed by single-cell RNA-seq analyses.

Gastric cancers are highly heterogeneous malignant tumors. To reveal the relationship between differentiation status of cancer cells and tumor immune microenvironments in gastric cancer, single-cell RNA-sequencing was performed on normal mucosa tissue, differentiated gastric cancer (DGC) tissue, poorly differentiated gastric cancer (PDGC) tissue and neuroendocrine carcinoma (NEC) tissue sampled from surgically resected gastric cancer specimens. We identified the signature genes for both DGC and PDGC, and found that signature genes of PDGC strongly enriched in the epithelial-mesenchymal transition (EMT) program. Furthermore, we found that DGC tends to be immune-rich type whereas PDGC tends to be immune-poor type defined according to the density of tumor-infiltrating CD8+ T cells. Additionally, interferon alpha and gamma responding genes were specifically expressed in the immune-rich malignant cells compared with immune-poor malignant cells. Through analyzing the mixed adenoneuroendocrine carcinoma, we identified intermediate state malignant cells during the trans-differentiation process from DGC to NEC, which showed double-negative expressions of both DGC marker genes and NEC marker genes. Interferon-related pathways were gradually downregulated along the DGC to NEC trans-differentiation path, which was accompanied by reduced CD8+ cytotoxic T-cell infiltration. In summary, molecular features of both malignant cells and immune microenvironment cells of DGC, PDGC and NEC were systematically revealed, which may partially explain the strong tumor heterogeneities of gastric cancer. Especially along the DGC to NEC trans-differentiation path, immune-evasion was gradually enhanced with the decreasing activities of interferon pathway responses in malignant cells.

Oat ß-glucan and L-arabinose synergistically ameliorate glucose uptake in insulin-resistant HepG2 cells and exert anti-diabetic activity via activation of the PI3K/AKT pathway in db/db mice.

Oat ß-glucan (OBG) and L-arabinose (LA) have exhibited positive effects on diabetes and its complications. However, it is unclear whether OBG and LA have a synergistic effect. We investigated the effect of variable compositions (OBG : LA = 1 : 1, 1 : 2, 1 : 4,1 : 6, 1 : 8, 1 : 10, 2 : 1, 4 : 1, 6 : 1, 8 : 1, 10 : 1) on glucose uptake in IR-HepG2 cells induced by dexamethasone (DEX) to find out the optimal composition showing synergistic effects. Furthermore, this study evaluated the anti-diabetic activity of the optimal composition in db/db mice. In vitro, the OBG : LA = 1 : 1 group showed the strongest synergistic effects among the varied compositions, outperforming OBG and LA alone. In vivo, there were more beneficial effects in the OBG : LA = 1 : 1 group compared with the OBG and LA single-dosing groups. OBG : LA = 1 : 1 supplementation markedly decreased the levels of fasting blood glucose (FBG) and insulin (INS) in serum, improved glucose tolerance and insulin sensitivity, lowered blood lipid levels, and reduced liver lipid accumulation. Moreover, the western blot results indicated that the OBG : LA = 1 : 1 group up-regulated the protein expression of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), and phospho-protein kinase B (p-AKT), while down-regulating the protein expression of phospho-phosphorylated insulin receptor substrate-1 (p-IRS1) to enhance insulin transduction in liver tissues. These findings suggest that OBG : LA = 1 : 1 synergistically ameliorated glucose metabolism disorders and alleviated insulin resistance by promoting the PI3K/AKT pathway in the liver.

Single-cell profiling reveals molecular basis of malignant phenotypes and tumor microenvironments in small bowel adenocarcinomas.

Small bowel adenocarcinomas (SBAs) are rare malignant tumors with a high mortality rate, and their molecular characteristics are still largely unexplored. Here we performed single-cell RNA sequencing for tumor samples from 12 SBA patients and predicted drug candidates for SBA. We identified four prevalent subtypes of malignant cells with distinct signatures including cell cycle program, mitochondria program, metabolism program and epithelial-mesenchymal transition (EMT) program. The progression relationships of these four subtypes of malignant cells were also revealed, which started from the cell cycle program, through the mitochondria program and then progressing into either the metabolism program or the EMT program. Importantly, ligand-receptor interaction pairs were found to be specifically enriched in pairs of EMT-program malignant cells and highly exhausted CD8+ T cells, suggesting that cancer cell subpopulations with EMT features may contribute most to the exhaustion of T cells. We also showed that the duodenal subtype of SBA exhibited molecular features more similar to gastric cancer whereas jejunal subtype of SBA more similar to colorectal cancer. Especially, we predicted specific drugs for SBA based on differential gene expression signatures between malignant cells and normal epithelial cells of SBA, and verified more potent inhibitory effects of volasertib and tozasertib for SBA cancer cells than conventional drugs of SBA at the same concentration, which provides new clues for treatments of SBA. In summary, our study provides a blueprint of the molecular signatures of both tumor cells and tumor microenvironment cells in SBA and reveals potential targets and drug candidates for its clinical treatments.

Prognostic significance of metastatic lymph node ratio in patients with gastric cancer after curative gastrectomy: a single-center retrospective study.

BACKGROUND: The objective of this study was to evaluate the prognostic value of Metastatic lymph node ratio (MLNR) after curative gastrectomy in patients with gastric cancer (GC) and the potential for new indicators to strengthen the current guidelines. METHODS: We retrospectively researched 3864 GC patients with curative gastrectomy between February 2011 and February 2016. The following clinical data were collected from the included patients: gender, type of gastrectomy, tumor location, T stage, N stage, ELN, tumor size, age at surgery, perineural invasion, vascular invasion, TNM stage, survival time and survival status. Patients were divided into low-MLNR (L-MLNR), and high-MLNR (H-MLNR) groups based on adjusted the X-tile cutoff-value of 0.25 for MLNR, the survival rates and clinicopathological characteristics of each group were compared. For the assessment of significant associations between clinicopathological characteristics and patients' survival, univariate and multivariate analyses were performed using the Kaplan-Meier method and Cox proportional hazards analysis. The log-rank test was used to examine the statistical significance of differences among different survival curves. Clinicopathological features significantly associated with MLNR were assessed by the Chi-square test and multinomial logistic regression. The discriminative ability was measured by calculating the Bayesian Information Criterion (BIC) values for each category. Assessment of the effect of clinicopathological features on MLNR for predicting prognosis of GC patients used stratum analysis through Kaplan-Meier analysis and Cox proportional risk Analysis. RESULTS: Survival analysis indicated that MLNR was negatively associated with overall survival (OS) (p < .001) and was an independent prognostic predictor in 3864 GC patients (p < .001). MLNR had significant prognostic significance in various subgroups with clinicopathological characteristics (gender, type of gastrectomy, tumor location, T stage, N stage, ELN, tumor size, age at surgery, perineural invasion, vascular invasion, and TNM stage) (p < .001). CONCLUSIONS: The MLNR may become a new indicator to assess the prognosis of GC patients who underwent curative gastrectomy. The results may have potential clinical implications that should be considered when developing clinical practice guidelines or the design of the future investigation.

Structural properties, antioxidant and immune activities of low molecular weight peptides from soybean dregs (Okara).

In this study, a method for preparing low molecular weight peptides (HPH-VAP) from okara using high-pressure homogenization assisted double enzymes was proposed. In order to explore its advantages, the effects of various methods on protein extraction rate and on the structure, antioxidant and immune properties of peptides were compared. The results showed that the protein extraction rate of this method was increased by 69% and 51% compared with other methods, and the structure only led to changes in the hydrogen bonds between peptide chains. HPH-VAP was screened out through functional characteristics, its structure was identified by HPLC-MS/MS, and further immunological activity analysis was carried out. The results showed that it promoted cell phagocytic ability, NO level and release of cytokines IL-6, IFN- γ, TNF-α. Therefore, this method is an effective and applicable method for industrial preparation of okara peptides, and has a positive effect on the reuse of okara resources.

Component Identification of Phenolic Acids in Cell Suspension Cultures of Saussureainvolucrata and Its Mechanism of Anti-Hepatoma Revealed by TMT Quantitative Proteomics.

Saussurea involucrata (S. involucrata) had been reported to have anti-hepatoma function. However, the mechanism is complex and unclear. To evaluate the anti-hepatoma mechanism of S. involucrata comprehensively and make a theoretical basis for the mechanical verification of later research, we carried out this work. In this study, the total phenolic acids from S. involucrata determined by a cell suspension culture (ESPI) was mainly composed of 4,5-dicaffeoylquinic acid, according to the LC-MS analysis. BALB/c nude female mice were injected with HepG2 cells to establish an animal model of liver tumor before being divided into a control group, a low-dose group, a middle-dose group, a high-dose group, and a DDP group. Subsequently, EPSI was used as the intervention drug for mice. Biochemical indicators and differences in protein expression determined by TMT quantitative proteomics were used to resolve the mechanism after the low- (100 mg/kg), middle- (200 mg/kg), and high-dose (400 mg/kg) interventions for 24 days. The results showed that EPSI can not only limit the growth of HepG2 cells in vitro, but also can inhibit liver tumors significantly with no toxicity at high doses in vivo. Proteomics analysis revealed that the upregulated differentially expressed proteins (DE proteins) in the high-dose group were over three times that in the control group. ESPI affected the pathways significantly associated with the protein metabolic process, metabolic process, catalytic activity, hydrolase activity, proteolysis, endopeptidase activity, serine-type endopeptidase activity, etc. The treatment group showed significant differences in the pathways associated with the renin-angiotensin system, hematopoietic cell lineage, etc. In conclusion, ESPI has a significant anti-hepatoma effect and the potential mechanism was revealed.

Integrated transcriptomics and epigenomics reveal chamber-specific and species-specific characteristics of human and mouse hearts.

DNA methylation, chromatin accessibility, and gene expression represent different levels information in biological process, but a comprehensive multiomics analysis of the mammalian heart is lacking. Here, we applied nucleosome occupancy and methylome sequencing, which detected DNA methylation and chromatin accessibility simultaneously, as well as RNA-seq, for multiomics analysis of the 4 chambers of adult and fetal human hearts, and adult mouse hearts. Our results showed conserved region-specific patterns in the mammalian heart at transcriptome and DNA methylation level. Adult and fetal human hearts showed distinct features in DNA methylome, chromatin accessibility, and transcriptome. Novel long noncoding RNAs were identified in the human heart, and the gene expression profiles of major cardiovascular diseases associated genes were displayed. Furthermore, cross-species comparisons revealed human-specific and mouse-specific differentially expressed genes between the atria and ventricles. We also reported the relationship among multiomics and found there was a bell-shaped relationship between gene-body methylation and expression in the human heart. In general, our study provided comprehensive spatiotemporal and evolutionary insights into the regulation of gene expression in the heart.

DNMTs Play an Important Role in Maintaining the Pluripotency of Leukemia Inhibitory Factor-Dependent Embryonic Stem Cells.

Naive pluripotency can be maintained in medium with two inhibitors plus leukemia inhibitory factor (2i/LIF) supplementation, which primarily affects canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these three supplements alone is sufficient to maintain naive self-renewal remains unclear. Here we show that LIF alone in medium is sufficient for adaptation of 2i/L-ESCs to embryonic stem cells (ESCs) in a hypermethylated state (L-ESCs). Global transcriptomic analysis shows that L-ESCs are close to 2i/L-ESCs and in a stable state between naive and primed pluripotency. Notably, our results demonstrate that DNA methyltransferases (DNMTs) play an important role in LIF-dependent mouse ESC adaptation and self-renewal. LIF-dependent ESC adaptation efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast stem cells, L-ESCs contribute to somatic tissues and germ cells in chimeras. L-ESCs cultured under such simple conditions as in this study would provide a more conducive platform to clarify the molecular mechanism of ESCs in in vitro culture.

Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum-Free Media.

Inhibitors of Mek1/2 and Gsk3ß, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium.

Publisher Correction: Single-cell multi-omics sequencing of human early embryos.

In the version of this Resource originally published, owing to a technical error an incorrect file was used for Supplementary Table 2; this has now been replaced with the correct file.

Single-cell multi-omics sequencing of human early embryos.

DNA methylation, chromatin states and their interrelationships represent critical epigenetic information, but these are largely unknown in human early embryos. Here, we apply single-cell chromatin overall omic-scale landscape sequencing (scCOOL-seq) to generate a genome-wide map of DNA methylation and chromatin accessibility at single-cell resolution during human preimplantation development. Unlike in mice, the chromatin of the paternal genome is already more open than that of the maternal genome at the mid-zygote stage in humans, and this state is maintained until the 4-cell stage. After fertilization, genes with high variations in DNA methylation, and those with high variations in chromatin accessibility, tend to be two different sets. Furthermore, 1,797 out of 5,155 (35%) widely open chromatin regions in promoters closed when transcription activity was inhibited, indicating a feedback mechanism between transcription and open chromatin maintenance. Our work paves the way for dissecting the complex, yet highly coordinated, epigenetic reprogramming during human preimplantation development.