Preparation and Properties of Partial-Degradable ZrO₂–Chitosan Particles–GelMA Composite Scaffolds

In the field of bone repair, the inorganic–organic composite scaffold is a promising strategy for mimicking the compositions of the natural bone. In addition, as implants for repairing load-bearing sites, an inert permanent bone substitute composites with bioactive degradable ingredients may make full use of the composite scaffold. Herein, the porous zirconia (ZrO2) matrix was prepared via the template replication method, and the partial degradable ZrO2–chitosan particles–GelMA composite scaffolds with different chitosan/GelMA volume ratios were prepared through the vacuum infiltration method. Dynamic light scattering (DLS) and the scanning electron microscope (SEM) were adopted to observe the size of the chitosan particles and the morphologies of the composites scaffold. The mechanical properties, swelling properties, and degradation properties of the composite scaffolds were also characterized by the mechanical properties testing machine and immersion tests. The CCK-8 assay was adopted to test the biocompatibility of the composite scaffold preliminarily. The results show that chitosan particles as small as 60 nm were obtained. In addition, the ratio of chitosan/GelMA can influence the mechanical properties and the swelling and degradation behaviors of the composites scaffold. Furthermore, improved cell proliferation performance was obtained for the composite scaffolds

Analysis of genomic copy number variations in human hepatocellular carcinoma cell lines HepG2 and Huh7

Objective To explore the effect of copy number variation on the occurrence and development of hepatocellular carcinoma by using copy number variation and transcriptome experiment of hepatocellular carcinoma combined with public clinical data. Methods The copy number variation found in hepatoma cell lines HepG2 and Huh7 was identified by optical genome mapping. The function of the copy number variant genes in the two cell lines was analyzed, and the protein interaction network was mapped according to the enrichment pathway. Key genes in the core network of two cell lines were selected to analyze the relationship between copy number variation and gene expression in hepatocellular carcinoma. The relationship between gene expression and clinical survival was analyzed by GEPIA database. RNA-seq assay and public data were used to verify gene expression levels. Results HepG2 cells mainly showed increased copy number, and related genes were enriched in estrogen signaling pathway and Staphylococcus aureus infection pathway. Huh7 cells showed both increased and decreased copy number, and related genes mainly concentrated in olfactory conduction and cytokine-cytokine receptor interaction pathways. The copy number of key genes SRC, MAPK3 and MAP3K7 was proportional to gene expression, and survival was significantly reduced in patients with high expression of these genes (P＜0.05). Compared with HEK293T cell line, the expression of SRC and MAP3K7 genes in the two hepatocellular carcinoma lines was significantly increased(P＜0.001), suggesting the specific variation of hepatocellular carcinoma. MAPK3 had no difference. Conclusions The expression of copy number variant genes SRC and MAP3K7 in hepatocellular carcinoma is significantly correlated with the prognosis of patients, and may significantly affect the development and heterogeneity of hepatocellular carcinoma

Calculating the spatial density of regulatory chromatin interactions using multi-modal datasets from the same cell line

Summary: Here, we present a protocol for calculating the spatial density of regulatory chromatin interactions (SD-RCI) using Hi-C, ATAC-seq, and ChIP-seq datasets from the same cell line. We describe steps for selecting and preprocessing datasets, training and predicting a model to obtain regulatory chromatin interactions, and evaluating model performance. We then detail calculation of SD-RCI and visualization of the correlation between SD-RCI and gene expression. This protocol is applicable to Hi-C, ATAC-seq, and ChIP-seq data from the human cell line.For complete details on the use and execution of this protocol, please refer to Gong et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Glucocorticoid receptor-mediated Nr1d1 chromatin circadian misalignment in stress-induced irritable bowel syndrome

Summary: Stress-elevated glucocorticoids cause circadian disturbances and gut-brain axis (GBA) disorders, including irritable bowel syndrome (IBS). We hypothesized that the glucocorticoid receptor (GR/NR3C1) might cause chromatin circadian misalignment in the colon epithelium. We observed significantly decreased core circadian gene Nr1d1 in water avoidance stressed (WAS) BALB/c colon epithelium, like in IBS patients. WAS decreased GR binding at the Nr1d1 promoter E-box (enhancer box), and GR could suppress Nr1d1 via this site. Stress also altered GR binding at the E-box sites along the Ikzf3-Nr1d1 chromatin and remodeled circadian chromatin 3D structures, including Ikzf3-Nr1d1 super-enhancer, Dbp, and Npas2. Intestinal deletion of Nr3c1 specifically abolished these stress-induced transcriptional alternations relevant to IBS phenotypes in BALB/c mice. GR mediated Ikzf3-Nr1d1 chromatin disease related circadian misalignment in stress-induced IBS animal model. This animal model dataset suggests that regulatory SNPs of human IKZF3-NR1D1 transcription through conserved chromatin looping have translational potential based on the GR-mediated circadian-stress crosstalk

Non-canonical STING–PERK pathway dependent epigenetic regulation of vascular endothelial dysfunction via integrating IRF3 and NF-κB in inflammatory response

Inflammation-driven endothelial dysfunction is the major initiating factor in atherosclerosis, while the underlying mechanism remains elusive. Here, we report that the non-canonical stimulator of interferon genes (STING)–PKR-like ER kinase (PERK) pathway was significantly activated in both human and mice atherosclerotic arteries. Typically, STING activation leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB)/p65, thereby facilitating IFN signals and inflammation. In contrast, our study reveals the activated non-canonical STING–PERK pathway increases scaffold protein bromodomain protein 4 (BRD4) expression, which encourages the formation of super-enhancers on the proximal promoter regions of the proinflammatory cytokines, thereby enabling the transactivation of these cytokines by integrating activated IRF3 and NF-κB via a condensation process. Endothelium-specific STING and BRD4 deficiency significantly decreased the plaque area and inflammation. Mechanistically, this pathway is triggered by leaked mitochondrial DNA (mtDNA) via mitochondrial permeability transition pore (mPTP), formed by voltage-dependent anion channel 1 (VDAC1) oligomer interaction with oxidized mtDNA upon cholesterol oxidation stimulation. Especially, compared to macrophages, endothelial STING activation plays a more pronounced role in atherosclerosis. We propose a non-canonical STING–PERK pathway-dependent epigenetic paradigm in atherosclerosis that integrates IRF3, NF-κB and BRD4 in inflammatory responses, which provides emerging therapeutic modalities for vascular endothelial dysfunction

The genetic and clinical characteristics of WFS1 related diabetes in Chinese early onset type 2 diabetes

Abstract Diabetes is one of the most common phenotypes of Wolfram syndrome owing to the presence of the variants of the WFS1 gene and is often misdiagnosed as other types of diabetes. We aimed to explore the prevalence of WFS1-related diabetes (WFS1-DM) and its clinical characteristics in a Chinese population with early-onset type 2 diabetes (EOD). We sequenced all exons of the WFS1 gene in 690 patients with EOD (age at diagnosis ≤ 40 years) for rare variants. Pathogenicity was defined according to the standards and guidelines of the American College of Medical Genetics and Genomics. We identified 33 rare variants predicted to be deleterious in 39 patients. The fasting [1.57(1.06–2.22) ng/ml] and postprandial C-peptide levels [2.8(1.75–4.46) ng/ml] of the patients with such WFS1 variations were lower than those of the patients without WFS1 variation [2.09(1.43–3.05) and 4.29(2.76–6.07) respectively, ng/ml]. Six (0.9%) patients carried pathogenic or likely pathogenic variants; they met the diagnostic criteria for WFS1-DM according to the latest guidelines, but typical phenotypes of Wolfram syndrome were seldom observed. They were diagnosed at an earlier age and usually presented with an absence of obesity, impaired beta cell function, and the need for insulin treatment. WFS1-DM is usually mistakenly diagnosed as type 2 diabetes, and genetic testing is helpful for individualized treatment