Duan-Fang Liao

More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.

Atherosclerosis (AS) is a chronic, progressive vascular disease marked by lipid deposition in the arterial intima, vascular wall thickening, luminal narrowing, and compromised blood flow. Although macrophage-derived foam cells are well-studied, vascular smooth muscle cells (VSMCs) also substantially contribute to AS, particularly when they transition into foam cells under oxidative stress. Accumulating evidence suggests that ferroptosis—an iron-dependent, regulated cell death mechanism characterized by lipid peroxidation—exacerbates AS pathology through oxidative damage and vascular dysfunction. Catechin, a potent antioxidant abundant in green tea, has demonstrated efficacy in reducing oxidative stress; however, its role in suppressing VSMC ferroptosis induced by oxidized low-density lipoprotein (ox-LDL) remains unclear. Here, we evaluated catechin's capacity to protect VSMCs against ox-LDL-induced ferroptosis, focusing on its modulation of the Nrf2/SLC7A11/GPX4 axis. Mouse vascular smooth muscle (MOVAS) cells were incubated with ox-LDL to induce foam cell formation and ferroptosis. We assessed intracellular iron, lipid peroxidation, reactive oxygen species (ROS), and antioxidant defenses and examined mitochondrial ultrastructure via transmission electron microscopy (TEM). Ferroptosis-related proteins were measured by Western blot, immunofluorescence, and qPCR. In vivo, ApoE^−/− mice on a high-fat diet (HFD) underwent partial carotid ligation with local catechin administration to investigate plaque formation and ferroptosis in arterial tissue. Our results show that catechin reduced intracellular Fe²⁺, decreased ROS and malondialdehyde (MDA) levels, and preserved mitochondrial integrity in ox-LDL-exposed MOVAS cells. Catechin also enhanced GSH and SOD levels and restored GPX4, SLC7A11, and Nrf2 expression, thereby reducing foam cell formation. In ApoE–/– mice, catechin reduced plaque size, mitigated lipid deposition, and upregulated GPX4, SLC7A11, and Nrf2 in the arterial wall. Collectively, these findings confirm that catechin prevents ox-LDL-induced ferroptosis in VSMCs by activating the Nrf2/SLC7A11/GPX4 pathway, highlighting its potential therapeutic value for atherosclerosis. This study provides additional evidence for the role of dietary polyphenols in regulating ferroptosis within VSMCs.

Bile acids and their corresponding intestinal epithelial receptors, the farnesoid X receptor (FXR), the G protein-coupled bile acid receptor (TGR5), play crucial roles in the physiological and pathological processes of intestinal epithelial cells. These acids and receptors are involved in the regulation of intestinal absorption, signal transduction, cellular proliferation and repair, cellular senescence, energy metabolism, and the modulation of gut microbiota. A comprehensive literature search was conducted using PubMed, employing keywords such as bile acid, bile acid receptor, FXR (nr1h4), TGR5 (gpbar1), intestinal epithelial cells, proliferation, differentiation, senescence, energy metabolism, gut microbiota, inflammatory bowel disease (IBD), colorectal cancer (CRC), and irritable bowel syndrome (IBS), with a focus on publications available in English. This review examines the diverse effects of bile acid signaling and bile receptor pathways on the proliferation, differentiation, senescence, and energy metabolism of intestinal epithelial cells. Additionally, it explores the interactions between bile acids, their receptors, and the microbiota, as well as the implications of these interactions for host health, particularly in relation to prevalent intestinal diseases. Finally, the review highlights the importance of developing highly specific ligands for FXR and TGR5 receptors in the context of metabolic and intestinal disorders.

Triple-negative breast cancer (TNBC) is a type of breast cancer characterized by high molecular heterogeneity. Owing to the lack of effective therapeutic strategies, patients with TNBC have a poor prognosis. Prunella vulgaris L. has the effects of reducing swelling, dissolving knots and treating breast carbuncles and mammary rocks. Modern pharmacological studies have reported that it can effectively inhibit the growth of breast cancer. The main active antitumor components of Prunella vulgaris are triterpenoids (PVT); however, the role and potential mechanism of PVT in TNBC remain unexplored. Our study aimed to further explore the inhibitory effects of PVT on TNBC and the associated mechanism. The results showed that 19 compounds associated with PVT were identified, 9 of which were triterpenoids. The percentages of ursolic acid and oleanolic acid in PVT were 34.51% and 11.32%, respectively. Triterpenes of Prunella vulgaris significantly inhibited the proliferation, migration and invasion of MDA-MB-231 cells and promoted their apoptosis in a concentration-dependent manner. PVT could also effectively downregulate the mRNA and protein expression levels of Ptp1b, Pi3k, Akt and mtor and upregulate the mRNA and protein expression levels of Il-24 in MDA-MB-231 cells. In mice with tumors of TNBC, PVT significantly reduced tumor growth and the expression levels of PTP1B, CXCL12, CXCR4, PI3K, AKT, mTOR and other proteins in TNBC tumor tissue and upregulated the expression of IL-24. This study showed that PVT played an anti-TNBC role by regulating the PTP1B/PI3K/AKT/mTOR signaling pathway and the IL-24/CXCL12/CXCR4 signaling axis.

Sensitive and reliable methylation assay is important for oncogentic studies and clinical applications. Here, a new methylation assay was developed by the use of adapter-dependent adapter in library preparation. This new assay avoids the use of bisulfite and provides a simple and highly sensitive scanning of methylation spectra of circulating free DNA and genomic DNA.

Hi-C sequencing is widely used for analyzing chromosomal interactions. In this study, we propose "superimposed Hi-C" which features paired EcoP15I sites in a linker to facilitate sticky-end ligation with target DNAs. Superimposed Hi-C overcomes Hi-C's technical limitations, enabling the identification of single cell's chromosomal interactions.