BACKGROUND: Dilated cardiomyopathy (DCM) and coronary heart disease (CHD) stand as two of the foremost causes of mortality. However, the comprehensive comprehension of the regulatory mechanisms governing DCM and CHD remains limited, particularly from the vantage point of single-cell transcriptional analysis.
METHODS: We used the GSE121893 dataset from the GEO database, analyzing single-cell expressions with tools like DropletUtils, Seurat, and Monocle. We also utilized the GSVA package for comparing gene roles in DCM and CHD, Finally, we conducted qRT-PCR and Western blot analyses to measure the expression levels of SMARCA4, Col1A1, Col3A1 and α-SMA, and the role of SMARCA4 on fibroblasts were explored by EdU and Transwell assay.
RESULTS: Our analysis identified six cell types in heart tissue, with fibroblasts showing the most interaction with other cells. DEGs in fibroblasts were linked to muscle development and morphogenesis. Pseudotime analysis revealed the dynamics of fibroblast changes in both the normal and disease groups and many transcription factors (TFs) potentially involved in this process. Among these TFs, SMARCA4 which was translated into protein BRG1, showed the most significantly difference. In vivo experiments have demonstrated that SMARCA4 indeed promoted fibroblasts proliferation and migration.
CONCLUSIONS: This study provides a clearer understanding of cell-type dynamics in heart diseases, emphasizing the role of fibroblasts and the significance of SMARCA4 in their function. Our results offer insights into the cellular mechanisms underlying DCM and CHD, potentially guiding future therapeutic strategies.

As a dominating air pollutant, atmospheric fine particulate matter within 2.5 μm in diameter (PM2.5) has attracted increasing attention from the researchers all over the world, which will lead to various adverse effects on the central nervous system (CNS), yet the potential mechanism is unclear. In this study, the microglia (BV2 cell line) were exposed to different concentrations of PM2.5 (5, 10 and 20 μg/cm2) for 24 h. It was found that PM2.5 could result in adverse effects on microglia such as decreased cell viability, structural damage and even cell death. And it was reported that long non-coding RNAs (lncRNAs) could participate in multitudinous neurological diseases. Therefore, the microarray analysis was conducted in order to disclose the underlying neurotoxicity mechanism of PM2.5 by ascertaining the differentially expressed lncRNAs (DElncRNAs). The consequences indicated that the DElncRNAs were enriched in various biological pathways, including ferroptosis, IL-17 signaling pathway and NOD-like receptor signaling pathway. Moreover, the cis- and trans-regulated mRNAs by DElncRNAs as well as the corresponding transcriptional factors (TFs) were observed, such as CEBPA, MYC, MEIS1 and KLF4. In summary, our study supplies some candidate libraries and potential preventive target against PM2.5-induced toxicity through targeting lncRNAs. Furthermore, the post-transcriptional regulation will contribute to the future research on PM2.5-induced neurotoxicity.

Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.

Bone acts as a self-healing organ, which undergoes continuous regeneration process that is tightly regulated by the cooperation of osteoclasts with the capability of bone resorption and osteoblasts with the capability of bone formation. Generally, bone marrow derived mesenchymal stem cells (BMSCs) differentiated to final osteoblasts have been considered as critical role in bone remodeling. In this regard, several transcription factors (TFs) whose binding sites are initially hidden deep within accessible chromatin that participate in modulating osteoblast differentiation and bone matrix mineralization. Then, it is necessary to explore further the dynamic changes about the epigenetic transcription machinery during osteoblastogenesis. Here, we performed the chromatin accessibility and transcriptomic landscape of osteoblast differentiation and mineralization by using transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-Seq). Our data found that global chromatin accessibility during osteoblastogenesis was extensively improved. Above this, it is shown that key target genes including Col6a3, Serpina3n, Ms4a4d, Lyz2, Phf11b and Grin3a were enriched in differential loci RNA-seq and ATAC-Seq peaks with continuous changed tendency during osteoblasts differentiation and mineralization. In addition, Analysis of Motif Enrichment (AME) was used to elucidate TFs which modulated these target genes. In this study, it was shown for the first time that these important TFs including MEF2A, PRRX1, Shox2 and HOXB13 could alter promoter accessibility of target genes during osteoblastogenesis. This helps us understand how TF binding motif accessibility influences osteoblast differentiation. In addition, it also suggests that modulating the chromatin accessibility of osteogenesis could be developed as the promising strategies to regulate bone regeneration.

BACKGROUND: Human embryonic stem cell (hESC)-derived endothelial cells (ECs) possess therapeutic potential in many diseases. Cytokine supplementation induction and transcription factor overexpression have become two mainstream methods of hESC-EC induction. Single-cell RNA-seq technology has been widely used to analyse dynamic processes during hESC-EC induction and components of induced endothelial cells. However, studies that used single-cell RNA-seq are mainly based on cytokine supplementation methods. In this study, we used a high-efficiency human embryonic stem cell-endothelial cell line (hESC-EC) called the \"FLI1-PKC system\" as a research model and employed single-cell RNA sequencing (scRNA-seq) to investigate the transcriptional landscape and cellular dynamics.
METHODS: The high-efficiency hESC-EC induction (FLI1-PKC) system was established in our previous study. We applied single-cell RNA sequencing (scRNA-seq) of the differentiated cells at different time points and investigated the gene expression profiles.
RESULTS: The FLI1-PKC induction system can directionally differentiate hESCs into mature endothelial cells with all the requisite functions. Unlike other hES-EC induction protocols, the FLI1-PKC method follows a different induction route; nonetheless, the transcriptome of induced endothelial cells (iECs) remains the same. The elevated number of activated transcription factors may explain why the FLI1-PKC system is more effective than other hES-EC protocols.
CONCLUSIONS: Our study has presented a single-cell transcriptional overview of a high-efficiency hESC-EC induction system, which can be used as a model and reference for further improvement in other hESC induction systems.

3-hydroxyacyl-CoA dehydratases 1 (Hacd1) is a critical enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The difference in expression of hacd1 might account for the stronger capacity of LC-PUFA biosynthesis in freshwater fish than in marine fish, but little is known about fish hacd1. Therefore, this study compared the responses of large yellow croaker and rainbow trout hacd1 to different oil sources or fatty acids, and also examined transcriptional regulation of this gene. In this study, hacd1 was highly expressed in the liver of large yellow croaker and rainbow trout, which is the main organ for LC-PUFA biosynthesis. Therefore, we cloned the hacd1 coding sequence, with a phylogenetic analysis showing that this gene is evolutionarily conserved. Its localization to the endoplasmic reticulum (ER), likely also indicates a conserved structure and function. The expression of hacd1 in the liver was significantly decreased after the substitution of soybean oil (SO) for fish oil but was not significantly affected after palm oil (PO) substitution. Linoleic acid (LA) incubation significantly promoted hacd1 expression in primary hepatocytes of large yellow croaker and eicosapentaenoic acid (EPA) incubation significantly promoted hacd1 expression in primary hepatocytes of rainbow trout. Transcription factors STAT4, C/EBPα, C/EBPβ, HNF1, HSF3 and FOXP3 were identified in both large yellow croaker and rainbow trout. HNF1 had a stronger activation effect in rainbow trout than in large yellow croaker. FOXP3 inhibited hacd1 promoter activity in large yellow croaker but had no effect in rainbow trout. Therefore, the differences between HNF1 and FOXP3 affected the expression of hacd1 in the liver thus being responsible for the high capacity of LC-PUFA biosynthesis in rainbow trout.

Sepsis remains a worldwide public health problem. This study aims to explore the role and mechanism of transcriptional factors (TFs) in sepsis-induced myocardial injury. Firstly, TF KLF13 was selected to explore its role in sepsis-induced myocardial injury. The caecal ligation and puncture (CLP) -induced sepsis mouse model was established and the septic mice were examined using standard histopathological methods. KLF13 expression was detected in the septic mouse heart and was also seen in a lipoploysaccharide (LPS) -induced cellular inflammation model. To explore this further both pro-apoptotic cleaved-caspase3/caspase3 and Bax levels and anti-apoptotic Bcl2 levels were examined, also in both models, In addition inflammatory cytokine (IL-1β, TNF-α, IL-8 and MCP-1) production and IκB-α protein level and p65 phosphorylation were examined in both septic mice and LPS-induced cells. Thus three parameters - cardiomyocyte apoptosis, inflammatory response and NF-κB pathway activation were evaluated under similar conditions. The septic mice showed significant oedema, disordered myofilament arrangement and degradation and necrosis to varying degrees in the myocardial cells. KLF13 was downregulated in both the septic mouse heart and the LPS-induced cellular inflammation model. Furthermore, both models showed abnormally increased cardiomyocyte apoptosis (increased cleaved-caspase3/caspase and Bax protein levels and decreased Bcl2 level), elevated inflammation (increased production of inflammatory cytokines) and the activated NF-κB pathway (increased p65 phosphorylation and decreased IκB-α protein level). KLF13 overexpression notably ameliorated sepsis-induced myocardial injury in vivo and in vitro. KLF13 overexpression protected against sepsis-induced myocardial injury and LPS-induced cellular inflammation and apoptosis via inhibiting the inflammatory pathways (especially NF-κB signalling) and cardiomyocyte apoptosis.

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Transposable elements (TEs) and transcription factors (TFs) are involved in the precise regulation of gene expression during the preimplantation stage. Activation of TEs is a key event for mammalian embryonic genome activation and preimplantation early embryonic development. TFs are involved in the regulation of drastic changes in gene expression patterns, but an inventory of the interplay between TEs and TFs during normal/abnormal human embryonic development is still lacking. Here we used single-cell RNA sequencing data generated from biparental and uniparental embryos to perform an integrative analysis of TE and TF expression. Our results showed that endogenous retroviruses (ERVs) are mainly expressed during the minor embryonic genome activation (EGA) process of early embryos, while Alu is gradually expressed in the middle and later stages. Some important ERVs (e.g., LTR5_Hs, MLT2A1) and Alu TEs are expressed at significantly lower levels in androgenic embryos. Integrative analysis revealed that the expression of the transcription factors CTCF and POU5F1 is correlated with the differential expression of ERV TEs. Comparative coexpression network analysis further showed distinct expression levels of important TFs (e.g., LEUTX and ZSCAN5A) in dizygotic embryos vs. parthenogenetic and androgenic embryos. This systematic investigation of TE and TF expression in human early embryonic development by single-cell RNA sequencing provides valuable insights into mammalian embryonic development.

BACKGROUND: Mesenchymal stem cells (MSCs) possess the potential to differentiate into chondrocytes, which makes them an ideal source for healing cartilage defects. Here, we seek to identify the essential genes participating in MSCs chondrogenesis.
METHODS: Human MSCs were induced for chondrogenesis for 7, 14, and 21 days using a high-density micromass culture system, and RNA was extracted for RNA-seq.
RESULTS: A total of 6247 differentially expressed genes (DEGs) were identified on day 7, and 85 DEGs were identified on day 14. However, no significant DEGs was identified on day 21. The top 30 DEGs at day 7, including COL9A3, COL10A1, and CILP2, are closely related to extracellular matrix organization. While the top 30 DEGs at day 14 revealed that inflammation-related genes were enriched, including CXCL8, TLR2, and CCL20. We also conducted protein-protein interaction (PPI) networks analysis using the search tool for the retrieval of interacting genes (STRING) database and identified key hub genes, including CXCL8, TLR2, CCL20, and MMP3. The transcriptional factors were also analyzed, identifying the top 5 TFs: LEF1, FOXO1, RORA, BHLHE41, and SOX5. We demonstrated one particular TF, RORA, in promoting early MSCs chondrogenesis.
CONCLUSIONS: Taken together, our results suggested that these DEGs may have a complex effect on MSCs chondrogenesis both synergistically and solitarily.