Search Results (325)

RNA modifications comprise a core epigenetic dimension of gene regulation; among these, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) have been most intensively investigated. While the functions of m6A in stem cell biology have been well characterized, the contributions of m5C remain comparatively less well defined. This review focuses on m5C modifications catalyzed by the NSUN family of RNA methyltransferases and their roles in regulating stem cell identity, pluripotency, and differentiation. Evidence from embryonic and mesenchymal stem cells, as well as animal models, demonstrates that NSUN-mediated m5C is deposited on diverse RNA substrates, including rRNA, tRNA, mRNA, mitochondrial RNA, and enhancer RNAs, thereby influencing processes such as self-renewal, cell cycle progression, RNA stability, metabolic activation, and lineage specification. Disruption of m5C regulation often leads to developmental defects, underscoring its essential role during embryogenesis. Collectively, these findings establish m5C as a versatile and dynamic regulator in stem cell biology and underscore the need for future studies to delineate the roles of the NSUN family in stem cells and define the RNA targets of m5C. In addition, its broader implications for development, regenerative medicine, and disease, including cancer, as well as its potential interplay with other RNA modifications such as m6A and pseudouridine, remain important areas for further investigation. Full article

EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), plays a critical role in neural development by regulating gene expression through the trimethylation of lysine 27 on histone H3 (H3K27me3), which promotes chromatin remodeling and transcriptional repression. Although PRC2 is known to regulate cell fate specification and gliogenesis, its in vivo functions during vertebrate neurodevelopment, particularly at the level of neuronal subtype differentiation, remain incompletely understood. Here, we investigated the consequences of ezh2 loss-of-function during zebrafish brain development, focusing on oligodendrocyte differentiation, cerebellar neurogenesis, and the formation of neurotransmitter-specific neuronal populations. Using whole-mount in situ hybridization, we found that ezh2 inactivation does not alter the expression of oligodendrocyte lineage markers, indicating that early oligodendrocyte precursor cell specification and myelination are preserved. However, a significant reduction in cerebellar proliferation was observed in ezh2-deficient larvae, as evidenced by the downregulation of pcna and cyclin A2, while other brain regions remained unaffected. Notably, the expression of atoh1c, a key marker of glutamatergic cerebellar progenitors, was strongly reduced at 5 days post fertilization, suggesting a selective role for ezh2 in maintaining cerebellar progenitor identity. This was associated with impaired differentiation of both glutamatergic granule cells and GABAergic Purkinje cells in specific cerebellar subregions. In contrast, the expression of markers for other major neurotransmitter systems remained unaffected, indicating a region-specific requirement for ezh2 in neuronal development. Finally, behavioral analysis revealed a hyperlocomotor phenotype in ezh2^−/− larvae, consistent with cerebellar dysfunction. Together, these findings identify ezh2 as a key regulator of progenitor maintenance and neuronal differentiation in the cerebellum, highlighting its crucial role in establishing functional cerebellar circuits. Full article

Arabinogalactan proteins (AGPs: hydroxyproline-rich glycoproteins) are ubiquitous in plants and play various functions in cases of development and reproduction. In Arabidopsis thaliana some AGPs can work as markers for gametophytic cell differentiation (among others embryological structures they mark generative cell wall and/or plasma membrane, and also sperm cells). However, apart from Arabidopsis, this labeling of generative cell and sperm cells in pollen grains has only been observed in a few flowering plant species belonging to dicotyledons. No such studies are available in monocotyledons. The main aim of our study was to see whether AGPs would be present at the generative cell–vegetative cell interface in different monocotyledons (representatives of Asparagaceae, Amarylidaceae and Liliaceae), and we also wanted to test whether they would be the same AGPs as in dicotyledons. For the study, we selected Gagea lutea (L.) Ker Gawl., Ornithogalum nutans L. and Galanthus nivalis L. species that differ in shape and size of generative cells. Antibodies against arabinogalactan proteins AGPs were used, including JIM8, JIM13, JIM14, MAC207, LM2, LM14, JIM15 and JIM4. The localization of the examined compounds was determined using immunohistochemistry techniques. The key finding was that AGPs (detected with JIM8 and JIM13 antibodies) consistently mark the boundary between the generative cell and the surrounding vegetative cytoplasm, suggesting their association with the generative cell–vegetative cell interface in all species studied. Identifying such molecular markers in male gametophyte may enhance the understanding of gametophytic cell fate, sperm cell identity and the molecular mechanisms underlying fertilization. Such labeling may also be useful in studies on pollen development, species comparisons, or responses to environmental stresses. Full article

Background/Objectives: Human ovarian granulosa cells (hGCs) are crucial to ovarian follicle development and function, exhibiting multipotency and the ability to differentiate into neuronal cells, chondrocytes, and osteoblasts in vitro. 3,4,5,4′-tetramethoxystilbene (DMU-212) is a methylated derivative of resveratrol, a natural polyphenol found in grapes and berries, with a wide spectrum of biological activities, including notable anticancer properties. Interestingly, DMU-212 exhibits cytotoxic effects predominantly on cancer cells while sparing non-cancerous ones, and evidence suggests that similar to resveratrol, it may also promote hGC differentiation. This study aimed to investigate the effects of the liposomal formulation of this methylated resveratrol analog—lipDMU-212—on the osteogenic differentiation ability of hGCs in a primary three-dimensional cell culture model. Methods: lipDMU-212 was formulated using the thin-film hydration method. GC spheroids’ viability was evaluated after exposure to lipDMU-212, an osteoinductive medium, or both. Osteogenic differentiation was confirmed using Alizarin Red staining and quantified by measuring Alkaline Phosphatase (ALP) activity on days 1, 7, and 15. RNA sequencing (RNA-seq) was performed to explore molecular mechanisms underlying lipDMU-212-induced differentiation. Results: lipDMU-212 promoted osteogenic differentiation of hGCs in the 3D cell culture model, as evidenced by increased mineralization and a ~4-fold increase in ALP activity compared with the control. RNA-seq revealed up-regulation of genes related to cell differentiation and cellular identity. Furthermore, JUN (+2.82, p = 0.003), LRP1 (+2.06, p = 0.05), AXIN1 (+3.02, p = 0.03), and FYN (+3.30, p = 0.01) were up-regulated, indicating modulation of the Wnt/β-catenin signaling pathway, a key regulator of osteoblast differentiation. Conclusions: The ability of GCs to differentiate into diverse tissue-specific cell types underscores their potential in regenerative medicine. This study contributes to the understanding of lipDMU-212’s role in osteogenic differentiation and highlights its potential in developing future therapies for degenerative bone diseases. Full article

The growth plate is a specialized cartilage structure near the ends of long bones that orchestrates longitudinal bone growth during fetal and postnatal stages. Within this region reside a dynamic population of growth plate skeletal stem cells (gpSSCs), primarily located in the resting zone, which possess self-renewal and multilineage differentiation capacity. Recent advances in cell-lineage tracing, single-cell transcriptomics, and in vivo functional studies have revealed distinct subpopulations of gpSSCs, which are defined by markers such as parathyroid hormone-related protein (PTHrP), CD73, axis inhibition protein 2 (Axin2), forkhead box protein A2 (FoxA2), and apolipoprotein E (ApoE). These stem cells interact intricately with their niche, particularly after the formation of the secondary ossification center, through stage-specific regulatory mechanisms involving several key signaling pathways. This review summarizes the current understanding of gpSSC identity, behavior, and regulation, focusing on how these cells sustain growth plate function through adapting to biomechanical and molecular cues. Full article

Senecavirus A (SVA) is an emerging threat to swine populations due to its potential to cause vesicular lesions, which are difficult to differentiate from other vesicular diseases of swine such as foot and mouth disease (FMD), requiring significant resources for differential diagnosis. The first Taiwanese isolate of SVA was identified in 2006, although the first clinical case was not reported until 2012. The genetic characteristics and seroprevalence of SVA in Taiwan remain unclear. This study aimed to assess the seroprevalence and genetic diversity of SVA in nursery/weaned swine and finisher swine on Taiwanese pig farms. Phylogenetic analysis of seven Taiwanese SVA isolates revealed clustering into groups I and II. The 2006 and 2012 isolates shared 95.5% and 95.7% identity, respectively, with an early USA strain (MT360258), while more recent strains collected between 2018 and 2022 exhibited 95.7–98.8% identity with a 2020 USA strain (MZ733977). Serological analysis of swine from 300 farms showed significantly higher herd-level seroprevalence in nursery/weaned swine (53%) than finisher swine (6.7%). Furthermore, comparative analysis of nine known B cell epitopes showed high sequence conservation across Taiwanese and global strains. These findings provide important baseline data on the genetic diversity and seroprevalence of SVA in Taiwan and support the development of improved surveillance strategies for this emerging swine pathogen. Full article

Chronic kidney disease (CKD) is a common and serious condition in felines. Accordingly, several cell therapies have been studied over the past decades for effective treatments. This study aimed to develop a new lineage of renal progenitor cells for use in cats with CKD. Metanephric and mesonephric progenitor cells were obtained from mesonephros and metanephros tissues of feline conceptuses at four distinct gestational stages. The cultured cells were characterized by their morphology, tumorigenic potential, immunophenotype determined by flow cytometry, and differentiation potential. We then conducted a pilot study in CKD-affected cats, comparing intraperitoneal injections of cultured metanephric progenitor cells (n = 4) to a placebo solution (n = 3). All four cell types exhibited adhesion and colony formation, but showed no tumorigenic potential. Cells tested positive for renal progenitor markers (CD117, Nephron, and WT1), confirming their identity. Treated cats showed no statistically significant differences (p ≤ 0.05) in any of the data analyzed. However, caregivers reported a voluntary increase in appetite after cell administration. Veterinarians confirmed this information during double-blind evaluations conducted after treatment. Although this data are qualitative, no clinical deterioration was observed in cats. Our results suggest that this new lineage of renal progenitor cells did not induce immediate adverse effects, thus supporting its potential for use in cell-based therapies. However, further studies are needed to evaluate its efficacy in treating renal diseases. Full article

Background: Cardiomyocyte death is a key factor in myocardial ischemia–reperfusion injury (MI/RI), and the expression patterns and molecular mechanisms of pyroptosis-related genes (PRGs) in ischemia–reperfusion injury are poorly understood. Methods: The mouse MI/RI injury-related datasets GSE61592 and GSE160516 were obtained from the Gene Expression Omnibus database, and differential expression analysis was performed on each to identify differentially expressed genes (DEGs). The DEGs were intersected with the PRGs obtained from GeneCards to identify differentially expressed PRGs in MI/RI. Enrichment analysis identified key pathways, while PPI network analysis revealed hub genes. The expression patterns and immune cell infiltration of hub genes were also investigated. The molecular docking prediction of key genes was performed using MOE software in conjunction with the ZINC small molecular compounds database. Key gene expression was validated in an external dataset (GSE4105), a mouse MI/RI model, and an HL-1 cell hypoxia/reoxygenation model via RT-qPCR. Results: A total of 29 differentially expressed PRGs were identified, which are primarily associated with pathways such as “immune system process”, “response to stress”, “identical protein binding”, and “extracellular region”. Seven key genes (Fkbp10, Apoe, Col1a2, Ppic, Tlr2, Fstl1, Serpinh1) were screened, all strongly correlated with immune infiltration. Seven FDA-approved small molecule compounds exhibiting the highest docking potential with each key gene were selected based on a comprehensive evaluation of S-scores and hydrogen bond binding energies. Apoe, Tlr2, and Serpinh1 were successfully validated across external datasets, the mouse MI/RI model, and the cardiomyocyte H/R model. Conclusions: Apoe, Tlr2, and Serpinh1 may be key genes involved in MI/RI-related pyroptosis. Targeting these genes may provide new insights into the treatment of MI/RI. Full article

Regulatory T (Treg) cells are essential for maintaining self-tolerance and regulating immune responses. In this study, we report the identification of Treg cell epitopes in human α-tubulin that were capable of enhancing IL-10-producing Foxp3⁺ Treg cells and LAG-3⁺CD49b⁺FoxP3⁻ Tr1 cells in vitro, using human peripheral blood mononuclear cells. Similarly, we also demonstrate that a peptide pool containing the identified Treg cell epitopes (αTBL pool) suppressed the T cell responses elicited by HLA class I- and class II-restricted T cell epitopes. Moreover, stimulation of naive CD4⁺ T cells with autologous monocyte-derived dendritic cells in the presence of the αTBL pool promoted the differentiation of functional FoxP3⁺ Treg cells, which suppressed the proliferation of CD3/CD28-activated T cells. Finally, we show that one of the identified epitopes, identical between human and mouse, also stimulated FoxP3⁺ Treg cells in splenocytes isolated from C57BL/6 mice. Considering the elevated expression of α-tubulin in all cell types, the presence of Treg cell epitopes in this protein may facilitate a broad mechanism of immune regulation. Moreover, α-tubulin Treg cell epitopes may prove useful in creating novel treatments for conditions marked by excessive or misdirected immune responses. Full article

Endometrial mesenchymal stem cells (eMSCs) are a novel and biologically potent source of multipotent stromal cells with potential beyond reproductive medicine. This study explored their phenotypic profile, trilineage differentiation, and the cytoprotective effects of their conditioned media (eMSCCM) on oxidatively stressed neonatal and adult chondrocytes. Canine eMSCs displayed typical fibroblast-like morphology and expressed high levels of mesenchymal surface markers CD29 and CD44, low hematopoietic markers CD34/CD45, and variable CD90, confirming a mesenchymal identity. Differentiation assays revealed osteogenic and chondrogenic differentiation, whereas adipogenic activity was limited. Using eMSCCM at 25% and 50% concentrations, chondrocyte viability was assessed after exposure to 200 µM H₂O₂. eMSCCM significantly enhanced the viability of H₂O₂-stressed chondrocytes in a dose-dependent manner, particularly at 50%, with marked effects at 24 and 48 h. Although metabolic activity declined at 72 h, the treated cells remained more metabolically active than untreated controls. These findings suggest that eMSCCM offers promising cytoprotective effects for cartilage-related oxidative stress conditions. Full article

Enhancer–promoter interactions (EPIs) play a key role in epigenetic regulation of gene expression, dominating cellular identity and functional diversity. Dissecting these interactions is crucial for understanding transcriptional regulatory networks and their significance in cell differentiation, development, and disease. Here, we propose a novel deep learning framework, EPIFBMC (Enhancer-Promoter Interaction prediction with FBMC network) that leverages DNA sequence and genomic features for accurate EPI prediction. The FBMC network consists of three key modules: the Four-Encoding module first encodes the DNA sequence in multiple dimensions to extract key sequence information; then the BESL (Balanced Ensemble Subset Learning) adopts an integrated subset learning strategy to optimize the feature-learning process of positive and negative samples; finally, the MCANet module completes the training of EPI prediction based on a Multi-channel Network. We evaluated EPIFBMC on three cell line datasets (HeLa, IMR90, and NHEK), and validated its generalizability across three independent datasets (K562, GM12878, HUVEC) through cross-cell-line experiments, comparing favorably with state-of-the-art methods. Notably, EPIFBMC balances genomic feature richness and computational complexity, significantly accelerating training speed. Ablation studies identified two key DNA sequence features—positional conservation and positional specificity score—which showed critical predictive value across a benchmark dataset of six diverse cell lines. The computational testing show that EPIFBMC shows excellent performance in the EPI prediction task, providing a powerful tool for decoding gene regulatory networks. It is believed that it will have important application prospects in developmental biology, disease mechanism research, and therapeutic target discovery. Full article

Introduction: Hypoxic cancers are radioresistant, but biomarkers based on expression of multiple genes can identify patients who will benefit from hypoxia modification. Most studies identifying relevant genes exposed cells in culture to 1% oxygen, which activates hypoxia-inducible factor (HIF). However, oxygen concentrations in hypoxic tumours are heterogeneous ranging from <0.1%. As lower oxygen levels would likely affect transcriptional responses, we aimed to investigate how gene selection at different oxygen levels affects the genes identified and their prognostic capability. Methods: Four MIBC (J82, T24, UMUC3, HT1376) and two non-MIBC (RT4, RT112) bladder cancer cell lines were exposed to varying oxygen levels (20%, 1%, 0.2% and 0.1% O₂) for 24 h and were then harvested and frozen. RNA was extracted and transcriptomes analysed using Clariom S microarrays. Differences in gene expression were investigated. Prognostic and predictive significance of a published 24-gene signature was compared with one generated from genes identified at lower oxygen levels. Results: The number of upregulated genes increased with decreasing O₂ level. The number of biological pathways involved also increased. Differences between cell lines dominated those due to hypoxia. Some genes were commonly upregulated in MIBC and NMIBC cells and others increased exclusively in either MIBC or NMIBC cells. The median expression of a published 24-gene bladder cancer hypoxia-associated signature increased with decreasing oxygen levels. Seventy-seven genes were upregulated in at least three cell lines by exposure to 0.1% O₂. The median expression of the 77 genes was of borderline prognostic significance in the bladder cancer cohort in the TCGA (The Cancer Genome Atlas). Five of the seventy-seven genes upregulated by hypoxia were present in the twenty-four-gene bladder hypoxia signature. The median expression of the 5 genes demonstrated identical prognostication to the 24-gene signature but failed to predict benefit from hypoxia modification. Conclusions: The number of genes upregulated by exposure of bladder cancer cells to hypoxia increases as O₂ level is decreased from 1% to 0.2% to 0.1%. Differential upregulation of gene expression by MIBC and NMIBC cells and the associated biological pathways may be useful in understanding the genetics of bladder cancer invasiveness. Based on a search of the literature, this is the first study that assessed the expression of genes in bladder cancer using three hypoxic concentration levels to identify biomarkers for disease progression and prognosis among differentially expressed bladder cancer genes. Full article

Fibro-adipogenic progenitor cells (FAPs) support muscle tissue homeostasis, regulate muscle growth, injury repair, and fibrosis, and activate muscle progenitor cell differentiation to promote regeneration. We aimed to investigate the effects of co-culturing FAPs with muscle satellite cells (MuSCs) on myogenic differentiation. Proteomic profiling of co-culture supernatants identified significant DCX, IMP2A, NUDT16L1, SLC38A2, and IL-6 upregulation. Comparative transcriptomics of mono-cultured versus co-cultured MuSCs revealed differential expression of oxidative stress-related genes (HMOX1, ALOX5, GSTM3, TRPM2, PADI1, and CTSL). Pathway enrichment analyses highlighted cell cycle regulation, TNF signaling, and ferroptosis. Gene ontology analysis of MuSCs indicated significant gene enrichment in myosin-related components. Combined transcriptomic and proteomic analyses demonstrated HO-1 downregulation at the transcriptional and translational levels, with altered pathways being predominantly related to myosin filament, muscle system process, and muscle contraction cellular components. HO-1 knockdown reduced intracellular iron accumulation in MuSCs, suppressing iron-dependent autophagy. This alleviated oxidative stress and promoted myogenic differentiation. Exogenous IL-6 (0.1 ng/mL) downregulated HO-1 expression, initiating an identical regulatory cascade, while HO-1 overexpression reversed the IL-6-mediated reduction in the expression of the autophagy markers LC3 and ATG5, suppressing myogenic enhancement. This establishes the co-culture-induced IL-6/HO-1 axis as a core regulator of iron-dependent oxidative stress and autophagy during myogenic differentiation. Full article

The impact of ionizing radiation (IR) with induction of various DNA damage is based not only on genetic but also on epigenetic effects. Epigenetic modifications determine the chromatin structure and DNA accessibility, thereby regulating cellular functions through the expression of individual genes or entire groups of genes. However, the influence of DNA repair processes on the restoration of local chromatin structures and global nuclear architectures is still insufficiently understood. In multicellular organisms, epigenetic mechanisms control diverse cellular functions of specific cell types through precise temporal and spatial regulation of gene expression and silencing. How altered epigenetic mechanisms regulate the pathophysiological function of cells, tissues, and ultimately entire organs following IR exposure remains to be investigated in detail. Radiation-induced epigenetic processes are particularly critical for immature cell populations such as tissue-specific stem and progenitor cells during development and differentiation of organ tissues. Genome-wide patterns of DNA and histone modifications are established cell types—specifically during the development and differentiation of organ tissues but can also be fundamentally altered in adult organism by stress responses, such as radiation-induced DNA damage. Following IR exposure, epigenetic factors are not always fully restored to their original state, resulting in epigenetic dysfunction that causes cells to lose their original identity and function. Moreover, severe radiation-induced DNA damage can induce premature senescence of cells in complex tissues, which ultimately leads to signs of aging and age-related diseases such as cancer. In this work, we provide an overview of the most important epigenetic changes following IR exposure and their pathophysiological significance for the development of acute and chronic radiation reactions. Full article

Tooth development (odontogenesis) is regulated by interactions between epithelial and mesenchymal tissues through signaling pathways such as Bone Morphogenetic Protein (BMP), Wingless-related integration site (Wnt), Sonic Hedgehog (SHH), and Fibroblast Growth Factor (FGF). Mesenchymal stem cells (MSCs) derived from dental tissues—including dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs)—show promise for regenerative dentistry due to their multilineage differentiation potential. Epigenetic regulation, particularly DNA methylation, is hypothesized to underpin their distinct regenerative capacities. This study reanalyzed publicly available DNA methylation data generated with Illumina Infinium HumanMethylation450 BeadChip arrays (450K arrays) from DPSCs, PDLSCs, and DFPCs. High-confidence CpG sites were selected based on detection p-values, probe variance, and genomic annotation. Principal Component Analysis (PCA) and hierarchical clustering identified distinct methylation profiles. Functional enrichment analyses highlighted biological processes and pathways associated with specific methylation clusters. Noncoding RNA analysis was integrated to construct regulatory networks linking DNA methylation patterns with key developmental genes. Distinct epigenetic signatures were identified for DPSCs, PDLSCs, and DFPCs, characterized by differential methylation across specific genomic contexts. Functional enrichment revealed pathways involved in odontogenesis, osteogenesis, and neurodevelopment. Network analysis identified central regulatory nodes—including genes, such as PAX6, FOXC2, NR2F2, SALL1, BMP7, and JAG1—highlighting their roles in tooth development. Several noncoding RNAs were also identified, sharing promoter methylation patterns with developmental genes and being implicated in regulatory networks associated with stem cell differentiation and tissue-specific function. Altogether, DNA methylation profiling revealed that distinct epigenetic landscapes underlie the developmental identity and differentiation potential of dental-derived mesenchymal stem cells. This integrative analysis highlights the relevance of noncoding RNAs and regulatory networks, suggesting novel biomarkers and potential therapeutic targets in regenerative dentistry and orthodontics. Full article