Search Results (530)

Caveolin-1 is a scaffolding protein of caveolae, flask-shaped membrane microdomains involved in diverse cellular processes. Caveolae are primarily localized to the plasma membrane, the trans-Golgi network, and mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs). Most enzymes involved in cholesterol biosynthesis reside in the ER, and although caveolin-1 avidly binds cholesterol, its role in cholesterol trafficking remains unclear. Acyl-coenzyme A:cholesterol acyltransferases (ACAT1 and ACAT2) convert free cholesterol into cholesteryl esters for storage, with ACAT1 serving as the predominant isoenzyme in most cell types. ACAT1 is an ER-resident protein, with a fraction associated with specialized ER subdomains, including the MAM. Here, we report that a subset of caveolin-1 molecules appears to be associated with a fraction of ACAT1 in ER subdomains. Using immunoprecipitation under detergent conditions, immunoadsorption of MAM-enriched membranes under detergent-free conditions, and electron microscopy, we provide evidence consistent with an association between a subset of caveolin-1 molecules and ACAT1. Functionally, in mouse embryonic fibroblasts, we show that genetic ablation of caveolin-1 significantly increases the esterification of low-density lipoprotein-derived cholesterol, suggesting that caveolin-1 may attenuate ACAT1 activity. Collectively, these findings indicate that caveolin-1 may modulate cholesterol esterification and contribute to the regulation of cholesterol distribution among cellular membranes. Full article

As emerging global environmental contaminants, organic ultraviolet absorbers (OUVAs) are widely used in personal care formulations and exhibit environmental persistence and potential bioaccumulation. Among these compounds, 2-ethylhexyl salicylate (EHS) and homosalate (HMS) are the most frequently used salicylate-type UV filters in cosmetic formulations. Although an increasing number of studies have demonstrated their environmental hazards, little is known about the molecular mechanisms underlying their cytotoxicity in mammalian systems, a fundamental knowledge gap for both human health protection and the development of more environmentally friendly consumer goods. In this study, we used mouse embryonic fibroblasts (MEFs, 3T6) and zebrafish as models to assess the toxicological phenotypes of EHS and HMS in vitro and in vivo, respectively. We found that both EHS and HMS induced cellular damage characterized by oxidative stress, disrupted intracellular calcium homeostasis, mitochondrial impairment, and DNA damage. Importantly, molecular analyses further suggested the concurrent activation of two distinct regulated cell death programs: pyroptosis, as suggested by Caspase-11-mediated GSDMD cleavage, and necroptosis, as suggested by ZBP1-RIPK3-Caspase-8-mediated MLKL phosphorylation. The in vitro data have been partially validated at the level of gene expression and in developmental toxicity in the zebrafish model, providing some in vivo phenotypic and molecular correlates. While the upstream events were experimentally verified, the causal links among them remain to be further elucidated. Taken together, this work suggested that OUVA-induced toxicity is not limited to isolated oxidative damage, but may also involve the activation of two different cell death programs. These findings provide important molecular clues to understanding the potential health and ecological risks of widely used UV filters and offer a scientific basis for their more environmentally friendly safety evaluation and regulatory management, which are crucial for advancing more sustainable chemistry and safer consumer goods. Full article

Candida albicans-associated denture stomatitis is a common inflammatory condition in denture wearers. Conventional tissue conditioners provide temporary relief but lack intrinsic antifungal activity, allowing persistent microbial colonization and biofilm formation. Functionalization with bioactive agents represents a promising preventive strategy. This study evaluated the antifungal efficacy and biocompatibility of pterostilbene (PTE), a natural stilbenoid compound, incorporated into a commercially available tissue conditioner. Antifungal activity of PTE against C. albicans ATCC 10231 was evaluated using broth microdilution and XTT biofilm assays. Tissue conditioner discs containing 1% and 2.5% (w/w) PTE were fabricated and tested after 24 h, 72 h, and 1 week using colony-forming unit (CFU) counts and metabolic activity assays. Biocompatibility was assessed by exposing mouse embryonic fibroblast (MEF) cells to conditioned eluates followed by an MTT viability assay. PTE inhibited biofilm formation in a concentration-dependent manner, with significant suppression observed at ≥8 µg/mL (p < 0.001). A time-dependent antifungal effect was observed over one week. PTE-functionalized tissue conditioners significantly reduced fungal adhesion compared with controls at all-time points (p < 0.001). Cell viability remained above 70%, meeting ISO 10993-5 criteria for non-cytotoxicity, indicating potential for localized prevention of denture stomatitis. Full article

The isolation of fibroblast-like cells from crocodile skeletal muscle provides a valuable platform for studies in comparative physiology, reptilian biology, regenerative medicine, and tissue engineering. In this article, we present an optimized protocol for isolating and characterizing fibroblast-like cells derived from the embryonic skeletal muscle of the Siamese crocodile (Crocodylus siamensis). The procedure improved cell yields and viability while maintaining phenotypic and genetic stability. Dorsal and tail skeletal muscle tissue was cultured in flasks pre-coated with collagen. The cells attached and began migrating from the explants within one day. Optimal culture conditions were achieved using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 or Minimum Essential Medium Alpha supplemented with 10% fetal bovine serum, 2% crocodile serum, and growth-promoting factors, incubated at 28 °C without CO₂. These conditions supported a shorter population doubling time and enhanced cell proliferation. The established cells displayed a spindle-shaped fibroblastic morphology, expressed the fibroblast-specific marker fibronectin, and maintained a stable karyotype through passage 20. The cell line remained stable and proliferative for at least 30 passages under standard culture conditions. Our study established the first standardized fibroblast-like cell line derived from C. siamensis, thus providing a foundation for future studies in reptilian physiology, cytogenetics, and regenerative biotechnology. Full article

Background/Objectives: Long non-coding RNAs (lncRNAs) are integral to the regulation of viral tumorigenesis. We have previously identified that the chicken lncRNA-803, which responds to Marek’s disease virus (MDV), inhibits apoptosis in the chicken embryonic fibroblast cell line DF-1, accompanied by changes in the expression of the p53 protein. Nonetheless, the molecular mechanism of lncRNA-803 in apoptosis has yet to be elucidated. Methods: In this study, through lentivirus-mediated overexpression and knockdown experiments, we determined that the overexpression of lncRNA-803 induces elevated expression levels of murine double minute 2 (MDM2), murine double minute 4 (MDM4), tumor protein p53 (p53), and tumor protein p53 binding protein 1 (TP53BP1) within the p53 signaling pathway. Results: This modulation subsequently leads to an upregulation of B-cell lymphoma-2 (Bcl-2) expression, while concurrently resulting in the downregulation of cysteinyl aspartate specific proteinase 8 (Caspase-8), cysteinyl aspartate specific proteinase 9 (Caspase-9), Bcl-2 associated protein X (Bax), and cysteinyl aspartate specific proteinase 9 (Caspase-3) in the apoptosis pathway. In terms of its mechanism, lncRNA-803 functions as a molecular sponge for miR-6555-3p. lncRNA-803 engages in competitive binding with miR-6555-3p, thereby diminishing its inhibitory effect on MDM4. Conclusions: These results elucidate that lncRNA-803 modulates apoptosis in DF-1 cells through a novel competing endogenous RNA mechanism involving the miR-6555-3p/MDM4/p53 axis. These findings provide new insights into the molecular pathogenesis of MDV. Full article

Sorghum is known for its anti-cancer, anti-inflammatory, and antioxidant properties, but its effect on cell growth is not well understood. First, the cytotoxicity of various sorghum extract (SE) concentrations was evaluated in C2C12 (murine myoblasts) and C3H10T1/2 (murine embryonic fibroblasts). The extent of DNA damage was then assessed, and the activation of the JAK2/STAT5b and IGF-1 pathways was observed. Studies on the transcriptional regulatory function of STAT5b revealed that SE increased STAT5b/DNA binding and transcriptional promoter activity. Consequently, STAT5b upregulation led to the increased expression of IGF-1. Moreover, other factors, such as growth hormone receptor and bone morphogenetic protein 7, were also upregulated. The results of these experiments suggest that sorghum may enhance muscle recovery or promote growth factors by stimulating the JAK2/STAT5b and IGF-1 pathways. Therefore, sorghum is expected to be an effective functional food for bone growth and muscle recovery, without inducing adverse side effects. Full article

The autophagy regulator ATG7 helps maintain cellular homeostasis and has been suggested to modulate aspects of antiviral immune responses. In Drosophila, ATG7-dependent autophagy contributes to host resistance to vesicular stomatitis virus (VSV), a negative-strand RNA virus of family Rhabdoviridae that is widely used for studying viral biology and developing vaccines and virotherapy. However, the role of ATG7 in mammalian cells, especially non-immune cell types, remains unclear. Herein, we systematically examined the impact of ATG7 on VSV infection using CRISPR-edited cell lines derived from murine embryonic fibroblast (MEF), HeLa, and Huh7.5 cells, in relation to its effect on the expression of antiviral interferon-stimulated genes (ISGs). We found that ATG7 deficiency blocked basal as well as VSV-induced LC3B lipidation, concomitant with moderate reductions in progeny virus yields, while the reconstitution of ATG7 reversed the phenotypes. Mechanistically, ATG7 did not affect viral entry but rather was associated with moderate upregulation of VSV RNA replication. Intriguingly, ATG7 inhibited baseline ISG expression, and this correlated with its pro-VSV effect in all three cell types, while its suppression of innate immune responses elicited post-VSV infection did not. Altogether, these data provide new insights into the role of ATG7 in regulating VSV replication and innate immunity and have implications for developing VSV-based prophylaxis/therapeutics. Full article

Introduction: Extensive thermal injury remains a formidable clinical challenge, primarily due to the profound deficit of autologous donor skin, which necessitates prolonged hospitalization and escalates healthcare expenditures. While human embryonic stem cells (hESCs) offer a theoretically inexhaustible source for regenerative therapy, optimizing their differentiation and engraftment remains critical for clinical translation. Methods: We used a three-stage protocol to induce the differentiation of hESCs into keratinocytes (KCs). To optimize the delivery of hESC-derived keratinocytes (EKCs), human dermal fibroblasts (HFBs) were utilized to provide essential extracellular matrix (ECM) and microenvironmental support. The two cell types could self-assemble into 3D spheroids. After optimizing the size and cell proportion, these spheroids were subsequently transplanted onto full-thickness dorsal wounds in immunodeficient mice to evaluate their regenerative capacity. Results: hESC-derived keratinocytes exhibited the expression of stage-specific epidermal markers, confirming high differentiation efficiency. In vitro, EKCs demonstrate the capacity to form stratified epidermal structures. By self-assembling into spheres with dermal fibroblasts, the EKCs demonstrated successful engraftment and sustained survival in vivo. The transplantation of these 3D spheroids significantly accelerated wound closure and re-epithelialization compared with controls. Conclusions: This study establishes a robust cell therapy approach characterized by a short preparation cycle with high differentiation efficiency and high transplantation survival rate, offering a novel strategy for the treatment of extensive skin defects. Full article

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation, development, and utilization. However, the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore, generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study, we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos, using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro, exhibiting high expression of key pluripotency markers, genomic stability, and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes. Full article

Late-stage metastatic cutaneous melanoma (MCM) and neuroblastoma (NB) are the most aggressive skin and childhood cancers with survival rates of <50%, mainly due to the emergence of resistance to available drugs, thus requiring an urgent solution. Quaternary phosphonium salts (QPSs) can exhibit strong anticancer effects, regardless of the developed resistance. Triphenyl (1) and diphenyl (3 and 4) phosphonium salts were synthesized, treating commercial triphenyl phosphine and synthesizing 11-diphenylphosphanyl-undecan-1-ol (2), respectively, with benzyl bromide. Upon full characterization, they were tested, for the first time, on MeTRAV (BRAF^V600D) and MeOV (BRAF^V600E) vemurafenib (PLX)-resistant MCM cells, etoposide (ETO)-sensitive (HTLA 230) and multidrug resistant (MDR) (HTLA ER) NB cells, non-tumorigenic human keratinocytes (HaCaT), and mouse embryonic fibroblasts (3T3), as well as red blood cells (RBCs). Viability of MeTRAV cells was decreased to 44.8% by administration of 1 (100 µM), in intermediate-time (48 h) treatments, while short-time exposure (24 h) to 3 (≥75 µM) and 4 (≥50 µM) was sufficient to reduce their viability to 33.6 and 32.2%. Viability of MeOV was decreased under 50% with 5 µM concentrations of 1 and 25 µM of 3 and 4, While cells were exterminated (26.9, 20.6, and 21.8%) with higher concentrations after 48 h exposure. Collectively, 1 was the better performing compound (IC₅₀ = 6.4 µM, 48 h). Viability of HTLA ER cells was decreased under 50% upon 72 h administrations of 1 at concentrations ≥ 50 µM, 48 h (≥75 µM) and 72 h (≥50 µM) of 3, and after 72 h (≥75 µM) of 4, but 72 h exposure and high concentrations of all compounds were necessary for their extermination (31.2, 28.7, and 29.7%). Viability of HTLA 230 cells was not <50% when 1 and 4 were administered for only 24 h, while their viability was <50% after administration of 3 at all times of exposure. At high concentrations, all compounds exterminated cells (33.6, 25.3%, 1, 48–72 h; 38.6, 30.2, and 24.7%, 3, 24–72 h; 33.2%, 4, 72 h). The best-performing compounds were 1 (IC₅₀ = 4.0 µM, HTLA 230) and 3 (IC₅₀ = 27.8 µM, HTLA ER) at 72 h exposure. The cytotoxic effects of compound 4 on MeTRAV cells, when exposed to 24/48 h treatments, were comparable to those of PLX on the same cells in 72 h treatments. Compound 1, in shorter 48 h treatments of PLX-R MeOV, was 2.5-fold more cytotoxic than PLX in 72 h ones. All compounds were not cytotoxic to 3T3 cells at all times of exposure; they had low cytotoxicity to HaCaT cells in 24 and 48 h treatments and were slightly cytotoxic to RBCs in 24 h ones. Compound 1 could be a promising platform to develop new intermediate-time therapies for PLX-R MeOV cells, while 4 could be used to develop 24 and 48 h treatments for PLX-R MeTRAV cells. Also, all compounds could be developed as new treatment options for both ETO-sensitive and MDR late-stage HR-NB cells, being even more effective than ETO by 1.2, 2.0, and 1.3 times (HTLA 230) and 3.2, 4.7, and 3.2 times (HTLA ER). All compounds have the potential to be developed as adjuvants in already existing anticancer cocktails to treat MCM and/or NB. Full article

The conversion of primed pluripotent stem cells to a naive-like state has emerged as a critical strategy for enhancing developmental potential and broadening applications in regenerative medicine. Conditioned media (CM)-based approaches provide a supportive microenvironment enriched with secreted factors that may facilitate this state transition without extensive genetic or chemical manipulation. In this study, we investigated the potential of human Wharton’s Jelly-derived mesenchymal stem cell-conditioned media (hWJ-MSCs-CM) and mouse embryonic fibroblasts CM (MEFs-CM) to support the conversion of primed rhesus monkey embryonic stem cells (rhESCs) into a naive-like state. The rhESCs were cultured under feeder-free and feeder conditions using both hWJ-MSCs-CM and MEFs-CM, exhibiting distinct morphological changes during conversion. Immunofluorescence analysis demonstrated the expression of pluripotency and naive markers under both conditions. Gene expression analysis further confirmed the upregulation of naive-specific genes and downregulation of primed markers, with statistically significant differences between groups. Additionally, epigenetic reprogramming was assessed, revealing differential effects of the CM sources on the reversion to a naive state. These findings highlight the potential of hWJ-MSCs-CM as a supportive system for naive-like state induction in primate ESCs. Full article

In vitro embryo production (IVP) has emerged as a crucial tool in assisted reproduction and animal biotechnology. A key factor in this process is in vitro oocyte maturation (IVM), a critical process preceding fertilization that directly influences embryo quality. FLI supplementation, composed of fibroblast growth factor (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), has been shown to facilitate the IVM process to mimic essential aspects of in vivo oocyte development, and therefore, promote higher rates of oocyte maturation, embryonic viability, blastocyst formation, and improve the number of live animals born after embryo transfer. Individually or together, these components participate in signaling pathways that are crucial for improving oocyte competence and early embryo development. This review highlights the individual and combined roles of FGF2, LIF, and IGF1 in maturation and embryo culture medium, their influence on subsequent embryonic development, and their signaling pathways. Additionally, the incorporation of antioxidants and amino acids as supplementary components in combination with FLI is explored as a strategy to mitigate oxidative stress and enhance metabolic support during IVM and embryo culture. Together, these elements can significantly improve IVP outcomes, providing a potential pathway for optimizing the efficiency of embryo production in various species. Full article

Objective: Rare-earth elements are extensively employed across diverse industrial sectors, increasingly raising concerns about their potential health hazards in both occupational and environmental contexts. Samarium oxide (Sm₂O₃), a routinely processed rare-earth product, reproducibly precipitates pulmonary fibrosis in experimental models, yet the molecular circuitry that transduces its fibrogenic signal remains almost entirely unmapped. This study aims to elucidate the role of miR-214-3p in Sm₂O₃-induced pulmonary fibrosis and to investigate its regulatory mechanism at the molecular level. Methods: A murine model of pulmonary fibrosis was established via intratracheal instillation of Sm₂O₃, and histopathological changes were assessed using hematoxylin and eosin (H&E) and Masson’s trichrome staining. RNA sequencing was performed on lung tissues to identify differentially expressed mRNAs. Leveraging our previously generated miRNA landscape of Sm₂O₃-exposed lungs, we subjected the dataset to Gene Ontology and KEGG enrichment analyses, which convergently identified miR-214-3p as the top-ranking candidate regulator of the fibrogenic MAPK axis. The direct targeting of MAP2K3 by miR-214-3p was validated using a dual-luciferase reporter assay. Expression levels of fibrotic markers (α-SMA, Collagen I) and key components of the MAPK signaling pathway (MAP2K3, p-MAPK14, MST1) were quantified in both in vivo and in vitro models using qRT-PCR and Western blotting. Gain- and loss-of-function studies, complemented by rescue assays, were performed in human embryonic lung fibroblasts (HELFs) via transient transfection of miR-214-3p mimics, inhibitors, or MAP2K3-overexpression plasmids. Cell proliferation was evaluated using the EdU assay, and TGF-β1 secretion was measured by ELISA. Results: Sm₂O₃ exposure induced significant pulmonary fibrosis in mice, accompanied by marked downregulation of miR-214-3p and upregulation of MAP2K3 in lung tissues. Overexpression of miR-214-3p or silencing of MAP2K3 effectively suppressed Sm₂O₃-induced fibroblast activation, including reduced cell proliferation, decreased expression of α-SMA and Collagen I, and inhibition of p38 MAPK phosphorylation. Notably, ectopic overexpression of MAP2K3 reversed the protective effects conferred by miR-214-3p, confirming a functional rescue. Conclusions: miR-214-3p directly silences MAP2K3, thereby blunting p38 MAPK-driven fibrogenesis after Sm₂O₃ exposure. Our data unveil a miR-214-3p–MAP2K3–p38 MAPK axis that constitutes a readily druggable target for rare-earth-element-induced pulmonary fibrosis. Full article

Naphthyridine derivatives have emerged as privileged scaffolds with diverse pharmacological activities, particularly in anticancer and antiviral drug discovery. In this study, a series of naphthyridine-based derivatives (1–10b) was designed, synthesized, and structurally characterized using IR, ¹H/¹³C NMR, and mass spectrometry, and evaluated as dual-function antiproliferative and anti-fowlpox virus agents supported by integrated computational analyses. The synthesized compounds were screened for in vitro antiproliferative activity against HeLa, HCT-116, and MCF-7 cancer cell lines, as well as normal WI-38 lung fibroblasts. Several derivatives exhibited potent cytotoxic activity with enhanced selectivity toward cancer cells. Compound 5b showed the highest activity against HeLa cells, compound 1 was most effective against HCT-116 cells, while compounds 7 and 8 displayed remarkable activity against MCF-7 cells, with compound 7 surpassing doxorubicin and compound 8 demonstrating excellent selectivity toward normal cells. Mechanistic investigations revealed that compounds 7 and 8 acted as dual topoisomerase I/IIβ inhibitors, inducing G2/M cell cycle arrest and intrinsic apoptosis associated with caspase-9 activation and downregulation of topoisomerase II protein expression. Selected derivatives were further evaluated for antiviral activity against fowlpox virus using in ovo and in vivo SPF embryonated chicken egg models, where compounds 2 and 9a exhibited the highest therapeutic indices, comparable to ribavirin, and compound 9a markedly suppressed viral replication and titers in vivo. ADMET profiling, molecular docking, molecular dynamics simulations, and DFT calculations supported the experimental findings and identified compound 10a as the most favorable theoretical candidate. Overall, this integrated experimental–computational approach establishes naphthyridine derivatives as a rationally designed multifunctional chemotype for simultaneous anticancer and antiviral drug development. Full article

NKAP (NF-kappa-B-activating protein) is a ubiquitously expressed nuclear protein involved in multiple biological processes. Males with missense NKAP mutations have been reported to present with marfanoid features and behavioral and musculoskeletal abnormalities. We have previously reported that a disruptive NKAP mutation resulted in extremely skewed X chromosome inactivation (XCI), leading to phenotypic manifestation of hemophilia A (HA) in a HA carrier. In this study, with the aim of exploring the phenotypic manifestations of deleterious NKAP mutations in males, as well as their involvement in the mechanism of XCI regulation in females, we generated NKAP mutant mice using CRISPR/Cas9 technology. Gait analysis studies conducted in male mice hemizygous for mutant NKAP by the CatWalk XT system revealed significant alterations in gait parameters, consistent with hypotonia reported in human mutant NKAP patients. By breeding mutant NKAP mice with HA mice, we generated a double heterozygous mutant NKAP/HA mouse model, i.e., female mice carrying mutant NKAP with a WT F8 copy on one X chromosome, and WT NKAP with a mutant F8 copy on the other X chromosome. XCI pattern analysis using methylation-sensitive restriction enzymes demonstrated that mutant NKAP/HA females exhibited significant XCI skewing of the X chromosome bearing the mutant NKAP copy. Furthermore, these females exhibited significantly reduced F8 mRNA levels and FVIII (factor VIII) antigen levels, as demonstrated by quantitative RT-PCR and ELISA, respectively. Murine embryonic fibroblasts (MEFs) derived from a hemizygous mutant NKAP embryo exhibited markedly reduced proliferation rate and increased senescence compared to WT NKAP MEFs, suggesting that XCI skewing induced by mutant NKAP results from secondary selection against cells with an active X chromosome bearing the mutant NKAP copy. Full article