Search Results (283)

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

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

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

This study aimed to establish a model for investigating X chromosome inactivation using transgenic mouse strains expressing green fluorescent protein (GFP). The D4/XGFP-Tg (XGFP) strain carries the GFP transgene on the X chromosome; therefore, due to random X chromosome inactivation, female offspring from crosses between XGFP males and CD-1 females exhibit mosaic GFP expression. In contrast, the B5/EGFP-Tg (EGFP) strain harbours autosomal integration of the same reporter construct, resulting in uniform GFP expression in progenies. Analysis of CD-1 × XGFP attached blastocysts revealed strong GFP expression in giant trophoblast cells and primordial germ cells (PGCs) at E6.5, demonstrating paternal X-chromosome reactivation. In 14.5-day-old CD-1 × XGFP female embryos and CD-1 × EGFP embryos, intense CAG promoter-driven GFP signals were detected in the brain, heart, gonads, somites, and limbs. In line with random X-chromosome inactivation, only 56% of embryonic fibroblast cells, derived from CD-1 × XGFP female embryos, exhibited GFP expression. These findings validate that CD-1 × XGFP mice represent a valuable in vivo model for studying X chromosome inactivation during early embryonic development and PGC specification. Furthermore, CD-1 × XGFP embryonic fibroblasts represent a valuable in vitro model for investigating the molecular mechanisms governing X-chromosome activation and inactivation. Full article

Background/Objectives: Cells derived from direct chemical reprogramming into osteoblasts represent a promising source for bone regeneration, but the efficiency needs improvement. Here, we systematically evaluated whether the natural compound psoralen (Psr) could enhance this process and explored its therapeutic potential and mechanism of action. Methods: Mouse embryonic fibroblasts (MEFs) were treated with a cocktail of forskolin and phenamil (FP), supplemented with Psr. In vitro differentiation was assessed by alkaline phosphatase and Alizarin Red S staining, reverse transcription quantitative PCR, immunofluorescence and Western blot. The bone-regenerative potential of the derived chemically induced osteoblast-like cells (ciOBs) was evaluated in critical-sized calvarial defects, femoral cortical defects and a subcutaneous ectopic implantation model, using micro-computed tomography and histology. Mechanistic insights of Psr were gained by analyzing the adenylyl cyclase 9 (ADCY9)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB) axis using inhibitor SQ22536. Results: Psr acted synergistically with the FP cocktail to drive efficient osteogenic reprogramming of MEFs. At an optimal concentration of 25 μM, Psr enabled the most robust induction of early osteogenic markers and generation of mature, mineralizing ciOBs in vitro. In vivo, FP + Psr-induced ciOBs repaired critical-sized calvarial and femoral cortical defects and generated substantial, vascularized bone tissue in ectopic sites. Mechanistically, Psr co-treatment potently activated the ADCY9/cAMP/PKA/CREB pathway, and pharmacological inhibition of this pathway completely abolished the pro-osteogenic effects of Psr. Conclusions: Psr acts as a potent synergistic enhancer of direct chemical reprogramming, generating functional osteoblast-like cells with robust bone-regenerative capacity via activation of the ADCY9/cAMP/PKA/CREB pathway. Full article

RhoB is an atypical Rho GTPase whose function is tightly linked to its subcellular localization and membrane trafficking, reflecting its unique post-translational modifications and association with endosomal membranes in addition to the plasma membrane. Despite its implication in membrane trafficking and cytoskeletal regulation, tools to directly monitor RhoB activity in space and time have been lacking. Here, we describe the development and validation of a single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensor that enables direct visualization of RhoB activity in living cells while preserving its native membrane-targeting determinants. The biosensor exhibits a large dynamic range and resolves spatially heterogeneous RhoB activity during leading-edge protrusion–retraction cycles in migrating mouse embryonic fibroblasts. To demonstrate the utility of this tool, we performed multiplex live-cell imaging with a previously developed near-infrared FRET biosensor for the exocytic Rho GTPase TC10. Quantitative morphodynamic and cross-correlation analyses reveal coordinated yet antagonistic spatiotemporal patterns of RhoB and TC10 activities at the leading edge and show that perturbation of TC10 regulation reorganizes their spatial coupling. Together, this work introduces a robust biosensor for RhoB and establishes a multiplex imaging framework to study the coordination of trafficking and signaling during cell migration. Full article

The purpose of our study is to explore the potential of a transcription factor-based strategy for directly converting mouse fibroblasts into photoreceptor-like cells. The mouse cDNAs of Ascl, Crx, Ngn1, Nrl, and Otx2 were cloned into a modified commercial adenoviral vector. Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos, and mouse postnatal fibroblasts (MPFs) were isolated from three-day-old mice. A pool of adenoviruses containing five genes was prepared to infect MEFs or MPFs once daily for two days. The MEFs or MPFs were incubated in a specific medium supplemented with forskolin and were changed every two days. After 7 or 14 days, the photoreceptor-like cells were assayed via immunofluorescence or polymerase chain reaction with reverse transcription (RT–PCR). The photoreceptor-like cells were then transplanted into adult C57BL/6 mouse retinas and were assessed by immunofluorescence 14 days following transplantation. Screening from a pool of five candidate genes, we reported that a combination of only three factors—Crx, Nrl, and Otx2—was sufficient to convert mouse embryonic and postnatal fibroblasts into photoreceptor-like cells. The induced photoreceptor-like cells expressed photoreceptor-specific proteins such as Recoverin, Rhodopsin, and Opsin and integrated into the outer nuclear layer of the retina following transplantation. This exploratory study provides preliminary evidence that fibroblasts can be directly converted into photoreceptor-like cells, suggesting a cellular model and potential source for future transplantation strategies aimed at retinal repair. Full article

The human WWOX gene resides on a common fragile site and is frequently deleted or altered during DNA replication. WWOX mutations are associated with various human diseases, including cancer, neurodegeneration, and developmental deficits. However, the regulation of WWOX expression remains largely unclear. We demonstrated that stress responses, including serum deprivation, oxidative stress, and anticancer drug treatment, increase WWOX expression in human SCC-15 cells and wild-type mouse embryonic fibroblasts (MEFs) through transcriptional activation. Serum deprivation induces higher levels of reactive oxygen species and cell death in Wwox^+/+ than Wwox^−/− MEFs. Anti-apoptotic Bcl-2 family proteins regulate mitochondrial homeostasis and prevent serum deprivation-induced oxidative stress and cell death. Our results showed that serum starvation decreases protein expression levels of Bcl-X_L and Mcl-1 in Wwox^+/+ but not in Wwox^−/− MEFs. Serum starvation also fails to downregulate Bcl-X_L and Mcl-1 protein expression in WWOX-knockdown SCC-15 cells. Replenishment of ectopic WWOX induces downregulation of Bcl-X_L and Mcl-1 protein levels in Wwox^−/− MEFs after serum starvation. We determined that WWOX-mediated downregulation of Bcl-XL and Mcl-1 is accomplished through a lysosome-dependent protein degradation pathway. Moreover, a decline in reactive oxygen species generation by pretreatment of Wwox^+/+ MEFs with an antioxidant N-acetyl-L-cysteine leads to decreased WWOX induction upon serum starvation. Taken together, our results suggest that stress stimuli trigger WWOX induction by elevating the production of reactive oxygen species in cells, which promotes the degradation of Bcl-XL and Mcl-1 proteins via a lysosome-mediated pathway, thereby further aggravating oxidative stress and cell death. Full article

Background/Objectives: Sex-specific differences in the mechanisms of acute liver injury remain poorly understood. CDK5 regulatory subunit-associated protein 3 (CDK5RAP3) is crucial for liver development and endoplasmic reticulum (ER) homeostasis. This study aimed to investigate sex-dependent changes in CDK5RAP3 expression in a carbon tetrachloride (CCl₄)-induced acute liver injury model and to explore the mechanisms underlying differential susceptibility between males and females. Methods: Acute liver injury was induced in male and female mice by CCl₄ administration. Liver injury was evaluated by serum biochemical parameters and histopathological analysis. CDK5RAP3 expression, inflammatory cytokines, and ER stress-related apoptotic markers were assessed. Hepatocyte apoptosis was examined by TUNEL staining. In addition, CDK5RAP3 was conditionally deleted in mouse embryonic fibroblasts (MEFs) using 4-hydroxytamoxifen to assess its direct role in regulating inflammatory and apoptotic responses in vitro. Results: CCl₄ exposure caused liver injury in both sexes, with male mice showing more severe biochemical and histological damage. CDK5RAP3 expression was significantly reduced after CCl₄ treatment, particularly in males. Inflammatory mediators and ER stress-associated apoptotic markers were upregulated, accompanied by increased hepatocyte apoptosis. A similar enhancement of inflammatory and apoptotic signaling was observed in CDK5RAP3-deficient MEFs. Conclusions: Downregulation of CDK5RAP3 is associated with ER stress, inflammation, and apoptosis, contributing to increased susceptibility of male mice to acute liver injury. These findings provide insight into sex-specific mechanisms of hepatic injury and highlight CDK5RAP3 as a potential therapeutic target. Full article

Polymethylmethacrylate (PMMA) bone cement is widely used in orthopedics, accounting for approximately 80% of knee joint replacements in the United States. While prosthesis designs and materials have evolved to improve performance and durability, PMMA cement has undergone minimal compositional changes. Carbon-based nanomaterials, particularly graphene oxide (GO), have attracted interest for their ability to enhance the mechanical and thermal properties of orthopedic cements. This study evaluated the effects of incorporating different GO concentrations into PMMA bone cement on its mechanical properties, cytocompatibility, and antibacterial activity. PMMA was modified with GO at 0.1, 0.25, and 0.5 weight percent (wt%) for mechanical and antibacterial tests, and at 1.0 wt% for cytocompatibility. Mechanical performance was assessed via four-point bending tests. Cytocompatibility was evaluated using mouse embryonic fibroblasts (NIH/3T3), and antibacterial activity was tested against Staphylococcus aureus using a modified direct contact assay. GO incorporation significantly increased Young’s modulus (0.1% and 0.25%, p = 0.009) and improved tensile strength (p = 0.0015) and flexural strength (p = 0.025) at 0.1%. Cytocompatibility remained comparable to the control (p = 0.873). Antibacterial activity was concentration dependent, with 0.25% and 0.5% GO maintaining significant bacterial inhibition up to 48 h, whereas 0.1% showed no sustained effect. Overall, 0.25 wt% GO provided the most suitable balance between mechanical integrity and antibacterial performance, indicating that PMMA–GO bone cements with this composition can combine enhanced mechanical properties with relevant antibacterial activity without compromising biocompatibility, and are therefore promising candidates for orthopedic applications. Full article

Poly(ethylene glycol) (PEG) and poly(2-hydroxy ethyl methacrylate) are polymers used for many biomedical applications due to their biocompatibility, non-toxicity, and antibiofouling properties. In this work, a new comb-like hydrogel based on 2-hydroxyethyl methacrylate (HEMA) grafted onto a polyethylene glycol network (net-PEG) was synthesized by gamma radiation from Co⁶⁰ in two steps. First, PEG (Mw = 20,000) was crosslinked at 30 kGy, and then HEMA was grafted, varying the concentration (5–20% v/v) and irradiation dose (2.5–15 kGy). Results of infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) confirmed the incorporation of HEMA onto net-PEG. Moreover, the properties of comb-like hydrogel (net-PEG)-g-HEMA were studied through swelling kinetics, drug loading and release, antimicrobial activity, and biocompatibility assays. The findings showed a different behavior in swelling kinetics and drug delivery depending on HEMA grafting. Comb-like hydrogel with 30 and 66% grafting could load more ciprofloxacin (2 mg g⁻¹) than net-PEG (1.5 mg g⁻¹) but only release 38 and 48% at 24 h, respectively. In addition, all drug-loaded hydrogels displayed inhibition for Gram-negative bacteria (E. coli) and a cell viability superior of 95% using mouse embryonic fibroblasts (BALT/T3). Comb-like hydrogel has potential application in the biomedical field such as in wound dressings or controlled drug delivery systems. Full article

Biodegradable Pickering emulsions are attracting increased appeal owing to their promising and diversifying therapeutic applications. In this study, for the first time, a novel therapeutic Pickering emulsion stabilized with ginger powder (GA4) was formulated, characterized, and tested for doxorubicin (DOX) delivery. GA4_Pes physicochemical characterization by DLS (Dynamic Light Scattering), POM (Polarized Optical Microscopy), Cryo-SEM (Cryo-Scanning Electron Microscopy), TEM (Transmission Electron Microscopy), and rheology testing confirmed stability for at least one month, solid-like gel properties, and multiple morphology even at a low concentration of stabilizer. In addition, the morphological, dimensional, and rheological properties of some GA4_Pe loaded with DOX (GA4_Pe@DOX) were examined. These formulations were of the w/o/w type, stable for at least 28 days, and showed efficient doxorubicin internalization. A 24 h in vitro release assay displayed a sustained and pH-dependent release, with 30% and 50% chemotherapeutic released at pH 7.4 and 5.6, respectively. Furthermore, in vitro cell viability assessment performed using GA4_Pe showed no toxicity on immortalized 3T3 mouse embryonic fibroblasts but a small significant inhibitory effect on human breast cancer cell line MCF7. Interestingly, the GA4_Pe@DOX emulsion exerted a cytotoxic effect on MCF7 cells very similar to that of the free DOX solution with the same doses of DOX loaded in the same emulsion. Therefore, the total biocompatibility/biodegradability, good drug entrapment, and high stability, as well as the prolonged release and anti-tumor efficacy maintenance of the loaded drug, suggest a feasible application of ginger powder-based Pickering emulsions for topical delivery as a selective therapeutic platform in targeted formulations of antineoplastic drugs. Full article