Search Results (673)

Titanium and its alloys are widely used for orthopedic implants, but their intrinsic bioinertness may hinder osseointegration. In this study, titanium dioxide nanotube (TNT) arrays were fabricated on Ti-6Al-4V scaffolds via anodization, and their effects on the adhesion behavior of human bone marrow mesenchymal stem cells (hBMSCs) were investigated. Surface characterization showed that anodization successfully generated ordered TNT layers, increased surface roughness, enhanced protein adsorption, and induced an apparent superhydrophilic wetting response. Compared to the untreated scaffold and TNT50, the small-diameter TNT10 surface significantly promoted hBMSC adhesion and proliferation. Microscope imaging further revealed enhanced cell spreading, F-actin organization, and vinculin expression on TNT surfaces, with the most prominent focal adhesion-related staining observed in TNT10. Quantitative proteomic analysis showed that TNT10 was associated with coordinated remodeling of adhesion- and cytoskeleton-related molecular programs, including focal adhesion, cell–substrate junction, and regulation of the actin cytoskeleton. In contrast, TNT50, despite supporting obvious cytoskeletal remodeling, was more compatible with a dynamic, higher-turnover adhesion state. Overall, these findings suggest that small-diameter TNTs provide a more favorable interfacial microenvironment for stable early hBMSC adhesion on porous titanium scaffolds. Full article

The aim of this study was to investigate which physicochemical and structural properties of cationic peptides P1–P6 may determine their selective anticancer activity against melanoma cells and their interactions with tumor cell membranes. An integrated approach was applied, including characterization in solution (osmotic pressure, NaCl stability, surface tension); cytotoxicity evaluation against Me45, B16F10, and HaCaT cells; analysis of interactions with phosphatidylglycerol (POPG) model membranes using isothermal titration calorimetry and steady-state fluorescence spectroscopy; membrane permeability assays; and F-actin staining. Anticancer activity depended on positively charged residues, hydrophobic amino acids, and sequence arrangement. Tryptophan-rich peptides P2 and P5 exhibited strong membrane interactions and high efficacy after 72 h. Highly hydrophobic P4, containing long C₁₂ chains with a relatively low net charge, caused nonselective lysis. P3 showed reduced activity due to insufficient amphipathicity, whereas P6, with excessive WWW and KKKK motifs, exhibited weak or nonselective effects. Thermodynamic and fluorescence analyses indicated that P2 and P5 initially bind POPG membranes via entropy-driven electrostatic interactions, followed by hydrophobic insertion of tryptophan residues, evidenced by increased fluorescence intensity and a blue shift of the emission maximum. P2, P4, and P5 induced actin cytoskeleton reorganization and increased membrane permeability, emphasizing the role of balanced amphipathicity and charge–hydrophobicity in designing selective anticancer peptides. Full article

The cuticular plate, a dense F-actin meshwork anchoring stereocilia in auditory hair cells (HCs), undergoes dynamic remodeling during development, but its structural transitions remain poorly understood. Here, we identified two distinct structural domains associated with this maturation. First, a transient F-actin-free area emerges within the lateral periphery of the developing cuticular plate, presenting as a crescent-shaped region that disappears upon HC maturation. Second, the lateral margin of the mature cuticular plate itself remodels into a persistent step-like structure, exhibiting cell-type-specific geometries in inner versus outer HCs. The consistent coincidence between Gαi-GPSM2 complex disruption and aberrant development of both structures in mutant mice implies a role for this complex in their formation. Additionally, microtubules spatially complemented F-actin distribution, suggesting coordinated cytoskeletal regulation. These findings revealed a sophisticated developmental program for cuticular plate maturation. Full article

Five previously undescribed steroids, chrysogenones A–E (1–5), were isolated from the deep-sea-derived Penicillium sp. MCCC 3A00121. Their chemical structures were unambiguously established through comprehensive spectroscopic analyses, density functional theory (DFT)-based electronic circular dichroism (ECD) calculations, and X-ray crystallography. Chrysogenones represent a class of oxidatively modified ergosterone-type derivatives, with 1, 2, and 5 featuring an uncommon malonyl substitution at C-12 of the ergosterone skeleton. Biologically, 1–5 exhibited varying degrees of inhibitory activity against renal fibrosis, as evidenced by the downregulation of the key fibrotic markers α-smooth muscle actin (α-SMA) and collagen I (COL1A1). Among them, chrysogenone B (2) emerged as the most promising candidate, demonstrating superior potency and pronounced inhibition of activated NRK-49F cell proliferation. Integrated network pharmacology analysis and molecular docking studies further suggested that the anti-renal fibrotic effects of compound 2 may be mediated through its interaction with putative molecular targets, including AKT1, HSP90AA1, and MDM2. Full article

Mechanical signals play key roles in the regulation of epidermal homeostasis and regeneration after injury. Integrins are key components of focal adhesions, and these complexes are major contributors to mechanotransduction. In keratinocytes, integrin-linked kinase (ILK) modulates essential processes for epidermal homeostasis and wound repair. However, its functions in the transduction of mechanical stimuli have remained virtually unexplored. In this study, we characterized epidermal tissues and primary keratinocytes from mice with epidermis-restricted inactivation of the Ilk gene (ILK-KO). ILK-deficient epidermis exhibits abnormalities in key components of mechanotransduction cascades, including disruptions in hemidesmosomal Collagen XVII immunoreactivity at the dermal–epidermal junction, and marked reduction in the nuclear localization of the mechanosensitive transcriptional regulator YAP. In wild-type (ILK+), but not in ILK-KO-cultured keratinocytes, exposure to cyclic bidirectional strain induced marked F-actin cytoskeletal rearrangements, characterized by the assembly of thick cortical actin bundles and stress fibers, as well as YAP nuclear translocation and transcriptional activity. Exposure to mechanical strain was additionally accompanied by differential changes in miRNA expression between ILK+ and ILK-KO cells. These findings reveal multiple and previously unappreciated key regulatory roles for ILK in epidermal keratinocyte responses to mechanical signals. Full article

Cervical Intervertebral Disc Degeneration (CIVDD) involves significant microenvironmental physical stiffening, forcing nucleus pulposus cells (NPCs) into a rigid phenotype via F-actin over-assembly. It remains unclear if cyclic tensile strain (CTS) can reverse this physical stiffening, particularly in severe degeneration. This study stratified 18 patients into Mild, Moderate, and Severe cohorts based on MRI. Primary NPCs were subjected to physiological 5% CTS (1 Hz, 24 h). Atomic Force Microscopy (AFM) and immunofluorescence were utilized to evaluate Young’s modulus and cytoskeletal remodeling. Results demonstrated that baseline cellular stiffness increased significantly with degeneration severity. Following CTS, all groups exhibited universal de-stiffening and F-actin depolymerization. Crucially, a “Degeneration Paradox” emerged: the Severe group displayed the highest relative elastic modulus recovery rate, significantly surpassing the Mild group. This microscopic recovery correlated inversely with preoperative disc height loss and range of motion. We conclude that severely degenerated cells are not metabolically quiescent but “physically locked” by a rigid cytoskeleton. Physiological CTS restores compliance via mechanical unloading, confirming that severe cells retain superior relative mechanoplasticity and may benefit from mechanotherapy-based “unlocking” strategies. Full article

Background: Glabridin, a natural compound derived from Glycyrrhiza glabra L., possesses skin-lightening effects. This study aims to further elucidate the depigmentation mechanism of glabridin by investigating its effects on melanogenesis and melanin transfer. Methods: We initially confirmed the anti-melanogenic effects of glabridin in MNT-1 human melanoma cells. Then, we investigated the mechanism of its anti-melanogenic effect by evaluating the protein expression of β-catenin and MITF via Western blot. To investigate melanin transfer, we compared glabridin’s efficacy with that of niacinamide, a recognized inhibitor of melanosome transfer and employed two complementary experimental models: (1) α-melanocyte-stimulating hormone (α-MSH)-stimulated MNT-1 cells to analyze dendrite formation, and (2) a UVB-irradiated co-culture system of MNT-1 cells and HaCaT keratinocytes to evaluate melanin transfer. Results: By measuring glabridin’s effects on melanin content, tyrosinase activity, and melanogenesis-related protein expression confirmed its inhibition of melanin synthesis. Further investigation demonstrated that glabridin suppresses melanogenesis by downregulating β-catenin and MITF, indicating inhibition of the Wnt/β-catenin pathway. Furthermore, in α-MSH-treated MNT-1 cells, both glabridin and niacinamide were found to suppress dendrite formation and elongation. In a UVB-exposed co-culture system, both glabridin and niacinamide inhibited melanin transfer to keratinocytes. Mechanistically, these effects were linked to the regulation of Rho GTPases (Rac1, RhoA, Cdc42) and suppression of F-actin reorganization. Conclusions: This study provides, for the first time, evidence that the skin-lightening effect of glabridin involves two complementary mechanisms: inhibition of melanogenesis through suppression of the Wnt/β-catenin pathway, and attenuation of both dendricity and melanin transfer via the influence of Rho family GTPases expression. Full article

Acanthamoeba castellanii, an opportunistic free-living amoeba, causes severe infections including Acanthamoeba keratitis. This exploratory study evaluated whether three non-steroidal anti-inflammatory drugs (NSAIDs)—acetylsalicylic acid, ibuprofen, and diclofenac (100 µM)—modulate pathogenicity-related processes in A. castellanii and explored the involvement of a gp63-like protein during encystment and adhesion. Trophozoites were continuously exposed to each drug and analyzed for adhesion, migration on host-derived discontinuous brain micropatterns, encystment efficiency, and parasite-induced cytoskeletal remodeling in MDCK epithelial cells. In silico docking was performed to assess potential drug–protein interactions. Drug exposure reduced adhesion with maximal inhibition at 60 min. After 1 h, migration decreased by 49%, 64%, and 38%, and encystment was reduced by 50%, 85%, and up to 90%, respectively, in cultures treated with acetylsalicylic acid, ibuprofen, and diclofenac. Co-incubation with untreated trophozoites lowered actin fluorescence to approximately 50%, whereas drug-treated co-cultures preserved fluorescence near control levels. Colocalization analysis showed increased spatial overlap between gp63-like protein and F-actin in cysts (~40%) and migrating trophozoites (~20%) compared with non-stimulated forms (~3.8%). Collectively, these findings suggest that NSAID-sensitive pathways influence host interaction, migration, and encystment in A. castellanii and allow for the proposal of gp63-like protein as a putative molecular component of the NSAIDs sensitive pathways. Full article

Background/Objectives: Diabetes mellitus (DM) is associated with a doubled prevalence of elevated intraocular pressure (IOP) caused by trabecular meshwork (TM) dysfunction. Chronic hyperglycaemia leads to oxidative stress and fibrotic remodeling of the TM. We previously identified the Sigma-1 receptor (S1R) as a novel anti-fibrotic target by demonstrating that its agonist, fluvoxamine (FLU), is protective in diabetes-related renal fibrosis. Here, we investigate its potential to mitigate ocular fibrosis. Methods: First, we wanted to verify in different in vivo models (high-fat diet/streptozotocin (HFD/STZ) rats, db/db mice) that type 2 DM (T2DM) leads to fibrotic remodeling of the TM. Then, in vitro, we assessed the effect of FLU (15 µM) on hyperglycaemia-induced (HG, 25 µM) fibrosis, oxidative stress and endogenous nitric oxide (NO) production. Results: In T2DM models, excessive accumulation of collagen, α-smooth muscle actin (αSMA), fibronectin (Fn) and F-actin was observed in the eyes. Ocular fibrosis was accompanied by IOP elevation (13.7 vs. 18.7 mmHg) in db/db mice. In human TM cells (HTM5), FLU decreased HG-induced cell proliferation (14% vs. 24%) and upregulated S1R protein expression. Furthermore, FLU suppressed the expressions of key fibrotic elements, including transforming growth factor-β2 (TGF-β2) by 37%, Fn by 49%, collagen type 1 (COL1A1) and type 4 (COL4A1) by 24% and 45%, respectively. FLU also reversed HG-induced F-actin accumulation by 39% and enhanced intracellular NO levels by 34%. Crucially, FLU decreased ROS generation by half, demonstrating its protective effect against HG-induced oxidative stress. Conclusions: These findings highlight the potential of S1R activation as a promising therapeutic target to alleviate hyperglycaemia-induced injury to the TM by modulating multiple molecular pathways. Full article

The silkworm, Bombyx mori, is a model organism with significant agricultural and economic importance, but it is threatened by Bombyx mori nucleopolyhedrovirus (BmNPV). A crucial chaperone, heat shock protein 90 (HSP90), can also facilitate the proliferation of viruses, and our previous quantitative acetylome analysis revealed that lysines 550 and 567 in the carboxyl-terminal domain (CTD) of Bombyx mori HSP90 (BmHSP90) were significantly deacetylated following BmNPV infection, but the underlying mechanism remained unknown. In this study, deacetylation-mimetic (K to R) mutants of BmHSP90 exhibited increased dimerization and chaperone activity compared with the wild-type. In addition, the mutants also exhibited higher affinity for actin, promoting F-actin polymerization. Collectively, these changes facilitated BmNPV replication and progeny virion production. This study reveals that the deacetylation of BmHSP90 at K550 and K567 mediates crucial host–virus interactions, providing novel insights into potential antiviral strategies. Full article

Ménière’s disease (MD) is thought to involve dysfunction of the blood–labyrinth barrier, but circulating mechanisms of endothelial injury remain poorly understood. The present study investigated whether cell-free DNA (cfDNA) and inflammatory mediators in plasma contribute to vascular stress and barrier disruption in MD. cfDNA levels were significantly elevated in plasma from patients compared with plasma from healthy controls. Exposure of primary human stria vascularis endothelial cell monolayers to plasma from MD patients led to decreased transepithelial electrical resistance and a significant increase in FITC-dextran permeability, indicating impaired barrier function. MD plasma also induced higher lactate dehydrogenase release and pronounced F-actin disorganization with reduced syndecan-1 expression, consistent with endothelial cytotoxicity and glycocalyx degradation. DNase I partially reversed these effects, implicating extracellular DNA as a key driver. Furthermore, IL-1β, CCL3 (MIP-1α), and CCL27 were elevated in MD plasma. Collectively, our data support a model in which cfDNA and inflammatory mediators cooperatively induce endothelial injury, cytoskeletal remodeling, and glycocalyx shedding, leading to blood–labyrinth barrier weakening. Targeting extracellular DNA or glycocalyx preservation may represent a novel strategy to protect inner ear vascular integrity and modify disease progression in MD, and cfDNA-related readouts may be promising biomarkers of endothelial damage. Full article

The Ras-related (RRAS) gene is a member of the Ras superfamily and remains largely uncharacterized compared to KRAS, NRAS, and HRAS. Its role in tumorigenesis remains poorly documented, as evidenced by its lack of canonical mutations in any cancer type. This study investigated the effects of the novel RRAS R78W and E63D mutants—identified in Filipino young-onset colorectal cancer (YO-CRC) patients—on cancer hallmarks. In silico analysis was performed to predict the effect of the mutations on RRAS structure. F-actin staining of transfected NIH3T3 cells displayed massive cytoskeletal remodeling and formation of migratory and invasive structures. RRAS R78W enhanced migration when compared to wild-type RRAS in NIH3T3 and HCT116 cells, whereas neither mutant affected invasive capacity. Both mutants did not abolish the pro-angiogenic ability of wild-type RRAS in endothelial tube formation assays. RRAS E63D conferred resistance to apoptosis in both cell lines. Both mutants had no effect on cellular proliferation in either cell line. Overexpression of both mutants did not increase Akt and Erk1/2 phosphorylation. In silico analysis further suggests that the mutations confer increased GEF-binding ability versus wild-type. Results of the study highlight the need to characterize Ras isoform- and mutation-specific phenotypic effects, which may have repercussions in CRC management. Full article

PIEZO1 are Ca²⁺-permeable mechanogated channels that play a crucial role in numerous fundamental cellular responses. Ca²⁺ influx via PIEZO1 could control the activity of various Ca²⁺-dependent molecules within the cells, thus activating Ca²⁺-dependent signaling processes and reactions. Previously, we demonstrated Ca²⁺-mediated coupling between PIEZO1 and KCa channels in the plasma membranes of transformed mouse fibroblasts, where a Ca²⁺ influx through PIEZO1 stimulates the activity of functionally co-localized KCa channels. Importantly, the selective PIEZO1 activator Yoda1 inhibited transformed fibroblast migration, induced F-actin assembly, and stress fiber formation. However, the impact of PIEZO1-KCa channel coupling on the observed effects remains unknown. Here, we performed the molecular identification of KCa channels in transformed mouse fibroblasts. Importantly, TRAM-34, a specific KCa3.1 channel blocker, abrogated the effect of Yoda1 on F-actin organization and fibroblast motility. We conclude that KCa3.1 channels in the plasma membrane are primary downstream effectors and critical contributors to the decrease in transformed fibroblast migration and F-actin assembly caused by selective PIEZO1 activation. Full article

Metastatic breast cancer (BC) remains a major clinical challenge, and identifying molecular mechanisms driving tumor cell migration and invasion is critical to develop effective therapeutic strategies. Clusterin (CLU), a secreted chaperone-like protein, is upregulated in BC and metastatic tissue; however, its functional contribution to tumor aggressiveness remains unclear. Here, we silenced CLU by siRNA in two BC cell lines with distinct aggressiveness and examined its impact on migration, invasion, and associated signaling pathways. Following CLU silencing, cell migration and invasion were assessed using transwell assays. Cytoskeletal organization was evaluated by F-actin staining, while downstream signaling pathways were analyzed by RT-PCR, Western blotting, and Rho GTPase pull-down. A comparative proteomic analysis was performed in CLU-expressing and CLU-silenced MDA-MB-231 cells. CLU knockdown significantly reduced migration and invasion in MDA-MB-231, concomitantly with loss of F-actin-rich membrane protrusions, reduced expression of MMP9, COL1A1, and COL4A1, and decreased activation of Akt, NF-κB, and RhoA. Proteomic profiling revealed extensive remodeling of pathways involved in cell adhesion, cytoskeletal dynamics, and extracellular matrix interactions. Differently, no or very mild effects were observed in CLU-silenced MCF-7 cells. These findings identify CLU as an upstream regulator of a pro-metastatic adhesion–cytoskeleton signaling in BC, selectively operative in EMT-engaged, basal-like cells, highlighting the importance of patient stratification for CLU-targeted therapeutic strategies. Full article

Cancer metastasis, the spread of tumour cells from the primary site to distant organs, is responsible for over 90% of cancer deaths, yet effective treatments remain elusive due to incomplete understanding of the molecular drivers involved. Podocalyxin (PODXL), a protein overexpressed in many aggressive cancers, links the cell membrane to the internal skeleton through its interaction with Ezrin, an actin cytoskeleton cross-linker. Despite its therapeutic relevance, the PODXL–Ezrin interface remains structurally uncharacterised and pharmacologically intractable. Here, we employed an integrated computational approach combining protein–protein docking, molecular dynamics (MD) simulations, and virtual screening to investigate the structural basis of the PODXL–Ezrin interaction. Using AlphaFold-predicted structures, we modelled PODXL and Ezrin complexes, revealing that PODXL’s cytoplasmic domain stabilises upon Ezrin binding, with Arg495 mediating temporally distinct electrostatic interactions essential for initial complex assembly. Particularly, we characterised the R495W missense mutation in PODXL’s Ezrin-binding domain, demonstrating that substitution of arginine with bulky, hydrophobic tryptophan may allosterically destabilise Ezrin’s dormant conformation. This mutation slightly increases the intramolecular distance between the F3 subdomain and C-terminal domain from 2.59 Å to 3.40 Å, thus leading to potential partial unmasking of the Thr567 phosphorylation site that could plausibly prime Ezrin for activation. Molecular dynamics simulations in the WT state with a total simulation time of 100 ns revealed enhanced structural rigidity and reduced radius of gyration fluctuations in the mutant complex, consistent with a potential “locked,” activation-prone state that amplifies oncogenic signalling. Through virtual screening, we identified NSC305787 as a selective destabiliser of the R495W mutant complex by disrupting key Trp495–pre-C-terminal loop Ezrin interactions and causing steric hindrance to PIP2 recruitment. Our findings identified mutation-dependent changes in drug binding that can guide the development and repurposing of compounds for targeting PODXL-related cancers and improve patient outcomes in PODXL-altered malignancies. Full article