Search Results (257)

Mechanical stimulation critically regulates mesenchymal stem cell (MSC) differentiation, yet its effects in three-dimensional (3D) environments remain poorly defined. Here, we developed a custom dynamic stretcher integrating poly(dimethylsiloxane) (PDMS) chambers to apply cyclic strain to human MSCs encapsulated in Fmoc-diphenylalanine (Fmoc-FF) peptide hydrogels—a fully synthetic, tunable extracellular matrix mimic. Finite element modeling verified uniform strain transmission across the hydrogel. Dynamic stretching at 0.5 Hz and 10% strain induced pronounced cytoskeletal alignment, enhanced actin stress fiber formation (coherency index $\approx 0.85$), and significantly increased proliferation compared to static or high-frequency (2.5 Hz, 1%) conditions (coherency index $\approx 0.6$). Quantitative image analysis confirmed strain-dependent increases in coherency index and F-actin intensity, indicating enhanced mechanotransductive remodeling. Biochemical assays and qRT–PCR revealed 2–3-fold upregulation of osteogenic markers—RUNX2, ALP, COL1A1, OSX, BMP, ON, and IBSP—under optimal strain. These results demonstrate that low-frequency, high-strain mechanical loading in 3D peptide hydrogels activates RhoA/ROCK and YAP/TAZ pathways, driving osteogenic differentiation. The integrated experimental–computational approach provides a robust platform for studying mechanobiological regulation and advancing mechanically tunable biomaterials for bone tissue engineering. Full article

Small-angle X-ray fiber diffraction has informed much of what we know regarding the molecular events during muscle contraction but robust tools for predicting X-ray fiber patterns from muscle have been lacking. A complication in formulating such tools is the dynamic, stochastic nature of the sarcomere structures during contraction where individual myofilaments undergo deformations due to nonuniform strain generated by the myosin crossbridges. Here, we address this need with a “forward problem” approach using a spatially explicit model (MUSICO) to predict the molecular configurations responsible for the observed muscle force and use these configurations to predict the diffraction patterns that can be compared to experiments. We combine this with a newly developed, rigorous formulation, presented here, for the calculation of 2D diffraction patterns from actin filaments under nonuniform strain. We compare all-atom predictions to coarse-grained simulations to show how much information is lost by coarse-graining, and discuss the results in the context of diffraction patterns currently obtainable experimentally. We show that most low-resolution coarse-grained models in the literature suffice for prediction of meridional peak shapes for the purposes of estimating force distributions in the actin filaments, but accurate prediction of layer line intensities require much higher resolution models, including the all-atom models as presented here. These developments represent an important step towards our long-term goal of using molecular simulations to interpret X-ray fiber diffraction patterns from striated muscle during active contraction. Full article

Background/Objectives: Amlodipine, a widely prescribed calcium channel blocker, has been associated with gingival enlargement, yet the mechanisms underlying this adverse effect remain unclear. The present study aimed to explore molecular and histopathological factors potentially contributing to gingival changes in patients receiving amlodipine therapy, with a particular focus on molecules implicated in extracellular matrix turnover and tissue remodeling. Methods: The study included three groups of participants: patients with amlodipine-induced gingival enlargement, patients with gingival enlargement of inflammatory origin, and amlodipine-treated patients without gingival overgrowth. Gingival tissue samples were analyzed using hematoxylin-eosin staining to assess inflammatory changes and general tissue architecture, and Picrosirius Red staining to visualize collagen fibers. Relative gene expression of alpha-smooth muscle actin (α-SMA), IL-13, MMP-1, and procollagen was determined by real-time PCR, while collagen content was quantified using ImageJ software. Results: Histopathological evaluation revealed a less pronounced inflammatory response in amlodipine-related gingival enlargement compared to those who did not use amlodipine. The highest expression of α-SMA was detected in patients who did not receive amlodipine, whereas IL-13 and procollagen expression were markedly elevated in the amlodipine-induced group compared to others. MMP-1 expression was significantly lower in amlodipine-treated patients relative to those who did not use amlodipine, suggesting impaired collagen degradation. These findings, together with our previous results indicating enhanced expression of profibrotic mediators, suggest that altered extracellular matrix metabolism is potentially dominant in this condition. Conclusions: Amlodipine-induced gingival enlargement appears to involve a multifactorial process characterized by a prominent fibrotic component, reduced matrix degradation, and secondary inflammation. Full article

Wool traits such as fiber diameter, fiber length, and greasy fleece yield are economically significant characteristics in sheep breeding programs. Traditional genome-wide association studies (GWAS) have identified relevant genomic regions but often fail to capture the non-linear and polygenic architecture underlying these traits. In this study, we implemented a two-stage machine learning (ML)-based GWAS framework to dissect the genetic basis of wool traits in Central Anatolian Merino sheep. Phenotypic records were collected from 228 animals, genotyped with the Illumina OvineSNP50 BeadChip. In the first stage, feature selection was conducted using LASSO, Ridge Regression, and Elastic Net, generating a consensus SNP panel per trait. In the second stage, association modeling with Random Forest and Support Vector Regression (SVR) identified the most predictive models (R² up to 0.86). Candidate gene annotation highlighted biologically relevant loci: MTHFD2L and EPGN (folate metabolism and keratinocyte proliferation) for fiber diameter; COL5A2, COL3A1, ITFG1, and ELMO1 (extracellular matrix integrity and actin remodeling) for staple length; and FAP, DPP4, PLCH1, and NPTX1 (extracellular matrix remodeling, proteolysis, and sebaceous gland function) for greasy fleece yield. These findings demonstrate the utility of ML-enhanced GWAS pipelines in identifying biologically meaningful markers and propose novel targets for genomic selection strategies to improve wool quality and yield in indigenous sheep populations. Full article

Aberrant wound healing in the retina can manifest as proliferative vitreoretinopathy (PVR), which involves the myofibroblast transdifferentiation of retinal pigment epithelial (RPE) cells. In this study, experiments were conducted to examine the structure–activity relationships of endocannabinoid-like compounds, N-acyl dopamines, on the myofibroblast transdifferentiation of RPE cells. The collagen matrix contraction assay was used to assess myofibroblast function. Western blot analysis and immunocytochemistry techniques were used to evaluate myofibroblast markers. N-palmitoyl dopamine (PALDA), N-oleoyl dopamine (OLDA), and N-arachidonoyl dopamine (NADA), in a concentration-dependent manner, inhibited contraction of collagen matrices mediated by either primary porcine RPE cells treated with TGF-β2, or human RPE cells treated with TGF-β2 plus TNFα (TNT). The rank order of potency was PLDA = OLDA > NADA. In contrast, the substitution of dopamine with other polar head groups led to a complete loss of their ability to inhibit myofibroblast transdifferentiation. Western blot analysis demonstrated that PALDA, OLDA, and NADA down-regulated the myofibroblast markers fibronectin and α-SMA. Immunocytochemistry experiments showed that these N-acyl dopamines reduced the incorporation of α-SMA into F-actin stress fibers. Overall, these structure–activity relationship studies demonstrate that the dopamine head group is crucial for N-acyl dopamine to inhibit myofibroblast transdifferentiation of RPE cell, whereas the fatty acid side chain determines the potency of it. This study points to the potential of N-acyl dopamines as a novel class of therapeutic agents for treating retinal fibrotic conditions, such as PVR. Full article

Receptor for advanced glycation end-products (RAGE) activation by hyperglycemia-induced AGE (advanced glycation end-products) accumulation is likely to play a crucial role in the development of complications such as diabetic peripheral neuropathy (DPN). RAGE signaling is mediated via its cytosolic tail. Through its cytosolic tail, RAGE recruits diaphanous-related formin 1 (Diaph1), a protein involved in actin filament organization. Disruption of RAGE–Diaph1 interactions using small molecules alleviates diabetic complications in mice; however, the role of Diaph1 in DPN progression has not been rigorously tested. In this study, we employed a Diaph1 knockout mouse (DKO) to investigate the role of Diaph1 in DPN progression. Herein, we demonstrate that, at the systemic level, CRISPR deletion of Diaph1 fails to ameliorate diabetes-induced weight loss in mice. Within the sciatic nerve (SCN), the lack of Diaph1 failed to prevent hyperglycemia-induced loss of β-actin in the nerve fibers. At a morphological level, the lack of Diaph1 leads to a partial rescue in DPN. While we observed improvements in axonal and fiber diameters in diabetic DKO mice, the g-ratio (an indicator of myelination) and myelin invaginations displayed incomplete rescue. Furthermore, the lack of Diaph1 failed to rescue motor or sensory nerve conduction defects resulting from hyperglycemia over 6 months. Overall, our data thus indicate that the complete loss of Diaph1 is insufficient to halt the progression of DPN. However, across a range of parameters including blood glucose levels, body weight measurements, axon and fiber diameters, and nerve conduction velocity, DKO diabetic mice show improvement when compared to wild-type diabetic mice. Full article

(1) Background: The extracellular matrix (ECM) is a natural scaffold for cells, creating a three-dimensional architecture composed of fibrous proteins (mainly collagen) and proteoglycans, which are synthesized by resident cells. In this study, a physiological hypoxic environment was utilized to enhance ECM production by human mesenchymal stem cells (hMSCs), a process relevant to tissue engineering and regenerative medicine. (2) Methods: hMSCs were treated with deferoxamine (DFO), a pharmaceutical hypoxia-mimetic agent that induces cellular responses similar to low-oxygen conditions through stabilization of hypoxia inducible factor-1α (HIF-1α). The time points 0 h 24 h, 3 h 24 h, and 24 h 24 h refer to DFO being added immediately after cell seeding (before cells adhesion), 3 h after cell seeding (during initial cells attachment), and 24 h after cell seeding (after focal adhesions formation and actin organization), respectively, to evaluate the influence of cell adhesion on ECM deposition. hMSCs incubated in culture media were subsequently exposed to DFO for 24 h. Samples were then subjected to cell viability tests, scanning electron microscopy (SEM), atomic force microscopy (AFM) and laser scanning confocal microscopy (CLSM) assessments. (3) Results: Viability tests indicated that DFO concentrations in the range of 0–300 µM were non-toxic over 24 h. The presence of collagen fibers in the DFO-derived ECM was confirmed with anti-collagen antibodies under CLSM. Increased ECM secretion was observed under the following conditions: 3 μM DFO (24 h 24 h), 100 μM DFO (0 h 24 h) and 300 μM DFO (3 h 24 h). SEM and AFM images revealed the morphology of various stages of collagen formation with both collagen fibrils and fibers identified. (4) Conclusions: Our preliminary study demonstrated enhanced ECM secretion by hMSC treated with DFO at concentrations of 3, 100, and 300 µM within a short cultivation period of 24–48 h without significant affecting cell viability. By mimicking physiological processes, it may be possible to stimulate endogenous tissue regeneration, for example, at an injury site. Full article

Background: Endothelial cells (EC), crucial components of the vascular system, are adaptable cells that maintain homeostasis and respond to pathological events through structural and functional plasticity. Hepatocyte growth factor (HGF) is a multifunctional cytokine that has been demonstrated to have protective and disruptive influence on the blood barrier function. In endothelial biology, its role is also poorly characterized. The present study explores the impact of supraphysiological concentrations of HGF on mouse brain endothelial cells (MBECs), scrutinizing how it alters their integrity and morphology. Methods: Two groups of MBECs—control (CTR) and experimental (EXP)—were analyzed at two time points: early passage (p5) and late passage (p41). The EXP-groups (p5 and p41) were treated with HGF at a concentration of 4 µL/mL. Cellular morphology was assessed with brightfield microscopy; protein expression and localization of the tight junction marker (ZO-1) and the endothelial marker (Factor VII related antigen/von Willebrand factor, vWf) were analyzed using Western blotting, immunocytochemistry, and confocal microscopy. Intercellular barrier function was estimated via Transendothelial Electric Resistance (TEER) and Transendothelial Dextran Permeability (TEDP) assays. Results: Microscopical analysis demonstrated a change in the morphology of the MBECs from a longitudinal, spindle-like shape to a rounded, more spheroid, cobblestone-like morphology under high-dose HGF treatment. Western blotting revealed a progressive decrease of ZO-1 expression in the EXP-groups. The expression of vWf did not show significant differences. Qualitative immunocytochemical staining: vWf showed consistent expression across all groups. ZO-1 displayed a punctate, well-defined membrane and cytoplasmic localization pattern in the CTR-groups at p5 and p41. In contrast, the p5 EXP-group demonstrated a shift to a more diffuse cytoplasmic pattern. At p41, the EXP-group displayed a markedly reduced ZO-1 signal with no clear-cut membrane localization. Confocal analysis: ZO-1: punctate membrane-associated localization in CTR-groups at p5 and 41. The EXP-groups at p5 and p41 confirmed the diffuse cytoplasmic ZO-1 distribution. Phalloidin: well-organized actin cytoskeleton in CTR-groups, but rearrangement and stress fiber disorganization in the EXP-groups, especially at p41. The merged images confirmed reduced co-localization of ZO-1 with actin structures. Barrier function: TEER values dropped significantly in HGF-treated cells. TEDP to small and medium molecular weight dextran increased markedly under HGF treatment. Conclusions: Our data demonstrate that supraphysiological doses of HGF in an in vitro MBEC-barrier-like model disrupt TJ organization, leading to morphological changes and functional weakening of the MBEC-barrier-like structure, as shown by uncoupling between ZO-1/F-actin cytoskeleton, reduced TEER, and increased size-selective paracellular permeability (TEDP). Full article

Background: Contact between blood and the large artificial surfaces within membrane lungs (MLs) is one reason for device-induced thrombus formation during extracorporeal membrane oxygenation (ECMO). Methods: Hemocompatibility testing of gas-exchange fibers (GFs) and heat-exchange fibers (HEs) from commercially available/non-used MLs (ML-type, coating: PLS, Bioline^®; Hilite7000LT, X.ELLENCE^®; Nautilus, Balance^®; EOS, PH.I.S.I.O^®) included static hemolysis and platelet adhesion/activation assays. Platelet activation of non-adherent platelets was identified after antibody (CD62P, PAC-1, CD61) and fibrinogen staining (flow cytometry). The surface coverage (%) of adherent platelets was quantified after F-actin filament-staining. Results: All materials were non-hemolytic and did not induce platelet activation. However, platelet adhesion (median (IQR)) depended on the type of surface coating of GFs made entirely of polymethylpentene. Both uncoated GFs (12 (7–19)%) and X.ELLENCE-coated GFs (Hilite-ML, 13 (8–19)%) showed a significantly higher surface coverage compared to Balance-coated GFs (Nautilus-ML, 3 (1–6)%), PH.I.S.I.O-coated GFs (EOS-ML, 2 (2–5)%) and Bioline-coated GFs (PLS-ML, 4 (1–8)%) (p < 0.001). HEs made of polyethyleneterephthalate (Hilite-ML, Nautilus-ML) that were coated with X.ELLENCE were covered with more platelets (5 (3–7)%) compared to Balance-coated HEs (3 (1–6)%), respectively (p = 0.029). Conclusions: In vitro testing disclosed fourfold higher platelet adhesion on X.ELLENCE-coated GFs (and HEs) from the Hilite-ML compared to other ECMO-materials. Additional hemocompatibility tests are necessary to assess the increased platelet adhesion on the materials from the Hilite-ML. Full article

Skeletal muscles, accounting for 40% of mammalian body mass, exhibit pronounced heterogeneity due to their distinct anatomical locations. Animal husbandry has focused excessively on longissimus dorsi (LDM) development while neglecting other muscles. In this study, we integrated Bulk RNA Sequencing (bulk RNA-seq) and Liquid Chromatography–Mass Spectrometry (LC-MS) analyses of Soleus (SOL), Gastrocnemius (GAS), and Psoas major muscles (PMM) across three key stages in Duroc pigs. We identified nine critical genes (S100A1, MBOAT2, CA3, GYG2, ACTN3, ENO3, SLC3A2, SLC16A10, and GAPDH) and eight metabolites potentially involved in regulating both skeletal muscle development and fiber-type transformation. The heterogeneity between SOL and GAS was low at birth but increased gradually during development. In contrast, PMM exhibited higher heterogeneity than SOL and GAS from birth. Notably, expression levels of MYH7, MYH1, and MYH4 displayed stage-specific and muscle type-dependent variations. Moreover, we observed a developmental shift from the MAPK signaling pathway (1–21 d) to the regulation of the actin cytoskeleton (21–120 d). Pairwise comparisons between the SOL, GAS, and PMM revealed that the signaling pathways were enriched in muscle fiber-type switching. Collectively, through the integration of bulk RNA-seq and LC-MS data, this study provides novel molecular breeding strategies for the genetic improvement of meat-producing animals. Full article

Pseudorabies virus (PRV), a highly pathogenic alphaherpesvirus, poses a potential threat to public health and safety due to its broad host range and risk of cross-species transmission. Viruses have evolved multiple strategies to exploit host factors for entry into and survival in host cells. Drebrin is an actin-binding protein that restricts rotavirus entry by inhibiting dynamin-mediated endocytosis. However, its role and mechanism in DNA virus infection, particularly in herpesviruses, remain unexplored. In this study, we investigated the role of Drebrin in PRV infection using pharmacological inhibition (BTP−2) and CRISPR-Cas9-mediated gene knockout. Both the Drebrin inhibitor BTP−2 and gene knockout significantly suppressed PRV replication. Intriguingly, Drebrin exhibited stage-specific effects on the viral life cycle: its inhibition enhanced viral internalization during early infection but impaired viral replication at later stages, suggesting that Drebrin plays a complex role in the regulation of PRV infection. PRV infection partially disrupted actin stress fibers and caused an increase in cell size. Drebrin knockout also altered the host-cell morphology, reduced the cell surface area, and induced actin cytoskeleton rearrangement, which was further modulated in PRV-infected cells. In summary, our data demonstrate that Drebrin functions as a critical host factor governing the entire PRV life cycle by regulating actin cytoskeleton reorganization. Full article

Background: Chronic exercise has been linked to positive effects on cognitive function and brain health. The aim of our study was to investigate how exercise affects cerebral resistance artery morphology, with an underlying focus on potential sex differences. Methods: Wistar rats were divided into male exercising (M.Ex; n = 6), female exercising (F.Ex; n = 5), male sedentary (M.Sed; n = 5), and female sedentary (F.Sed; n = 5) groups. After a 12-week swimming program, histological examinations of the intracerebral and pial arterioles were performed. SMA-DAB (smooth muscle actin) and resorcin-fuchsin (elastica) stained brain coronal sections were used for quantitative colorimetric analysis. Results: Investigating the effect of exercise, we found that in both pial and intracerebral arterioles, the elastic fiber density increased in both female and male exercising animals compared to the sedentary groups (p < 0.05 (M.Sed vs. M.Ex); p < 0.0001 (F.Sed vs. F.Ex)). As sex differences, we found that in female animals’ pial arterioles, the density of elastic fiber was increased compared to the male exercising group (p < 0.001 (M.Ex vs. F.Ex)). In pial arterioles, the smooth muscle density was higher in the male sedentary animals (p < 0.01 (M.Sed vs. F.Sed)); in intracerebral arterioles, the smooth muscle density increased with exercise in the male animals as well (p < 0.0001 (M.Ex vs. F.Ex)). Conclusions: Our results demonstrate that the increase in vascular elasticity is more pronounced overall in female animals. Full article

Migrastatic strategies are considered as candidate therapeutic approaches to suppress cancer invasion into local surrounding tissues and metastatic spread. The F-actin cytoskeleton is responsible for key properties regulating (cancer) cell migration. The cortical F-actin network controls cell stiffness, which, in turn, determines cell migration strategies and efficiency. Moreover, the dynamic remodeling of F-actin networks mediating filopodia, lamellipodia, and F-actin stress fibers is crucial for cell migration. Here, we have used a conditional knockout approach to delete cofilin, an F-actin-binding protein that controls severing. We find that the deletion of cofilin prevents the migration of cancer cells from tumoroids into the surrounding extracellular matrix without affecting their viability. This identifies cofilin as a candidate target to suppress metastatic spread. Pharmacological inhibitors interfering with F-actin dynamics have been developed but their effects are pleiotropic, including severe toxicity, and their impact on 3D tumor cell migration has not been tested or separated from this toxicity. Using concentration ranges of a panel of inhibitors, we select cytochalasins based on the suppression of 2D migration at non-toxic concentrations. We then show that these attenuate the escape of tumor cells from tumoroids and their migration into the surrounding extracellular matrix without toxicity in 3D cultures. This effect is accompanied by suppression of cell stiffness at such non-toxic concentrations, as measured by acoustic force spectroscopy. These findings identify cytochalasins B and D as candidate migrastatic drugs to suppress metastatic spread. Full article

Polarized fluorescence microscopy of “ghost” muscle fibers, containing fluorescently labeled F-actin, tropomyosin, and myosin, has provided new insights into the molecular mechanisms underlying muscle contraction. At low Ca²⁺, the troponin-induced overtwisting of the actin filament alters the configuration of myosin binding sites, preventing actin–myosin interactions. As Ca²⁺ levels rise, the actin filament undergoes untwisting, while tropomyosin becomes overtwisted, facilitating the binding of myosin to actin. In the weakly bound state, myosin heads greatly increase both the internal twist and the bending stiffness of actin filaments, accompanied by the untwisting of tropomyosin. Following phosphate (Pi) release, myosin induces the untwisting of overtwisted actin filaments, driving thin-filament sliding relative to the thick filament during force generation. Point mutations in tropomyosin significantly alter the ability of actin and tropomyosin filaments to respond to Pi release, with coordinated changes in twist and bending stiffness. These structural effects correlate with changes in actomyosin ATPase activity. Together, these findings support a model in which dynamic filament twisting is involved in the molecular mechanisms of muscle contraction together with the active working stroke in the myosin motor, and suggest that impairment of this ability may cause contractile dysfunction. Full article

This study evaluated the effects of mechanical agitation of Aloe vera Barbadensis Miller solution at different concentrations using passive ultrasonic irrigation (PUI), XP Endo Finisher (XPF), XP Clean (XPC), and Easy Clean (ECL), compared to conventional endodontic irrigation (CIE), on bond strength and adhesive failure patterns in the cervical, middle, and apical thirds of the root canal. Aloe vera solutions at 1%, 3%, and 5% were tested to reverse collagen fiber collapse induced by hypochlorous acid, a free radical released by 2.5% sodium hypochlorite, which impairs dentin hybridization and the light curing of resin cement. Fiberglass posts were cemented using an etch-and-rinse adhesive system (Ambar; FGM) and conventional dual resin cement (Allcem Core) in root dentin across all thirds. Human teeth underwent chemical–mechanical preparation, and the Aloe vera solution was agitated using the CIE, PUI, XPF, XPC, or ECL protocols. Slices from each root third were evaluated under a stereomicroscope at 10× magnification and subjected to the push-out test. Cytotoxicity was assessed by applying various Aloe vera concentrations to stem cells from the apical papilla (SCAPs) for 24 h, followed by analysis of cell metabolism (Alamar Blue), viability (Live/Dead), and proliferation (F-actin). Aloe vera demonstrated significant biological activity and enhanced bond strength, particularly at 3% and 5%, irrespective of the agitation method or root third. Thus, it can be concluded that using Aloe vera solution is an alternative for pre-treatment before the cementation of fiberglass posts with conventional dual-cure resin cement in endodontically treated dentin. Full article