Search Results (689)

Molecule Interacting with CasL 1 (MICAL1) is a flavoprotein monooxygenase that promotes filamentous actin (F-actin) depolymerization. Transcriptomic studies have linked MICAL1 downregulation to skeletal muscle atrophy and muscular dystrophy, yet its functional contribution to myogenesis remains unexplored. We found that MICAL1 protein increased progressively during myogenic differentiation of C2C12 cells, reaching a maximum on day 5 in parallel with myosin heavy chain (MyHC). siRNA-mediated MICAL1 silencing produced an ~1.7-fold accumulation of F-actin, while total β-actin protein remained unchanged, indicating a shift in the G-/F-actin equilibrium toward polymerization rather than altered actin expression. The accumulated F-actin reduced YAP1 phosphorylation, promoted its nuclear translocation, and increased the expression of the YAP1 target gene CTGF. MICAL1 depletion also enhanced myoblast proliferation: EdU incorporation and cell viability increased, and PCNA, CCNB1, and CCND1 protein expression was upregulated, while the cell cycle distribution shifted toward the G2/M phase, with a reciprocal loss in G0/G1. Concurrently, MICAL1 knockdown suppressed MyoD, Myogenin, and MyHC throughout differentiation and severely impaired myotube formation, with reductions in the fusion index, myotube area, and length. We conclude that MICAL1 is required for the proliferation-to-differentiation switch in myoblasts and that its activity restrains F-actin-driven YAP1 signaling to permit timely myogenic commitment. MICAL1 may therefore represent a candidate for further investigation in muscle-wasting diseases. Full article

Nanoplastic particles are increasingly detected in aquatic environments, yet whether cellular stress responses persist after exposure cessation remains unclear. Here, Lateolabrax maculatus rostral (LMR) cells from spotted sea bass were continuously exposed for up to 30 passages (105 days) to nominal 20 nm carboxylated fluorescent polystyrene particles (20 μg/mL), followed by 10 particle-free passages (recovery). Long-term exposure was associated with reduced proliferation and pronounced changes in cell surface morphology and ultrastructure. Although NP-associated fluorescence became undetectable during recovery, several nuclear-associated alterations persisted throughout the 10-passage recovery period, including TEM-observed nuclear-envelope alterations, filamentous actin (F-actin) reorganization, and sustained elevation of phosphorylated H2AX (γ-H2AX) foci. Several nuclear pore complex (NPC) genes were downregulated during exposure and rebounded after particle removal, whereas the nuclear-to-cytoplasmic distribution of proliferating cell nuclear antigen (PCNA) remained shifted. Together, these findings indicate that prolonged exposure to this nanoscale polystyrene particle formulation was associated with nuclear stress responses that did not fully resolve within the 10-passage recovery window in fish cells. Because bulk-polymer and chemical-extract (leachate) controls were not included, nanoscale-specific effects cannot be distinguished from contributions of polymer chemistry, surface functionalization, fluorescent dye, or other formulation-related effects. Full article

The herpes simplex virus 1 (HSV-1) US3 is a multifunctional serine/threonine kinase that promotes HSV-1 replication and spread. But its role and the mechanisms by which US3 regulates actin cytoskeletal remodeling remain poorly defined. We combined flow cytometry, confocal microscopy, immunoprecipitation-mass spectrometry (IP-MS), protein complex mapping, and machine learning to characterize US3-mediated F-actin dynamics. Flow cytometry and confocal microscopy showed that wild-type HSV-1 induces significant F-actin remodeling, while the ΔUS3 mutant displays F-actin levels comparable to uninfected cells, identifying US3 as a key regulator. IP-MS identified 47 high-confidence US3 interactors enriched in conserved actin regulatory complexes, including Arp2/3 nucleation machinery, formin-associated assemblies, cofilin severing complexes, and Rho-family GTPase modules. Mapping interactors to the CORUM database revealed clustering within actin nucleation, polymerization, and severing complexes, indicating that US3 operates through organized cellular machines. Machine-learning classifiers trained on label-independent mass-spectrometry features were used to prioritize interactors resembling known actin regulators; under protein-group-aware cross-validation, logistic regression performed best (average precision 0.24; ROC-AUC 0.66), and the analysis was interpreted as prioritization rather than de novo discovery. Pharmacological inhibition of Arp2/3 and formin pathways significantly attenuated US3-dependent F-actin remodeling, supporting the functional involvement of these pathways. Together, these findings are consistent with an inferred hierarchical axis in which US3 modulates Rho GTPase signaling and cofilin activation to promote F-actin disassembly, coordinating cytoskeletal remodeling required for efficient viral egress and spread. Full article

Quantum Dots (QDs) are nanoparticles that are highly desirable for biomedical applications such as drug delivery, cellular tracking, and imaging due to their fluorescent and tunable optical properties. However, the biochemical mechanism of their interaction with intracellular proteins that regulate cytoskeletal organization remains poorly understood. While previous studies have shown QDs’ ability to interact with actin and alter actin dynamics, their impacts on actin-binding proteins have not been explored. In this study, we investigated the interaction between CdSe/ZnS QDs and the actin-binding protein, α-actinin, and assessed its impact on actin cytoskeletal organization. Our results demonstrated a strong interaction between QDs and α-actinin, which impeded an α-actinin-mediated filamentous actin (F-actin) bundling, as well as compromised the activity of α-actinin in preventing actin depolymerization. Furthermore, the physics-based modeling and simulations carried out at physiological temperatures supported these findings by identifying stable interaction surfaces between QDs and α-actinin. This study provides mechanistic insight into nanoparticle–protein interactions and highlights the potential cytoskeletal toxicity associated with it. Full article

RNA interference (RNAi) represents a promising alternative to chemical insecticides, but its efficacy depends on efficient double-stranded RNA (dsRNA) uptake, a process poorly characterized in thrips. To enable sufficient dsRNA production for functional studies, we first optimized an E. coli expression system by constructing a vector containing three tandem terminators, which substantially enhanced dsRNA yield by approximately 11-fold. Using this optimized production system, this study identified a conserved muscle actin fragment for dsRNA synthesis and evaluated RNAi responses in Megalurothrips usitatus and Frankliniella occidentalis. Insect mortality, target-gene suppression, and transcriptomic responses were evaluated via RT-qPCR and RNA-seq analyses using artificial diets supplemented with muscle actin dsRNA. The designed dsactin shared > 97% sequence identity between the two species. Oral ingestion of 1500 ng µL⁻¹ dsRNA caused concentration-dependent mortality (72% in M. usitatus, 48% in F. occidentalis) and significant down-regulation of muscle actin mRNA within 72 h. Transcriptomic analysis in M. usitatus revealed upregulation of genes associated with clathrin-mediated endocytosis and SID-1-like transmembrane transport, suggesting a potential dual-pathway model for dsRNA uptake. These findings provide correlative insights into RNAi efficiency in thrips. Full article

Background/Objectives: Accurate preoperative assessment of peritoneal dissemination is essential in ovarian cancer because it influences surgical strategy and the achievement of complete gross resection. However, [¹⁸F]FDG-PET may be limited in detecting lesions with low glycolytic activity and in differentiating malignancy from inflammatory changes. This case series evaluated the clinical relevance of [¹⁸F]FAPI-74 PET/CT for preoperative assessment of peritoneal dissemination in ovarian cancer. Methods: Four patients underwent [¹⁸F]FAPI-74 PET/CT as part of preoperative evaluation, with comparison to [¹⁸F]FDG-PET/CT when available. Imaging findings were correlated with intraoperative observations and histopathological results, including immunohistochemical assessment of fibroblast activation protein and α-smooth muscle actin. Results: FAPI-PET detected peritoneal dissemination not identified by FDG-PET in several cases, including occult metastasis confirmed histologically and additional lesions after neoadjuvant chemotherapy. FAPI-avid lesions showed stromal activation on immunohistochemistry, supporting the biological basis of FAPI uptake. In one case, additional FAPI uptake may have been partly influenced by inflammatory changes associated with bloody ascites. Conclusions: FAPI-PET may provide complementary information by visualizing stromal components of ovarian cancer and may support preoperative mapping of peritoneal dissemination, although interpretation should consider inflammatory conditions. Full article

Based on their domain organisation, four proteins from the protist Dictyostelium discoideum have been assigned to the IQGAP family of scaffold proteins. Although these proteins are shorter than animal IQGAPs, their involvement in the regulation of the actin cytoskeleton in cell motility, macroendocytosis, cytokinesis, and adhesion appears to be broadly conserved between these evolutionarily distant organisms. In this article, we show that the putative three-dimensional structure of Dictyostelium IQGAP-related proteins, as predicted by AlphaFold 3, closely corresponds to the C-terminal half of human IQGAP1, thus supporting their common origin. IqgD is the largest IQGAP-related protein in Dictyostelium, with an overall domain organisation similar to human IQGAPs. IqgD is localised in the cell cortex, interacts with F-actin and Rac1 GTPases, and primarily supports cell adhesion to the underlying surface and cell growth on bacterial lawns. DGAP1 and GAPA are truncated proteins that have retained a 700-residue-long C-terminal region of homology compared to their animal relatives. They play important, yet opposite, roles in regulating contractile cortical assemblies comprising F-actin, myosin II, and the actin-bundling proteins cortexillins, which are especially important for cytokinesis and epithelial morphogenesis. Finally, IqgC, although structurally resembling other IQGAPs, turns out to be more closely related to GAP1 proteins from fungi. This multifaceted protein carries RasGAP activity, interacts with several other small GTPases, and positively regulates macroendocytosis and cell–substratum adhesion. Full article

Background/Objectives: The early identification of skin cancer by standard RGB dermoscopy is a clinical difficulty because of the complex visual differences between impacted lesions and healthy tissue. Methods: For the biomedical challenge, a novel approach to signal processing and image reconstruction is introduced in this study, called the spectrum-aided visual enhancer (SAVE). The proposed SAVE mechanism aims at reconstructing the diagnostically relevant spectral information from the conventional RGB dermoscopic images using the principles of hyperspectral imaging (HSI) and band selection (BS). After quality control and pre-processing, the images in the ISIC2019 dataset were selected, with 865 images that contain basal cell carcinoma (BCC), seborrheic keratosis (SK), and actinic keratosis (AK) lesions. To reduce data leakage, the dataset was split into training, validation, and testing subsets of 70%, 20%, and 10%, respectively. Five supervised deep learning object detection models were trained and tested on the conventional RGB image dataset and on the SAVE-enhanced dataset. Five supervised deep learning object detection models, namely, YOLOv8, YOLOv10, YOLOv11, SSDLite, and SSD, were trained and tested on the conventional RGB image dataset and the SAVE-enhanced dataset. Additional repeated experimental assessments and statistical comparisons were also carried out to evaluate the improvement in performance. Results: The experimental results showed that the SAVE-based pre-processing always yielded better performance in terms of lesion detection than conventional RGB image processing. The SAVE framework for SSD was evaluated and compared with all other evaluated models and was found to be the most successful, with an accuracy of 96%, a precision of 97%, a recall of 96%, and an F1 score of 96%. Conclusions: The results indicate that the proposed SAVE framework could be a promising RGB-compatible spectral enhancement technique for boosting skin cancer detection and computer-aided dermatologic analysis with the aid of AI. Full article

Bovine viral diarrhea virus (BVDV), a significant global pathogen threatening cattle industries worldwide, presents substantial challenges for disease control. Its ability to infect cattle across all age groups, coupled with incompletely understood transmission mechanisms, complicates prevention and treatment strategies. We previously reported that BVDV induced tunneling nanotubes (TNTs)—F-actin-rich cytoplasmic connections between adjacent cells—and utilizes these structures for intercellular transmission. In this study, we used lentiviral transfection to express various structural and non-structural proteins of BVDV and identified NS5A as a critical viral protein that induces the formation of TNTs. RNA-seq analysis revealed that CKAP2, a host protein, plays a key role in TNT generation, with the PI3K/AKT/mTOR signaling pathway being essential for this process. Further investigation demonstrated that CKAP2 interacts with BVDV NS5A, triggering the activation of the PI3K/AKT/mTOR pathway, thereby promoting TNT formation and enhancing viral dissemination. Our data highlight a previously unknown mechanism of BVDV spreading and replication, which could have significant implications for within-host spread and immune evasion. Full article

Tropomyosins (Tpm) are the family of actin-binding proteins encoded by four genes in humans. Missense mutations in the TPM1 gene associated with cardiomyopathies have been studied in the sarcomeric isoform Tpm1.1. The cardiomyopathy-causing mutations E40K and E54K are located in exon 2b of the TPM1 gene and may be expressed in non-muscle cytoplasmic Tpm isoforms, including Tpm1.7, which is associated with early tissue development. In the present work, we investigate the effects of mutations E40K and E54K on the properties of Tpm1.7. The E40K and E54K mutations caused destabilization of the Tpm1.7 molecule at the N- and C-termini parts. Neither mutation affected the Tpm1.7 affinity for filamentous actin (F-actin). The bending stiffness of F-actin/Tpm1.7 E40K filaments was lower compared to F-actin/Tpm1.7 WT (wild-type). The interplay of Tpm1.7 and motor proteins was studied in an in vitro motility assay with skeletal myosin. Tpm1.7 WT reduced the sliding velocity of F-actin by half; the velocity of F-actin with Tpm1.7 E54K did not differ from that of bare F-actin; and Tpm1.7 E40K decreased the F-actin velocity by approximately threefold. While Tpm1.7 E40K did not affect the protective effect of Tpm1.7 against F-actin severing by cofilin-1, the E54K mutation enhanced protection against cofilin-1. Thus, cardiomyopathic mutations in the TPM1 gene can affect the properties of non-muscle Tpm isoforms, which indicates that this should be taken into account when studying the molecular mechanisms of the pathogenesis of these diseases. Full article

Computational clearing (CC) enhances widefield (WF) fluorescence microscopy by suppressing out-of-focus haze and autofluorescence, yielding semi-confocal quality images suitable for segmentation and image-based phenotyping. Here, we propose an “image tracing” workflow for inflammatory mouse intestinal organoids (mIOs) using paired CC and WF images to generate a differential signal (CC − WF). mIOs were derived from intestinal crypts of Lgr5-EGFP stem cell reporter mice and expanded under epidermal growth factor, Noggin, and R-spondin (ENR) conditions. Inflammation was induced by dextran sulfate sodium (DSS) treatment. CC processing enhanced phalloidin-stained apical F-actin and improved EGFP signals by reducing background noise, enabling robust segmentation and quantitative extraction of image morphometrics including area, circularity, and perimeter. CC-WF vectors derived from three-dimensional area–perimeter–circularity plots sensitively captured DSS-induced epithelial disruption analogous to a leaky-epithelium phenotype. Transcriptomic analysis by RNA-seq of DSS-treated mIOs revealed upregulation of inflammatory pathways including TNF-α signaling via NF-κB and IL-6/JAK/STAT3, aligning with microscopy findings. In a proof-of-concept demonstration using phalloidin-stained fluorescence images, ROC analysis of the CC-WF workflow achieved an AUC = 0.95 with 87.5% sensitivity and 92.9% specificity in distinguishing intact from injured mIOs. Full article

Chronic exercise induces functional adaptations in skeletal muscle microcirculation, but whether these are accompanied by sex-specific histological remodeling of arterioles remains unclear. This study examined gracilis muscle arterioles in trained (Ex) and sedentary (Se) female (F) and male (M) Wistar rats after a 12-week intensive swimming program (Mex = 6, FEx = 6; MSed = 6, FSed = 5). Histological remodeling was assessed by quantitative colorimetry analysis of resorcin-fuchsin (elastica; n = 661) and SMA-DAB (smooth muscle actin; n = 692) staining, focusing on elastic fiber density, internal elastic lamina (IEL) characteristics, and smooth muscle density in intramuscular and intermuscular vessels. Elastic fiber density and IEL thickness were generally greater in female animals than in males (p < 0.05). IEL staining intensity showed significant effects of sex (p = 0.043), exercise (p = 0.021), and a significant sex-by-exercise interaction (p = 0.037), with exercised females exhibiting the highest IEL staining intensity. Smooth muscle density did not differ significantly by sex or training status, although it was higher in intermuscular than intramuscular arterioles (p < 0.001), and increased with vessel diameter primarily in the intermuscular group. These findings demonstrate exercise-induced, sex-specific histological remodeling of skeletal muscle arterioles, primarily affecting elastic components, with more pronounced elastic adaptation in females. Full article

We developed a multi-channel and multilayered polydimethylsiloxane (PDMS) microfluidic platform that integrates cyclic mechanical stimulation, independent reagent delivery, and real-time optical observation within a single device. The platform employs a four-layer architecture comprising a pneumatic valve control layer, an observation channel for cell culture and imaging (24 mm × 4 mm), a medium perfusion layer with independent inlet ports, and a vacuum actuation layer that deforms a 200 μm PDMS membrane under −20 kPa cyclic pressure at 1 Hz. Cyclic membrane strain of 10% was calibrated using fluorescent bead tracking and image analysis. Finite element analysis based on nonlinear Föppl–von Kármán plate theory confirmed that the central cell culture region (60% of membrane area) exhibits a mean von Mises strain of 14.2% with a uniformity of 81.3% (CV = 18.7%), validating relatively uniform mechanical stimulation across the culture surface. As a proof-of-concept, human aortic smooth muscle cells (CRL-1999) cultured under cyclic strain showed significant upregulation of HIF-1$\alpha$ expression (2.5-fold, $p<0.01$) and pronounced F-actin stress fiber alignment visualized by fluorescence microscopy, confirming the platform’s capability for mechanotransduction studies and real-time cellular observation. The multi-channel architecture enables multi-condition parallel testing by simultaneously introducing different reagent concentrations through independent inlet ports while maintaining identical mechanical parameters across all channels, providing a versatile tool for systematic investigation of cellular responses under controlled biomechanical conditions. Full article

Chemotaxis enables eukaryotic cells to detect and migrate along extracellular chemoattractant gradients spanning several orders of magnitude. This remarkable dynamic range relies on adaptation, a process that allows cells to reset their signaling machinery while preserving sensitivity to incremental changes in stimulus intensity. Although numerous actin-dependent feedback mechanisms have been characterized, the molecular basis of adaptation within an actin-independent core gradient-sensing module remains poorly understood. Here, we identify the Ras GTPase-activating protein, C2GAP1, as a critical F-actin-independent effector of the heterotrimeric G protein, Gα2, in Dictyostelium discoideum. Using cytoskeleton-free gradient-sensing cells, quantitative imaging, biochemical assays, FRET-based G-protein activation measurements, and structural modeling, we demonstrate that C2GAP1 controls concentration-dependent adaptation during gradient sensing. Mechanistically, C2GAP1 directly associates with Gα2 in both GDP- and GTP-bound states, with preferential binding to activated Gα2, thereby sustaining membrane recruitment and locally attenuating Ras and downstream signaling. Loss of C2GAP1 enhances G-protein activation, disrupts local inhibition, and impairs rapid reorientation in dynamic gradients. These findings define a direct coupling between heterotrimeric G proteins and the RasGAP, C2GAP1, as a core adaptive module that enables gradient sensing across a wide concentration range. Full article

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with a poor prognosis. While sorafenib serves as the first-line therapy for advanced HCC, its efficacy is frequently hampered by side effects and the development of drug resistance, necessitating the development of novel agents to enhance HCC sensitivity to sorafenib. In this study, we demonstrate that the antihistamine astemizole significantly enhanced the antitumor efficacy of sorafenib in HCC cell lines. This combination treatment cooperatively inhibited HCC cells’ proliferation and induced cell cycle arrest at the G1 phase, as evidenced by decreased cyclin D1 and p-Rb levels and increased p27 expression. Furthermore, the combination of astemizole and sorafenib synergistically inhibited HCC cells’ migration, invasion, and adhesion. It also reduced F-actin polymerization and the expression of metastasis-regulating proteins, including p-Integrinβ1, FAK, and MMP1. Additionally, the combination treatment suppressed tube formation in HUVECs, accompanied by downregulation of HIF-1α and reduced VEGF secretion. Co-inhibition of Eag1 and the ERK/MAPK signaling pathway may underlie the enhanced anti-HCC effects of sorafenib by astemizole. Collectively, these findings indicate that astemizole significantly enhanced the antitumor activity of sorafenib by inhibiting proliferation, metastasis, and angiogenesis in HCC cells, suggesting its potential as a promising adjuvant to improve sorafenib-based therapy in HCC. Full article