Search Results (161)

Cellular mechanical properties are critical indicators of cellular state and promising disease biomarkers. This study introduces a novel microhand system, featuring chopstick-like plate-shaped end-effectors, designed for stable and high-precision single-cell mechanical characterization. First, we automated the force sensor calibration to overcome the inefficiency and unreliability of conventional manual methods. To validate the system’s sensitivity, we precisely quantified the mechanical contributions of subcellular components, such as the actin cytoskeleton and chromatin, by measuring stiffness reductions after treatment with Cytochalasin D and Trichostatin A, respectively. Notably, when applied to a cellular model of Hutchinson–Gilford progeria syndrome, we successfully captured disease-induced mechanical alterations. A distinct population of high-stiffness cells was detected in progerin-overexpressing cells, a feature not observed in the control groups. Furthermore, by controlling the indentation speed and depth, the mechanical properties of the cytoplasm and nucleus could be distinctly evaluated. These results demonstrate that our microhand system is a highly sensitive and robust platform, capable of detecting subtle, disease-related changes and elucidating the contributions of specific subcellular structures to cell mechanics. Full article

Fungi of the genus Parengyodontium (Ascomycota, Hypocreales, Cordycipitaceae) are emerging as promising sources of secondary metabolites with significant biotechnological potential. While traditionally understudied, species such as Parengyodontium album, Parengyodontium torokii and Parengyodontium americanum have been isolated from diverse and sometimes extreme environments—including deep-sea sediments, mangroves, and NASA clean rooms—suggesting remarkable ecological adaptability. This review presents a comprehensive synthesis of current knowledge on the chemical diversity, biological activities, and potential industrial applications of secondary metabolites produced by fungi belonging to the genus. A wide variety of compounds have been identified, including polyketides (e.g., engyodontiumones, alternaphenol B2), terpenoids (e.g., cytochalasin K), alkaloids, and torrubielline derivatives. These metabolites exhibit cytotoxic, antibacterial, and antifouling properties, with promising anticancer and antimicrobial activities. In addition, recent evidence points to the genus’s role in bioremediation, particularly through the degradation of polyethylene by P. album. Despite the advances highlighted here, challenges remain in scaling production, elucidating biosynthetic pathways, and confirming in vivo efficacy. This review underscores the value of integrating chemical, genomic, and metabolomic approaches to fully unlock the biotechnological potential of Parengyodontium species. Additionally, we broaden the perspective by comparing trends in secondary metabolites among Cordycipitaceae, highlighting lifestyle-related chemical compounds that serve as a reference for the Parengyodontium profile. Full article

Duck enteritis virus (DEV) is responsible for duck viral enteritis, a contagious and lethal disease in waterfowls. The host proteins targeted by DEV are unknown. In this study, we developed a recombinant DEV rVP26-Flag and identified 17 host proteins that interact with VP26 in infected chicken embryo fibroblast cells using co-immunoprecipitation in conjunction with liquid chromatography–tandem mass spectrometry (Co-IP-MS/MS). The 17 potential targets of VP26 proteins include Xirp1, TMOD3, DCN, ATP5PD, AP3M1, MYO5A, MYH10, MYH9 (non-muscle myosin IIA heavy chain), and GSN. Most of these proteins are microfilament or cytoskeletal proteins with functions such as cytoskeletal protein binding, actin filament interaction, microfilament motor activity, and myosin II interaction. Using the Search Tool for the Retrieval of Interacting Genes analysis, we predicted a functional network of microfilament cytoskeletal proteins interacting with VP26. Interaction between DEV VP26 and the carboxyl-terminus domain of MYH9 (1651–1960 aa) was verified via co-localization and Co-IP assays. We also demonstrated that the inhibition of actin polymerization with cytochalasin D and latrunculin A reduced the DEV titer. Furthermore, siRNA-mediated knockdown of MYH9, which has intrinsic ATPase activity, also resulted in a reduced viral titer. A targeted inhibitor of myosin II ATPase, (-)-Blebbistatin, significantly suppressed DEV infection both in vitro and in vivo. These results suggest that the actin–myosin II network plays a crucial role in DEV proliferation, with MYH9 being an important host factor influencing DEV infection. Full article

Pathogenic bacteria have developed different ways to cause infections. One strategy involves using components from host cells. This study looks at the role of the cytoskeleton in the human colon adenocarcinoma Caco-2 and neonatal non-transformed epithelial H4 cell lines during bacterial invasion. The bacteria studied include Cronobacter malonaticus, Cronobacter sakazakii, and E. coli K1, as they are associated with known diseases. Salmonella enteritidis 358 served as a positive control and E. coli K12 as a negative control for the invasion experiments. Before the invasion experiments, cell lines were treated with microfilament inhibitors, specifically Cytochalasin D, and microtubule inhibitors, such as Colchicine, Nocodazole, Vinblastine, and Taxol. The results showed that Cytochalasin D reduced about 60–80% of Cronobacter invasion into H4 cells and 50% of E. coli K1 invasion. In contrast, Colchicine reduced the invasion of some strains to just 2% compared to untreated cells. Meanwhile, Nocodazole and Taxol increased the invasion of C. sakazakii 709 and C. malonaticus 1569 into H4 cells by about 140% and 160%, respectively, while slightly inhibiting other strains. In Caco-2 cells, certain strains exhibited increased invasion due to Cytochalasin D, Vinblastine, and Colchicine treatment. This led to increases of up to 500%, 227%, and 248% compared to untreated cells. However, Nocodazole and Taxol decreased invasion into Caco-2 cells, with only E. coli K1 showing an increase of about 150% in Taxol-treated cells. The findings with eukaryotic cytoskeleton inhibitors on neonatal H4 cells suggest that bacterial invasion mainly relies on microfilaments or microfilament-dependent. No specific dependence on the cytoskeleton was seen in Caco-2 cells. In conclusion, cytoskeletal inhibitors significantly affected bacterial invasion, specifically Cronobacter, compared to untreated cells. This suggests that invasion methods may vary by strain and are influenced by how each inhibitor alters cytoskeleton behavior. Therefore, the invasion process, both with and without cytoskeletal inhibitors, is crucial for understanding how bacteria manipulate cell components during infection. Full article

Focal adhesions (FAs) are multi-protein complexes that mediate cell attachment to the extracellular matrix. Their formation and maturation depend on intracellular tension generated by actin filaments interacting with phosphorylated myosin II. Using live-cell and confocal microscopy, we investigated how FA dynamics are regulated by actin polymerization and myosin II-driven contractility. We found that knockdown of myosin II resulted in complete and irreversible disassembly of FAs. However, partial inhibition of myosin II, through either ROCK or myosin light chain kinase (MLCK) inhibitors, leads to gradual FA shrinkage. In contrast, complete inhibition of myosin II phosphorylation causes disassembly of existing FAs, followed by the formation of new, small FAs at the cell periphery. In both cases, FAs formed after inhibition of myosin II phosphorylation exhibited significantly longer lifespans than FAs in control cells. Similarly, partial inhibition of actin polymerization using nanomolar concentrations of latrunculin B or cytochalasin D also promoted the formation of small FAs. Complete and irreversible FA disassembly occurred only when actin filaments were fully disrupted, leading to cell lamella retraction. These findings suggest that actin polymerization at the cell edge is the minimal and sufficient requirement for the assembly of small FAs. Notably, our data demonstrate for the first time that perturbation of the actin–myosin system results in stabilization and prolonged lifespan of small FAs, whereas larger FAs, formed in the presence of myosin II activity, are more dynamic. Together, these results emphasize the essential role of cortical actin organization and myosin II phosphorylation in the maintenance and turnover of FAs. 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

In the marine red alga Pyropia yezoensis, filamentous phases of the life cycle, e.g., the conchocelis (sporophyte) and conchosporangium (conchosporophyte), proliferate by tip growth. In this study, we investigated the possible involvement of phosphoinositide turnover and actin polymerization in the spontaneous initiation and tip growth of new branches in isolated single-celled conchocelis cells using pharmacological treatments. Treatment with LY294002 and U73122, specific inhibitors of phosphoinositide-phosphate 3-kinase and phospholipase C, respectively, reduced side-branch formation and inhibited the elongation of branches. In addition, two inhibitors of the actin cytoskeleton, cytochalasin B (CCB) and latrunculin B (LAT-B), had similar effects on tip growth. However, CCB did not alter the branching rate of single-celled conchocelis, whereas LAT-B did. As CCB and LAT-B affect actin polymerization through different mechanisms, this result suggests differences in the contributions of actin polymerization to branch initiation versus tip growth. These findings demonstrate the critical and diverse functional roles played by phosphoinositide turnover and actin polymerization in the regulation of the initiation and maintenance of tip growth in the conchocelis phase of P. yezoensis. Full article

Combining biosynthetic gene cluster analysis with the OSMAC strategy, fractionation of the fermentation extract of Aspergillus templicola from the sponge Agelas sp. led to the isolation of four novel cytochalasins, colachalasins J–M (1–4), a novel cyclic pentapeptide, avellanin P (5), together with five known compounds (6–10). The structures of 1–9 were elucidated using spectroscopic data, single crystal X-ray diffraction, and Marfey’s analysis. Compound 2 exhibited potent anti-inflammatory activity in zebrafish assays. Additionally, Compounds 4 and 6 showed modest cytotoxicity against several human cancer cell lines with IC₅₀ values ranging from 2.6 to 11.2 μm. Full article

Phlebopus portentosus is an edible and medicinal ectomycorrhizal mushroom with delicious and high nutritional value. However, the mechanism of secondary metabolite biosynthesis in P. portentosus is still unclear. In this study, the genomics, metabolomics, and transcriptomics were integrated to reveal the biosynthesis mechanism of secondary metabolites in P. portentosus under different cultivation conditions. The 31.4 Mb genome of P. portentosus YAF023 with 15 scaffolds was assembled by Illumina and Nanopore sequencing and annotated, and 206 cytochrome P450s, 201 carbohydrate-active enzymes, 186 transcription factors, 18 terpene synthases (TPSs), and 5 polyketide synthases (PKSs) were identified. Multi-omics analysis showed that PpPKS1 is probably involved in the biosynthesis of Ethyl orsellinate; PpPKS2 and PpPKS5 are probably involved in the synthesis of 6-Methylsalicylic acid and Cytochalasin Z5, respectively; PpTRI5 was involved in the tetracyclic sesquiterpene β-type trichodiene compounds; and PpSTCs was involved in the synthesis of β-copaene analogs or derivatives. Co-expression network analysis and binding site prediction of the promoter regions suggested that PpHOX4 and PpHSF1 regulated the gene expression of PpPKS1, and Ppzf-C2H2 32 and PpHSF5 regulated the gene expression of PpSTCs 8, and PpSTCs 3, respectively. This study will provide an important foundation for further development and utilization of secondary metabolites of P. portentosus. Full article

Cow mastitis is a major challenge in dairy farming, significantly affecting both milk quality and cow health. Cytochalasin B (CB) is a fungal toxin and an actin cytoskeleton depolymerizing agent that exhibits anti-inflammatory and antitumor properties; however, its mechanism in cow mastitis remains unclear. In this study, we systematically evaluated the effects of CB on mastitis using an LPS-induced inflammation model in bovine mammary epithelial cells (MAC-T) and a mouse mastitis model. The techniques employed included Real-time quantitative PCR detecting system (qPCR), Western blot, HE staining, immunofluorescence (IF), and immunohistochemistry (IHC). The results demonstrated that CB significantly alleviated LPS-induced mastitis by downregulating the expression of pro-inflammatory factors IL-1β, TNF-α, and the NLRP3 inflammasome while also reducing cell apoptosis. Further mechanistic investigations revealed that CB mitigates the inflammatory response by inhibiting the expression of ARPC3, ARPC4, and HSP70, thereby disrupting cytoskeletal rearrangement and the activation of the NLRP3 inflammasome. Overall, this study reveals the potential therapeutic role of CB in cow mastitis and provides a theoretical foundation for developing novel intervention strategies. Full article

Chaetoglobosin A (CheA), a typical structure of the cytochalasin family, exhibits outstanding efficacy against a variety of tumor cells and plant pathogens. However, its low yield and high production cost are major obstacles limiting its wide application. In order to increase CheA yield, an engineered strain was established by overexpressing CgMfs, the gene encoding the MFS family’s efflux pump, on chassis cells lacking CgXpp1, which have been shown to act as a negative regulator of CheA biosynthesis. As expected, the engineered strain significantly boosted CheA production from 63.19 to 265.93 mg/L after incubation in PDA medium for 10 d, whereas the yield of the engineered strain was remarkably enhanced 2.93-fold compared with the wild type, following 10 d of cultivation utilizing potato starch industrial waste. The addition of metal ions had a positive effect on CheA production, with Cu²⁺ being the most effective and improving production to 176.92 mg/L. The optimal fermentation conditions were determined by response surface optimization, and under the optimal conditions, the engineered strain could stably produce CheA with a yield of 197.58 mg/L. This study provided the conditions for reducing production costs while increasing CheA production, as well as new strategies and insights for the production of the target compound. Full article

The secondary metabolome of Nemania diffusa, isolated as an ash endophytic fungus, was analyzed in detail. From its cultures, a previously undescribed cytochalasin 1 was isolated using preparative HPLC, together with six known congeners: 18-dehydroxy-cytochalasin E (2), cytochalasins Z₇ (3), Z₈ (4), and E (5), 18-dehydroxy-17-didehydro-cytochalasin E (6), and K Steyn (7). The structures of these compounds were determined using data from high-resolution mass spectrometry (HR-MS), in combination with 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Metabolites 1–4 share a characteristic 12-membered lactone moiety, placing them within a rarely examined cytochalasin subclass. Thus, the compounds were incorporated into our ongoing screening campaign to study the structure–activity relationship of this metabolite family. We initially determined their cytotoxicity in eukaryotic mouse fibroblast L929 cells using an MTT-based colorimetric assay, and further investigated their effect on the cellular actin dynamics of the human osteosarcoma cell line U-2OS in detail. Unexpectedly, we discovered a high number of irreversible compounds (1, 2, and 4). Additionally, we highlighted specific structural features within the 12-membered cytochalasin subclass that may play a role in directing the reversibility of these compounds. Full article

Vanillic acid (VA) is a dietary benzoic acid derivative, flavoring agent, and food stabilizer. In this study, the antioxidant and anti-inflammatory potential of VA was explored in vitro and ex vivo in human immune cells and non-cellular models. In neutrophils, VA significantly downregulated the fMLP-induced oxidative burst and the generation of reactive oxygen species (ROS); it also suppressed the release of pro-inflammatory cytokines (TNF-α, IL-8) and the tissue-remodeling enzyme elastase-2 (ELA-2) in cells stimulated with LPS and fMLP+cytochalasin B. Additionally, VA showed good biocompatibility with human neutrophils and peripheral blood mononuclear cells (PBMCs) across the tested concentrations of 1–50 µg/mL. Furthermore, VA at 1–5 μg/mL enhanced the non-enzymatic antioxidant capacity of human plasma (NEAC) and prevented oxidative and nitrative damage to plasma proteins by protecting tyrosine moieties and thiols from peroxynitrite. VA also inhibited lipid peroxidation and the formation of thiobarbituric acid-reactive substances (at 50 μg/mL) and protein-bound carbonyls (at 5–50 μg/mL) in peroxynitrite-treated plasma. In non-cellular tests, VA acted as a hypochlorous acid and hydrogen peroxide scavenger and inhibited non-enzymatic protein glycation, outperforming the references Trolox and aminoguanidine. Along with existing data from animal models and studies on polyphenol intake, these results might support the synergic role of VA in dietary protection against chronic diseases related to oxidative stress and inflammation. Full article

Malbranchea circinata SDU050, a fungus derived from deep-sea sediment, is a prolific producer of diverse secondary metabolites. Genome sequencing revealed the presence of at least 69 biosynthetic gene clusters (BGCs), including 30 encoding type I polyketide synthases (PKSs). This study reports the isolation and identification of four classes of secondary metabolites from wild-type M. circinata SDU050, alongside five additional metabolite classes, including three novel cytochalasins (7–9), obtained from a mutant strain through the metabolic blockade strategy. Furthermore, bioinformatic analysis of the BGC associated with the isocoumarin sclerin (1) enabled the deduction of its biosynthetic pathway based on gene function predictions. Bioactivity assays demonstrated that sclerin (1) and (−)-mycousnine (10) exhibited weak antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus subtilis. These findings underscore the chemical diversity and biosynthetic potential of M. circinata SDU050 and highlight an effective strategy for exploring marine fungal metabolites. Full article

Neurosteroids are undeniably regarded as neuroprotective mediators, regulating brain function by rapid non-genomic actions involving interference with microtubules. Conversely, hyperphosphorylated Tau is considered responsible for the onset of a plethora of neurodegenerative diseases, as it dissociates from microtubules, leading to their destabilization, thus impairing synaptic vesicle transport and neurotransmission. Consequently, we aimed to investigate the effects of neurosteroids, specifically allopregnanolone (Allo) and dehydroepiandrosterone (DHEA), on the levels of total and phosphorylated at Serine 404 Tau (p-Tau) in C57BL/6 mice brain slices. In total tissue extracts, we found that neurosteroids elevated both total and p-Tau levels without significantly altering the p-Tau/Tau ratio. In addition, the levels of several enzymes implicated in Tau phosphorylation did not display significant differences between conditions, suggesting that neurosteroids influence Tau distribution rather than its phosphorylation. Hence, we subsequently examined the mitochondria-enriched subcellular fraction where, again, both p-Tau and total Tau levels were increased in the presence of neurosteroids. These effects seem actin-dependent, as disrupting actin polymerization by cytochalasin B preserved Tau levels. Furthermore, co-incubation with high [Ca²⁺] and neurosteroids mitigated the effects of Ca²⁺ overload, pointing to cytoskeletal remodeling as a potential mechanism underlying neurosteroid-induced neuroprotection. Full article