Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved.
METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment.
RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation.
CONCLUSIONS: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

BACKGROUND: Bicuspid aortic valves (BAV) are frequently associated with ascending aortic aneurysms. The etiology is incompletely understood, but genetic factors, in addition to flow perturbations, are likely involved. Since loss of contractility and elaboration of extracellular matrix in the vessel wall are features of BAV-associated aortopathy, phenotypic modulation of smooth muscle cells (SMCs) may play a role.
METHODS: Ascending aortic tissue was collected intra-operatively from 25 individuals with normal (i.e., tricuspid) aortic valves (TAV) and from 25 individuals with BAVs. For both TAV and BAV, 10 patients had non-dilated (ND) and 15 patients had dilated (D) aortas. SMCs were isolated and cultured from a subset of patients from each group. Aortic tissue and SMCs were fluorescently immunolabeled for SMC phenotypic markers (i.e., alpha-smooth muscle actin (ASMA, contractile), vimentin (synthetic) and p16INK4a and p21Cip1 (senescence). SMCs were also analyzed for replicative senescence in culture.
RESULTS: In normal-sized and dilated BAV aortas, SMCs switched from the contractile state to either synthetic or senescent phenotypes, as observed by loss of ASMA (ND: P = 0.001, D: P = 0.002) and associated increases in vimentin (ND: P = 0.03, D: P = 0.004) or p16/p21 (ND: P = 0.03, D: P<0.0001) compared to TAV. Dilatation of the aorta exacerbated SMC phenotypic switching in both BAV and TAV aortas (all P<0.05). In SMCs cultured from normal and dilated aortas, those isolated from BAV reached replicative senescence faster than those from TAV aortas (all P = 0.02). Furthermore, there was a stark inverse correlation between ASMA and cell passage number in BAV SMCs (ND: P = 0.0006, D: P = 0.01), but not in TAV SMCs (ND: P = 0.93, D: P = 0.20).
CONCLUSIONS: The findings of this study provide direct evidence from cell culture studies implying that SMCs switch from the contractile state to either synthetic or senescent phenotypes in the non-dilated BAV aorta. In cultured SMCs from both non-dilated and dilated aortas, we found that this process may precede dilatation and accompany aneurysm development in BAV. Our findings suggest that therapeutically targeting SMC phenotypic modulation in BAV patients may be a viable option to prevent or delay ascending aortic aneurysm formation.

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1\'s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

OBJECTIVE: Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing\'s sarcoma (ES), and osteosarcoma (OS). The current study investigated the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines, focusing on the actin cytoskeleton.
METHODS: The CASY Cell Counter and Analyzer was used to study cell proliferation and determine the optimal concentrations of fetal calf serum to maintain viability without stimulation of cell proliferation. CellTiter-BlueCell viability assay was used to determine the effects of CPP on the viability of bone sarcoma cells. The Radius assay was used to determine cell migration. Staining for Deoxyribonuclease I, G-actin, and F-actin was used to assay for the effects on the cytoskeleton.
RESULTS: Reductions in cell viability and motility were observed across all cell lines following CPP treatment. CPP induced changes in the actin cytoskeleton, leading to decreased cell motility.
CONCLUSIONS: CPP effectively reduces the motility of bone sarcoma cells by altering the actin cytoskeleton. These findings underscore CPP\'s potential as a therapeutic tool for bone sarcomas and highlight the need for further research in this area.

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.

The overall 5-year survival rate of ovarian cancer (OC) is generally low as the disease is often diagnosed at an advanced stage of progression. To save lives, OC must be identified in its early stages when treatment is most effective. Early-stage OC causes the upregulation of lysophosphatidic acid (LPA), making the molecule a promising biomarker for early-stage detection. An LPA assay can additionally stage the disease since LPA levels increase with OC progression. This work presents two methods that demonstrate the prospective application for detecting LPA: the electromagnetic piezoelectric acoustic sensor (EMPAS) and a chemiluminescence-based iron oxide nanoparticle (IONP) approach. Both methods incorporate the protein complex gelsolin-actin, which enables testing for detection of the biomarker as the binding of LPA to the complex results in the separation of gelsolin from actin. The EMPAS was characterized with contact angle goniometry and atomic force microscopy, while gelsolin-actin-functionalized IONPs were characterized with transmission electron microscopy and Fourier transform infrared spectroscopy. In addition to characterization, LPA detection was demonstrated as a proof-of-concept in Milli-Q water, buffer, or human serum, highlighting various LPA assays that can be developed for the early-stage detection of OC.

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.

Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct β- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that β- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that β-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by intense deposition of fat globules in the hepatic parenchyma that could potentially progress to liver cirrhosis and hepatocellular carcinoma. Here, we evaluated a rat model to study the molecular pathogenesis of the spectrum of MASLD and to screen therapeutic agents. SHRSP5/Dmcr rats were fed a high-fat and cholesterol (HFC) diet for a period of 12 weeks and evaluated for the development of steatosis (MASLD), steatohepatitis, fibrosis and cirrhosis. A group of animals were sacrificed at the end of the 4th, 6th, 8th and 12th weeks from the beginning of the experiment, along with the control rats that received normal diet. Blood and liver samples were collected for biochemical and histopathological evaluations. Immunohistochemical staining was performed for α-SMA and Collagen Type I. Histopathological examinations demonstrated steatosis at the 4th week, steatohepatitis with progressive fibrosis at the 6th week, advanced fibrosis with bridging at the 8th week and cirrhosis at the 12th week. Biochemical markers and staining for α-SMA and Collagen Type I demonstrated the progression of steatosis to steatohepatitis, hepatic fibrosis and liver cirrhosis in a stepwise manner. Control animals fed a normal diet did not show any biochemical or histopathological alterations. The results of the present study clearly demonstrated that the HFC diet-induced model of steatosis, steatohepatitis, hepatic fibrosis and cirrhosis is a feasible, quick and appropriate animal model to study the molecular pathogenesis of the spectrum of MASLD and to screen potent therapeutic agents.