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Cancer cells mechanically interact with the tumor microenvironment during cancer development. Mechano-reciprocity has emerged as a crucial factor affecting anti-cancer drug resistance during adjuvant therapy. Here, we investigated the focal adhesion kinase (FAK)/Yes-associated protein (YAP) signaling axis as a prospective strategy for circumventing cisplatin resistance in ovarian cancer (OC). The Cancer Genome Atlas (TCGA) data analysis revealed that FAK overexpression significantly correlated with unfavorable clinical outcomes in patients with ovarian cancer. AFM indentation experiments showed that cell elasticity depends on FAK activity. Notably, the combination of FAK inhibition and cisplatin treatment led to a 69 % reduction in the IC 50 of cisplatin. This combined treatment also increased apoptosis compared to the individual treatments, along with the upregulation of the pro-apoptotic factor BAX and cleaved PARP. Suppressing FAK expression sequestered YAP in the cytosol, potentially reducing cellular proliferation and promoting apoptosis. Moreover, reduced FAK expression sensitized drug-resistant OC cells to cisplatin treatment owing to a decrease in nuclear tension, allowing the relocation of YAP to the cytosol. In a mouse model, the co-administration of an FAK inhibitor and cisplatin significantly suppressed tumor growth and increased apoptotic events and DNA fragmentation. Our findings suggest that drug resistance can be attributed to the perturbation of mechanosensing signaling pathways, which drive the mechanical reinforcement of cancer cells. OC cells can restore their sensitivity to cisplatin treatment by strategically reducing YAP localization in the nucleus through FAK downregulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCμ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm 2 ), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured. Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading., Competing Interests: Declaration of competing interest The authors state that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Drug resistance is arguably one of the biggest challenges facing cancer research today. Understanding the underlying mechanisms of drug resistance in tumor progression and metastasis are essential in developing better treatment modalities. Given the matrix stiffness affecting the mechanotransduction capabilities of cancer cells, characterization of the related signal transduction pathways can provide a better understanding for developing novel therapeutic strategies. In this review, we aimed to summarize the recent advancements in tumor matrix biology in parallel to therapeutic approaches targeting matrix stiffness and its consequences in cellular processes in tumor progression and metastasis. The cellular processes governed by signal transduction pathways and their aberrant activation may result in activating the epithelial-to-mesenchymal transition, cancer stemness, and autophagy, which can be attributed to drug resistance. Developing therapeutic strategies to target these cellular processes in cancer biology will offer novel therapeutic approaches to tailor better personalized treatment modalities for clinical studies.

Synthetic signaling receptors enable programmable cellular responses coupling with customized inputs. However, engineering a designer force-sensing receptor to rewire mechanotransduction remains largely unexplored. Herein, we introduce nongenetically engineered artificial mechanoreceptors (AMRs) capable of reprogramming non-mechanoresponsive receptor tyrosine kinases (RTKs) to sense user-defined force cues, enabling de novo-designed mechanotransduction. AMR is a modular DNA-protein chimera comprising a mechanosensing-and-transmitting DNA nanodevice grafted on natural RTKs via aptameric anchors. AMR senses intercellular tensile force via an allosteric DNA mechano-switch with tunable piconewton-sensitive force tolerance, actuating a force-triggered dynamic DNA assembly to manipulate RTK dimerization and activate intracellular signaling. By swapping the force-reception ligands, we demonstrate the AMR-mediated activation of c-Met, a representative RTK, in response to the cellular tensile forces mediated by cell-adhesion proteins (integrin, E-cadherin) or membrane protein endocytosis (CI-M6PR). Moreover, AMR also allows the reprogramming of FGFR1, another RTK, to customize mechanobiological function, for example, adhesion-mediated neural stem cell maintenance., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The extracellular matrix (ECM) shapes the stem cell fate during differentiation by exerting relevant biophysical cues. However, the mechanism of stem cell fate decisions in response to ECM-backed complex biophysical cues has not been fully understood due to the lack of versatile ECMs. Here, we designed two versatile ECMs using colloidal self-assembly technology to probe the mechanisms of their effects on mechanotransduction and stem cell fate regulation. Binary colloidal crystals (BCC) with a hexagonally close-packed structure, composed of silica (5 μm) and polystyrene (0.4 μm) particles as well as a polydimethylsiloxane-embedded BCC (BCCP), were fabricated. They have defined surface chemistry, roughness, stiffness, ion release, and protein adsorption properties, which can modulate the cell adhesion, proliferation, and differentiation of human adipose-derived stem cells (hASCs). On the BCC, hASCs preferred osteogenesis at an early stage but showed a higher tendency toward adipogenesis at later stages. In contrast, the results of BCCP diverged from those of BCC, suggesting a unique regulation of ECM-dependent mechanotransduction. The BCC-mediated cell adhesion reduced the size of the focal adhesion complex, accompanying an ordered spatial organization and cytoskeletal rearrangement. This morphological restriction led to the modulation of mechanosensitive transcription factors, such as c-FOS, the enrichment of transcripts in specific signaling pathways such as PI3K/AKT, and the activation of the Hippo signaling pathway. Epigenetic analyses showed changes in histone modifications across different substrates, suggesting that chromatin remodeling participated in BCC-mediated mechanotransduction. This study demonstrates that BCCs are versatile artificial ECMs that can regulate human stem cells' fate through unique biological signaling, which is beneficial in biomaterial design and stem cell engineering.

Cisplatin, a highly effective chemotherapeutic drug for various human cancers, induces irreversible sensorineural hearing loss as a side effect. Currently there are no highly effective clinical strategies for the prevention of cisplatin-induced ototoxicity. Previous studies have indicated that short-term cisplatin ototoxicity primarily affects the outer hair cells of the cochlea. Therefore, preventing the entry of cisplatin into hair cells may be a promising strategy to prevent cisplatin ototoxicity. This study aimed to investigate the entry route of cisplatin into mouse cochlear hair cells. The competitive inhibitor of organic cation transporter 2 (OCT2), cimetidine, and the sensory mechanoelectrical transduction (MET) channel blocker benzamil, demonstrated a protective effect against cisplatin toxicity in hair cells in cochlear explants. Sensory MET-deficient hair cells explanted from Tmc1 Δ ;Tmc2 Δ mice were resistant to cisplatin toxicity. Cimetidine showed an additive protective effect against cisplatin toxicity in sensory MET-deficient hair cells. However, in the apical turn, cimetidine, benzamil, or genetic ablation of sensory MET channels showed limited protective effects, implying the presence of other entry routes for cisplatin to enter the hair cells in the apical turn. Systemic administration of cimetidine failed to protect cochlear hair cells from ototoxicity caused by systemically administered cisplatin. Notably, outer hair cells in MET-deficient mice exhibited no apparent deterioration after systemic administration of cisplatin, whereas the outer hair cells in wild-type mice showed remarkable deterioration. The susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on the sensory MET channel both ex vivo and in vivo. This result justifies the development of new pharmaceuticals, such as a specific antagonists for sensory MET channels or custom-designed cisplatin analogs which are impermeable to sensory MET channels., Competing Interests: Declarations of competing interest There is no conflict of interest in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Increased intercellular tension is associated with enhanced cell proliferation and tissue growth. Here, we present evidence for a force-transduction mechanism that links mechanical perturbations of epithelial (E)-cadherin (CDH1) receptors to the force-dependent activation of epidermal growth factor receptor (EGFR, ERBB1)-a key regulator of cell proliferation. Here, coimmunoprecipitation studies first show that E-cadherin and EGFR form complexes at the plasma membrane that are disrupted by either epidermal growth factor (EGF) or increased tension on homophilic E-cadherin bonds. Although force on E-cadherin bonds disrupts the complex in the absence of EGF, soluble EGF is required to mechanically activate EGFR at cadherin adhesions. Fully quantified spectral imaging fluorescence resonance energy transfer further revealed that E-cadherin and EGFR directly associate to form a heterotrimeric complex of two cadherins and one EGFR protein. Together, these results support a model in which the tugging forces on homophilic E-cadherin bonds trigger force-activated signaling by releasing EGFR monomers to dimerize, bind EGF ligand, and signal. These findings reveal the initial steps in E-cadherin-mediated force transduction that directly link intercellular force fluctuations to the activation of growth regulatory signaling cascades., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Most lung development occurs in the context of cyclic stretch. Alteration of the mechanical microenvironment is a common feature of many pulmonary diseases, with congenital diaphragmatic hernia (CDH) and fetal tracheal occlusion (FETO, a therapy for CDH) being extreme examples with changes in lung structure, cell differentiation, and function. To address limitations in cell culture and in vivo mechanotransductive models, we developed two mouse lung organoid (mLO) mechanotransductive models using postnatal day 5 (PND5) mouse lung CD326-positive cells and fibroblasts subjected to increased, decreased, and cyclic strain. In the first model, mLOs were exposed to forskolin (FSK) and/or disrupted (DIS) and evaluated at 20 h. mLO cross-sectional area changed by +59%, +24%, and -68% in FSK, control, and DIS mLOs, respectively. FSK-treated organoids had twice as many proliferating cells as other organoids. In the second model, 20 h of 10.25% biaxial cyclic strain increased the mRNAs of lung mesenchymal cell lineages compared with static stretch and no stretch. Cyclic stretch increased TGF-β and integrin-mediated signaling, with upstream analysis indicating roles for histone deacetylases, microRNAs, and long noncoding RNAs. Cyclic stretch mLOs increased αSMA-positive and αSMA-PDGFRα-double-positive cells compared with no stretch and static stretch mLOs. In this PND5 mLO mechanotransductive model, cell proliferation is increased by static stretch, and cyclic stretch induces mesenchymal gene expression changes important in postnatal lung development.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca 2+ and Na + influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca 2+ -dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca 2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

Action potentials depend on voltage-gated sodium channels (Na V 1s), which have nine α subtypes. Na V 1 inhibition is a target for pathologies involving excitable cells such as pain. However, because Na V 1 subtypes are widely expressed, inhibitors may inhibit regulatory sensory systems. Here, we investigated specific Na V 1s and their inhibition in mouse esophageal mechanoreceptors-non-nociceptive vagal sensory afferents that are stimulated by low threshold mechanical distension, which regulate esophageal motility. Using single fiber electrophysiology, we found mechanoreceptor responses to esophageal distension were abolished by tetrodotoxin. Single-cell RT-PCR revealed that esophageal-labeled TRPV1-negative vagal neurons expressed multiple tetrodotoxin-sensitive Na V 1s: Na V 1.7 (almost all neurons) and Na V 1.1, Na V 1.2, and Na V 1.6 (in ∼50% of neurons). Inhibition of Na V 1.7, using PF-05089771, had a small inhibitory effect on mechanoreceptor responses to distension. Inhibition of Na V 1.1 and Na V 1.6, using ICA-121341, had a similar small inhibitory effect. The combination of PF-05089771 and ICA-121341 inhibited but did not eliminate mechanoreceptor responses. Inhibition of Na V 1.2, Na V 1.6, and Na V 1.7 using LSN-3049227 inhibited but did not eliminate mechanoreceptor responses. Thus, all four tetrodotoxin-sensitive Na V 1s contribute to action potential initiation from esophageal mechanoreceptors terminals. This is different to those Na V 1s necessary for vagal action potential conduction, as demonstrated using GCaMP6s imaging of esophageal vagal neurons during electrical stimulation. Tetrodotoxin-sensitive conduction was abolished in many esophageal neurons by PF-05089771 alone, indicating a critical role of Na V 1.7. In summary, multiple Na V 1 subtypes contribute to electrical signaling in esophageal mechanoreceptors. Thus, inhibition of individual Na V 1s would likely have minimal effect on afferent regulation of esophageal motility.

Cell-matrix interactions govern cell behavior and tissue function by facilitating transduction of biomechanical cues. Engineered tissues often incorporate these interactions by employing cell-adhesive materials. However, using constitutively active cell-adhesive materials impedes control over cell fate and elicits inflammatory responses upon implantation. Here, an alternative cell-material interaction strategy that provides mechanotransducive properties via discrete inducible on-cell crosslinking (DOCKING) of materials, including those that are inherently non-cell-adhesive, is introduced. Specifically, tyramine-functionalized materials are tethered to tyrosines that are naturally present in extracellular protein domains via enzyme-mediated oxidative crosslinking. Temporal control over the stiffness of on-cell tethered 3D microniches reveals that DOCKING uniquely enables lineage programming of stem cells by targeting adhesome-related mechanotransduction pathways acting independently of cell volume changes and spreading. In short, DOCKING represents a bioinspired and cytocompatible cell-tethering strategy that offers new routes to study and engineer cell-material interactions, thereby advancing applications ranging from drug delivery, to cell-based therapy, and cultured meat., (© 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

There is a need to develop a novel analgesic for pain associated with interstitial cystitis/painful bladder syndrome (IC/PBS). The use of the conventional μ-opioid receptor agonists to manage IC/PBS pain is controversial due to adverse CNS effects. These effects are attenuated in benzylideneoxymorphone (BOM), a low-efficacy μ-opioid receptor agonist/δ-opioid receptor antagonist that attenuates thermal pain and is devoid of reinforcing effects. We hypothesize that BOM will inhibit bladder pain by attenuating responses of urinary bladder distension (UBD)-sensitive afferent fibers. Therefore, the effect of BOM was tested on responses of UBD-sensitive afferent fibers in L6 dorsal root from inflamed and non-inflamed bladder of rats. Immunohistochemical (IHC) examination reveals that following the induction of inflammation there were significant high expressions of μ, δ, and μ-δ heteromer receptors in DRG. BOM dose-dependently (1-10 mg/kg, i.v) attenuated mechanotransduction properties of these afferent fibers from inflamed but not from non-inflamed rats. In behavioral model of bladder pain, BOM significantly attenuated visceromotor responses (VMRs) to UBD only in inflamed group of rats when injected either systemically (10 mg/kg, i.v.) or locally into the bladder (0.1 ml of 10 mg/ml). Furthermore, oxymorphone (OXM), a high-efficacy μ-opioid receptor agonist, attenuated responses of mechanosensitive bladder afferent fibers and VMRs to UBD. Naloxone (10 mg/kg, i.v.) significantly reversed the inhibitory effects of BOM and OXM on responses of bladder afferent fibers and VMRs suggesting μ-opioid receptor-related analgesic effects of these compounds. The results reveal that a low-efficacy, bifunctional opioid-based compound can produce analgesia by attenuating mechanotransduction functions of afferent fibers innervating the urinary bladder., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1., (© 2021. The Author(s).)

Mechanotransduction, the interplay between physical and chemical signaling, plays vital roles in many biological processes. The state-of-the-art techniques to quantify cell forces employ deformable polymer films or molecular probes tethered to glass substrates. However, the applications of these assays in fundamental and clinical research are restricted by the planar geometry and low throughput of microscopy readout. Herein, we develop a DNA-based microparticle tension sensor, which features a spherical surface and thus allows for investigation of mechanotransduction at curved interfaces. The micron-scale of μTS enables flow cytometry readout, which is rapid and high throughput. We applied the method to map and measure T-cell receptor forces and platelet integrin forces at 12 and 56 pN thresholds. Furthermore, we quantified the inhibition efficiency of two anti-platelet drugs providing a proof-of-concept demonstration of μTS to screen drugs that modulate cellular mechanics., (© 2021 Wiley-VCH GmbH.)

Objective: To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated., Design: Laboratory studies., Setting: Translational science laboratory., Patient(s)/animal(s): Human fibroid cell lines and patient-matched fibroid and myometrial cell lines., Intervention(s): Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632)., Main Outcome Measure(s): Progesterone-responsive reporter activation., Result(s): Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4., Conclusion(s): Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation., (Copyright © 2020. Published by Elsevier Inc.)

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli 's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic., Competing Interests: The authors declare no competing interest.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases. NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Red blood cell (RBC) deformability has vital importance for microcirculation in the body, as RBCs travel in narrow capillaries under shear stress. Deformability can be defined as a remarkable cell ability to change shape in response to an external force which allows the cell to pass through the narrowest blood capillaries. Previous studies showed that RBC deformability could be regulated by Ca 2+ /protein kinase C (PKC) signaling mechanisms due to the phosphorylative changes in RBC membrane proteins by kinases and phosphatases. We investigated the roles of Ca 2+ /PKC signaling pathway on RBC mechanical responses and impaired RBC deformability under continuous shear stress (SS). A protein kinase C inhibitor Chelerythrine, a tyrosine phosphatase inhibitor Calpeptin, and a calcium channel blocker Verapamil were applied into human blood samples in 1 micromolar concentration. Samples with drugs were treated with or without 3 mM Ca 2+ . A shear stress at 5 Pa level was applied to each sample continuously for 300 s. RBC deformability was measured by a laser-assisted optical rotational cell analyzer (LORRCA) and was calculated as the change in elongation index (EI) of RBC upon a range of shear stress (SS, 0.3-50 Pa). RBC mechanical stress responses were evaluated before and after continuous SS through the parameterization of EI-SS curves. The drug administrations did not produce any significant alterations in RBC mechanical responses when they were applied alone. However, the application of the drugs together with Ca 2+ substantially increased RBC deformability compared to calcium alone. Verapamil significantly improved Ca 2+ -induced impairments of deformability both before and after 5 Pa SS exposure (p < 0.0001). Calpeptin and Chelerythrine significantly ameliorated impaired deformability only after continuous SS (p < 0.05). Shear-induced improvements of deformability were conserved by the drug administrations although shear-induced deformability was impaired when the drugs were applied with calcium. The blocking of Ca 2+ channel by Verapamil improved impaired RBC mechanical responses independent of the SS effect. The inhibition of tyrosine phosphatase and protein kinase C by Calpeptin and Chelerythrine, respectively, exhibited ameliorating effects on calcium-impaired deformability with the contribution of shear stress. The modulation of Ca 2+ /PKC signaling pathway could regulate the mechanical stress responses of RBCs when cells are under continuous SS exposure. Shear-induced improvements in the mechanical properties of RBCs by this signaling mechanism could facilitate RBC flow in the microcirculation of pathophysiological disorders, wherein Ca 2+ homeostasis is disturbed and RBC deformability is reduced., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanosensory neurons use mechanotransduction (MET) ion channels to detect mechanical forces and displacements. Proteins that function as MET channels have appeared multiple times during evolution and occur in at least four different families: the DEG/ENaC and TRP channels, as well as the TMC and Piezo proteins. We found twelve putative members of MET channel families in two spider transcriptomes, but detected only one, the Piezo protein, by in situ hybridization in their mechanosensory neurons. In contrast, probes for orthologs of TRP, ENaC or TMC genes that code MET channels in other species did not produce any signals in these cells. An antibody against C. salei Piezo detected the protein in all parts of their mechanosensory cells and in many neurons of the CNS. Unspecific blockers of MET channels, Ruthenium Red and GsMTx4, had no effect on the mechanically activated currents of the mechanosensory VS-3 neurons, but the latter toxin reduced action potential firing when these cells were stimulated electrically. The Piezo protein is expressed throughout the spider nervous system including the mechanosensory neurons. It is possible that it contributes to mechanosensory transduction in spider mechanosensilla, but it must have other functions in peripheral and central neurons.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

This study aimed to determine the mechanosensing role of angiotensin II type 1 receptor (AT 1 R) in flow-induced dilation (FID) and oxidative stress production in middle cerebral arteries (MCA) of Sprague-Dawley rats. Eleven-week old, healthy male Sprague-Dawley rats on a standard diet were given the AT 1 R blocker losartan (1 mg/mL) in drinking water (losartan group) or tap water (control group) ad libitum for 7 days. Blockade of AT 1 R attenuated FID and acetylcholine-induced dilation was compared with control group. Nitric oxide (NO) synthase inhibitor N ω -nitro-l-arginine methyl ester (l-NAME) and cyclooxygenase inhibitor indomethacin (Indo) significantly reduced FID in control group. The attenuated FID in losartan group was further reduced by Indo only at Δ100 mmHg, whereas l-NAME had no effect. In losartan group, Tempol (a superoxide scavenger) restored dilatation, whereas Tempol + l-NAME together significantly reduced FID compared with restored dilatation with Tempol alone. Direct fluorescence measurements of NO and reactive oxygen species (ROS) production in MCA, in no-flow conditions revealed significantly reduced vascular NO levels with AT 1 R blockade compared with control group, whereas in flow condition increased the NO and ROS production in losartan group and had no effect in the control group. In losartan group, Tempol decreased ROS production in both no-flow and flow conditions. AT 1 R blockade elicited increased serum concentrations of ANG II, 8-iso-PGF2α, and TBARS, and decreased antioxidant enzyme activity (SOD and CAT). These results suggest that in small isolated cerebral arteries: 1 ) AT 1 receptor maintains dilations in physiological conditions; 2 ) AT 1 R blockade leads to increased vascular and systemic oxidative stress, which underlies impaired FID. NEW & NOTEWORTHY The AT 1 R blockade impaired the endothelium-dependent, both flow- and acetylcholine-induced dilations of MCA by decreasing vascular NO production and increasing the level of vascular and systemic oxidative stress, whereas it mildly influenced the vascular wall inflammatory phenotype, but had no effect on the systemic inflammatory response. Our data provide functional and molecular evidence for an important role of AT 1 receptor activation in physiological conditions, suggesting that AT 1 receptors have multiple biological functions.

Sensorineural hearing loss is a health problem with global prevalence. Aminoglycoside antibiotics, for instance gentamicin, may cause ototoxicity in mammals as a result of apoptosis and elevated oxidative stress in cochlear hair cells. Our study aimed to examine the potential effects of theophylline, an HDAC2 agonist, on gentamicin-induced cytotoxicity to sensory hair cells. Mouse cochlear explants and HEI-OC1 cells were in vitro cultured and challenged by gentamicin to induce ototoxicity, with or without theophylline. Cochlear hair cells were evaluated by fluorescent microscopy, and their mechanotransduction was assessed by electrophysiology. Expression levels of HDAC2 and apoptosis pathway factors were also evaluated following gentamicin and theophylline treatments. The functional role of HDAC2 in this setting was investigated by siRNA targeted silencing. Theophylline protected cochlear hair cells from ototoxicity induced by gentamicin, in terms of preserving cochlear structure and mechanotransduction ability, and preventing the activation of the intrinsic apoptosis pathway dose-dependently. HDAC2 expression was downregulated by gentamicin, which could be restored by theophylline. HDAC2 silencing in HEI-OC1 cells negated the beneficial effect of theophylline against gentamicin-induced growth defect and apoptosis activation. Theophylline protects sensory hair cells from gentamicin ototoxicity by maintaining HDAC2 expression. Our study thereby discovers a critical role of HDAC2 in gentamicin-induced ototoxicity, which could shine light on potential therapeutic options for treatment against sensorineural hearing loss., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

High dietary salt increases arterial pressure partly through activation of magnocellular neurosecretory cells (MNC VP ) that secrete the antidiuretic and vasoconstrictor hormone vasopressin (VP) into the circulation. Here, we show that the intrinsic and synaptic excitation of MNC VP caused by hypertonicity are differentially potentiated in two models of salt-dependent hypertension in rats. One model combined salty chow with a chronic subpressor dose of angiotensin II (AngII-salt), the other involved replacing drinking water with 2% NaCl (salt loading, SL). In both models, we observed a significant increase in the quantal amplitude of EPSCs on MNC VP . However, model-specific changes were also observed. AngII-salt increased the probability of glutamate release by osmoreceptor afferents and increased overall excitatory network drive. In contrast, SL specifically increased membrane stiffness and the intrinsic osmosensitivity of MNC VP . These results reveal that dietary salt increases the excitability of MNC VP through effects on the cell-autonomous and synaptic osmoresponsiveness of MNC VP ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In hepatitis, activated hepatic stellate cells (HSCs) produce collagens, causing liver fibrosis. Microenvironmental stiffness is a known trigger of HSC activation and is communicated through mechanotransduction. Cell proliferation, alpha smooth muscle actin (α-SMA) and collagen type Iα (Col1α) are indicative of activated HSCs. We hypothesized that certain compounds could interfere with the HSC's recognition of microenvironmental stiffness by blocking cell adhesion signaling. To verify the potential of mechanotransduction, and in particular of focal adhesion proteins, as liver fibrosis drug targets, we evaluated existing drugs. We examined the effects of the integrin antagonist, BS-1417; the focal adhesion kinase (FAK) inhibitor, defactinib; the cyclin-dependent kinase (CDK) inhibitor, roscovitine; and two microtubule modulators, paclitaxel and colchicine, on stiffness-induced HSC activation. To determine the extent of transforming growth factor β (TGF-β) participation in mechanotransduction, we measured gene expression levels of α-SMA and Col1α. We also measured ATP levels to determine cell number. Results revealed that interestingly, although TGF-β did not show additional HSC activation after stiffness stimulation, the TGF-β receptor inhibitor, SB525334, markedly suppressed stiffness-induced α-SMA and Col1α mRNA expression. BS-1417, roscovitine, defactinib and colchicine suppressed α-SMA and Col1α mRNA expression as well as the number of HSCs. Paclitaxel also suppressed stiffness-induced α-SMA mRNA expression and the number of HSCs, but mildly reduced that of Col1α mRNA. Together, these results show that an integrin antagonist and mechanotransduction-targeting drugs reduced stiffness-induced HSC activation in a dose-dependent fashion. The targeting of focal adhesion proteins involved in mechanotransduction is promising in liver fibrosis drug development.

Hypertrophic scar (HS) formation is a skin fibroproliferative disease that occurs following a cutaneous injury, leading to functional and cosmetic impairment. To date, few therapeutic treatments exhibit satisfactory outcomes. The mechanical force has been shown to be a key regulator of HS formation, but the underlying mechanism is not completely understood. The Piezo1 channel has been identified as a novel mechanically activated cation channel (MAC) and is reportedly capable of regulating force-mediated cellular biological behaviors. However, the mechanotransduction role of Piezo1 in HS formation has not been investigated. In this work, we found that Piezo1 was overexpressed in myofibroblasts of human and rat HS tissues. In vitro, cyclic mechanical stretch (CMS) increased Piezo1 expression and Piezo1-mediated calcium influx in human dermal fibroblasts (HDFs). In addition, Piezo1 activity promoted HDFs proliferation, motility, and differentiation in response to CMS. More importantly, intradermal injection of GsMTx4, a Piezo1-blocking peptide, protected rats from stretch-induced HS formation. Together, Piezo1 was shown to participate in HS formation and could be a novel target for the development of promising therapies for HS formation.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes 1-3 . However, how exactly they sense mechanical force remains under investigation 4 . The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels 4-8 , but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states 9-11 . Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation 4,11 .

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca 2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca 2+ -dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca 2+ -dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.

ATP is a major energy source in the mammalian cells, but it is an extracellular chemical messenger acting on P2 purinergic receptors. A line of evidence has shown that ATP is released from many different types of cells including neurons, endothelial cells, and muscle cells. In this review, we described the distribution of P2 receptor subtypes in the cardiac cells and their physiological and pathological roles in the heart. So far, the effects of external application of ATP or its analogues, and those of UTP on cardiac contractility and rhythm have been reported. In addition, specific genetic alterations and pharmacological agonists and antagonists have been adopted to discover specific roles of P2 receptor subtypes including P2X4-, P2X7-, P2Y2- and P2Y6-receptors in cardiac cells under physiological and pathological conditions. Accumulated data suggest that P2X4 receptors may play a beneficial role in cardiac muscle function, and that P2Y2- and P2Y6-receptors can induce cardiac fibrosis. Recent evidence further demonstrates P2Y1 receptor and P2X4 receptor as important mechanical signaling molecules to alter membrane potential and Ca 2+ signaling in atrial myocytes and their uneven expression profile between right and left atrium.

Gentamicin is a potent broad-spectrum aminoglycoside antibiotic whose use is hampered by ototoxic side-effects. Hospital gentamicin is a mixture of five gentamicin C-subtypes and several impurities of various ranges of nonexact concentrations. We developed a purification strategy enabling assaying of individual C-subtypes and impurities for ototoxicity and antimicrobial activity. We found that C-subtypes displayed broad and potent in vitro antimicrobial activities comparable to the hospital gentamicin mixture. In contrast, they showed different degrees of ototoxicity in cochlear explants, with gentamicin C2b being the least and gentamicin C2 the most ototoxic. Structure-activity relationships identified sites in the C4'-C6' region on ring I that reduced ototoxicity while preserving antimicrobial activity, thus identifying targets for future drug design and mechanisms for hair cell toxicity. Structure-activity relationship data suggested and electrophysiological data showed that the C-subtypes both bind and permeate the hair cell mechanotransducer channel, with the stronger the binding the less ototoxic the compound. Finally, both individual and reformulated mixtures of C-subtypes demonstrated decreased ototoxicity while maintaining antimicrobial activity, thereby serving as a proof-of-concept of drug reformulation to minimizing ototoxicity of gentamicin in patients., Competing Interests: Competing interest statement: R.G., J.P.M., Z.S., and D.L. are employed by Nanosyn.

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca 2+ responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y 1 receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca 2+ responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca 2+ signals via P2Y 1 receptors and Ca 2+ recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits. SIGNIFICANCE STATEMENT There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca 2+ responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion., (Copyright © 2020 Turovsky et al.)

The role viscoelasticity in fibrotic disease progression is an emerging area of interest. Here, a fast-relaxing hydrogel system is exploited to investigate potential crosstalk between calcium signaling and mechanotransduction. Poly(ethylene glycol) (PEG) hydrogels containing boronate and triazole crosslinkers are synthesized, with varying ratios of boronate to triazole crosslinks to systematically vary the extent of stress relaxation. Valvular interstitial cells (VICs) encapsulated in hydrogels with the highest levels of stress relaxation (90%) exhibit a spread morphology by day 1 and are highly aligned (80 ± 2%) by day 5. Key myofibroblast markers, including α-smooth muscle actin (αSMA) and collagen 1a1 (COL1A1), are significantly elevated. VIC myofibroblast activation decreases by 42 ± 18% through inhibition of mechanotransduction, independently of VIC morphology and alignment. Calcium signaling through a transient receptor potential vanilloid 4 (TRPV4) is found to regulate VIC spreading, alignment, and activation in a time dependent manner. Inhibition of calcium signaling at early time points results in disturbed cell alignment, decreased mechanotransduction, and diminished activation, while inhibition at later time points only causes partially reduced myofibroblast activation. These results suggest a potential crosstalk mechanism, where calcium signaling acts upstream of mechanosensing and can regulate VIC myofibroblast activation independently of mechanotransduction., (© 2020 Wiley-VCH GmbH.)

Background: Nanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO 2 (RITC)), even at 0.1 μg/μL. These protruded structures are involved in a cell's rigidity sensing, but how these NPs affect rigidity sensing is unknown., Results: Here, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 μg/μL of MNPs@SiO 2 (RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 μm in height, 24.21 nN/μm in stiffness k) under MNPs@SiO 2 (RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO 2 (RITC)., Conclusions: Our results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Loss of the sense of touch in fingertips and toes is one of the earliest sensory dysfunctions in patients receiving chemotherapy with anti-cancer drugs such as vincristine. However, mechanisms underlying this chemotherapy-induced sensory dysfunction is incompletely understood. Whisker hair follicles are tactile organs in non-primate mammals which are functionally equivalent to human fingertips. Here we used mouse whisker hair follicles as a model system and applied the pressure-clamped single-fiber recording technique to explore how vincristine treatment affect mechanoreceptors in whisker hair follicles. We showed that in vivo treatment of mice with vincristine impaired whisker tactile behavioral responses. The pressure-clamped single-fiber recordings made from whisker hair follicle afferent nerves showed that mechanical stimulations evoked three types of mechanical responses, rapidly adapting response (RA), slowly adapting type 1 response (SA1) and slowly adapting type 2 response (SA2). Vincristine treatment significantly reduced SA1 responses but did not significantly affect RA and SA2 responses. Our findings suggest that SA1 mechanoreceptors were selectively impaired by vincristine leading to the impairment of in vivo whisker tactile behavioral responses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress β1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies., Objective: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells., Study Design: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 μM, or control) for 48 hours. Protein and mRNA levels of β1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction., Results: We found that simvastatin significantly reduced the protein expression of β1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in β1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1., Conclusion: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor β receptor I (TGFβRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a "resting" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation., Competing Interests: A.D.M. is a co-founder of and has an equity interest in Insilicomed Inc. and an equity interest in Vektor Medical, Inc. He serves on the scientific advisory board of Insilicomed, and as scientific advisor to both companies. Some of his research grants have been identified for conflict of interest management based on the overall scope of the project and its potential benefit to these companies. The author is required to disclose this relationship in publications acknowledging the grant support; however, the research subject and findings reported in this study did not involve the companies in any way and have no relationship with the business activities or scientific interests of either company. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies. We declare that the competing interest statements by A.D.M. does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: The ability of heart valve cells to respond to their mechanical environment represents a key mechanism by which the integrity and function of valve cusps is maintained. A number of different mechanotransduction pathways have been implicated in the response of valve cells to mechanical stimulation. In this study, we explore the expression pattern of several mechanosensitive ion channels (MSC) and their potential to mediate mechanosensitive responses of human valve interstitial cells (VIC)., Methods: MSC presence and function were probed using the patch clamp technique. Protein abundance of key MSC was evaluated by Western blotting in isolated fibroblastic VIC (VICFB) and in VIC differentiated towards myofibroblastic (VICMB) or osteoblastic (VICOB) phenotypes. Expression was compared in non-calcified and calcified human aortic valves. MSC contributions to stretch-induced collagen gene expression and to VIC migration were assessed by pharmacological inhibition of specific channels., Results: Two MSC types were recorded in VICFB: potassium selective and cation non-selective channels. In keeping with functional data, the presence of both TREK-1 and Kir6.1 (potassium selective), as well as TRPM4, TRPV4 and TRPC6 (cationic non-selective) channels was confirmed in VIC at the protein level. Differentiation of VICFB into VICMB or VICOB phenotypes was associated with a lower expression of TREK-1 and Kir6.1, and a higher expression of TRPV4 and TRPC6. Differences in MSC expression were also seen in non-calcified vs calcified aortic valves where TREK-1, TRPM4 and TRPV4 expression were higher in calcified compared to control tissues. Cyclic stretch-induced expression of COL I mRNA in cultured VICFB was blocked by RN-9893, a selective inhibitor of TRPV4 channels while having no effect on the stretch-induced expression of COL III. VICFB migration was blocked with the non-specific MSC blocker streptomycin and by GSK417651A an inhibitor of TRPC6/3., Conclusion: Aortic VIC express a range of MSC that play a role in functional responses of these cells to mechanical stimulation. MSC expression levels differ in calcified and non-calcified valves in ways that are in part compatible with the change in expression seen between VIC phenotypes. These changes in MSC expression, and associated alterations in the ability of VIC to respond to their mechanical environment, may form novel targets for intervention during aortic valvulopathies., Competing Interests: The authors have declared that no competing interests exist.

Mechanotransduction is the process in which cells can convert extracellular mechanical stimuli into biochemical changes within a cell. While this a normal process for physiological development and function in many organ systems, tumour cells can exploit this process to promote tumour progression. Here we summarise the current state of knowledge of mechanotransduction in osteosarcoma (OSA), the most common primary bone tumour, referencing both human and canine models and other similar mesenchymal malignancies (e.g., Ewing sarcoma). Specifically, we discuss the mechanical properties of OSA cells, the pathways that these cells utilise to respond to external mechanical cues, and mechanotransduction-targeting strategies tested in OSA so far. We point out gaps in the literature and propose avenues to address them. Understanding how the physical microenvironment influences cell signalling and behaviour will lead to the improved design of strategies to target the mechanical vulnerabilities of OSA cells.

Myelin loss in the brain is a common occurrence in traumatic brain injury (TBI) that results from impact-induced acceleration forces to the head. Fast and abrupt head motions, either resulting from violent blows and/or jolts, cause rapid stretching of the brain tissue, and the long axons within the white matter tracts are especially vulnerable to such mechanical strain. Recent studies have shown that mechanotransduction plays an important role in regulating oligodendrocyte progenitors cell differentiation into oligodendrocytes. However, little is known about the impact of mechanical strain on mature oligodendrocytes and the stability of their associated myelin sheaths. We used an in vitro cellular stretch device to address these questions, as well as characterize a mechanotransduction mechanism that mediates oligodendrocyte responses. Mechanical stretch caused a transient and reversible myelin protein loss in oligodendrocytes. Cell death was not observed. Myelin protein loss was accompanied by an increase in intracellular Ca 2+ and Erk1/2 activation. Chelating Ca 2+ or inhibiting Erk1/2 activation was sufficient to block the stretch-induced loss of myelin protein. Further biochemical analyses revealed that the stretch-induced myelin protein loss was mediated by the release of Ca 2+ from the endoplasmic reticulum (ER) and subsequent Ca 2+ -dependent activation of Erk1/2. Altogether, our findings characterize an Erk1/2-dependent mechanotransduction mechanism in mature oligodendrocytes that de-stabilizes the myelination program., (© 2020 Wiley Periodicals, Inc.)

Ammonia (including NH 3 and NH 4 + ) is a major pollutant of freshwater environments. However, the toxic effects of ammonia on the early stages of fish are not fully understood, and little is known about the effects on the sensory system. In this study, we hypothesized that ammonia exposure can cause adverse effects on embryonic development and impair the lateral line system of fish. Zebrafish embryos were exposed to high-ammonia water (10, 15, 20, 25, and 30 mM NH 4 Cl; pH 7.0) for 96 h (0-96 h post-fertilization). The body length, heart rate, and otic vesicle size had significantly decreased with ≥15 mM NH 4 Cl, while the number and function of lateral-line hair cells had decreased with ≥10 mM NH 4 Cl. The mechanoelectrical transduction (MET) channel-mediated Ca 2+ influx was measured with a scanning ion-selective microelectrode technique to reveal the function of hair cells. We found that NH 4 + (≥5 mM NH 4 Cl) entered hair cells and suppressed the Ca 2+ influx of hair cells. Neomycin and La 3+ (MET channel blockers) suppressed NH 4 + influx, suggesting that NH 4 + enters hair cells via MET channels in hair bundles. In conclusion, this study showed that ammonia exposure (≥10 mM NH 4 Cl) can cause adverse effects in zebrafish embryos, and lateral-line hair cells are sensitive to ammonia exposure., Competing Interests: Declaration of competing interest The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The principle of mechanopharmacology of airway smooth muscle (ASM) is based on the premise that physical agitation, such as pressure oscillation applied to an airway, is able to induce bronchodilation by reducing contractility and softening the cytoskeleton of ASM. Although the underlying mechanism is not entirely clear, there is evidence to suggest that large-amplitude stretches are able to disrupt the actomyosin interaction in the crossbridge cycle and weaken the cytoskeleton in ASM cells. Rho-kinase is known to enhance force generation and strengthen structural integrity of the cytoskeleton during smooth muscle activation and plays a key role in the maintenance of force during prolonged muscle contractions. Synergy in relaxation has been observed when the muscle is subject to oscillatory length change while Rho-kinase is pharmacologically inhibited. In this review, inhibition of Rho-kinase coupled to therapeutic pressure oscillation applied to the airways is explored as a combination treatment for asthma., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rho GTPase-activating protein (Rho-GAP) and Rho GDP dissociation inhibitor (Rho- GDI) are two main negative regulators of Rho GTPase. Our previous work has found that Rho-GDI and Rho GTPase are involved in the response of human periodontal ligament (PDL) cells to mechanical stress. However, whether Rho-GAP also has a role in this process remains unknown. Here, we attempted to find the Rho-GAP gene that may be involved in pathological stretch-induced apoptosis of PDL cells. Human PDL fibroblasts were exposed to 20% cyclic strain for 6 hours or 24 hours, after which the expression levels of ARHGAP10, ARHGAP17, ARHGAP21, ARHGAP24 and ARHGAP28 were determined. Results showed that ARHGAP17 expression decreased the most obviously after treatment of stretch. In addition, ARHGAP17 overexpression abolished 20% cyclic strain-induced apoptosis. Therefore, ARHGAP17 has an important role in pathological stretch-induced apoptosis of human PDL fibroblasts. Moreover, we found that ARHGAP17 overexpression also alleviated cyclic strain-induced activation of Rac1/Cdc42, a major downstream target of ARHGAP17. Furthermore, two Rac1 inhibitors, NSC23766 and EHT 1864, both attenuated ARHGAP17 knockdown-mediated apoptosis in human PDL fibroblasts. Collectively, our data demonstrate that ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human PDL fibroblasts through inactivating Rac1/Cdc42. This study highlights the importance of Rho signalling in the response of human PDL fibroblasts to mechanical stress., (© 2020 John Wiley & Sons Australia, Ltd.)

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery 1 . Although the response of epidermal cells to stretching has been studied in vitro 2,3 , it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.

C. elegans uses specialized mechanoreceptor neurons to sense various mechanical cues. However, whether other tissues and organs in C. elegans are able to perceive mechanical forces is not clear. In this study, with a whole-cell patch-clamp recording, we show that body wall muscles (BWMs) in C. elegans convert mechanical energy into ionic currents in a cell-autonomous manner. Mechano-gated ion channels in BWMs are blocked in amiloride or cation-free solutions. A further characterization of physiological properties of mechano-gate ion channels in BMWs and a genetic screening show that mechanosensation in BMWs is not dependent on UNC-105 and well-defined mechano-gated ion channels MEC-4 and TRP-4 in C. elegans. Taken together, our results demonstrate that BWMs in C. elegans function as mechanoreceptors to sense mechanical stimuli with an amiloride-sensitive, non-selective cation channel., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.

The mechanical component in the pathophysiology of dry eye disease (DED) deserves attention as an important factor. The lubrication deficit induced impaired mechano-transduction of lid pressure to the ocular surfaces may lead to the dysregulation of homeostasis in the epithelium, with sensations of pain and secondary inflammation. Ocular pain is possibly the first sign of attrition and may occur in the absence of visible epithelial damage. Attrition is a process which involves the constant or repeated challenge of ocular surface tissues by mechanical shear forces; it is enhanced by the thinning of corneal epithelium in severe DED. As a highly dynamic process leading to pain and neurogenic inflammation, the identification of the impact of attrition and its potential pathogenic role could add a new perspective to the current more tear film-oriented models of ocular surface disease. Treatment of DED addressing lubrication deficiencies and inflammation should also consider the decrease of attrition in order to stimulate epithelial recovery and neural regeneration. The importance of hyaluronic acid, its molecular characteristics, the extracellular matrix and autoregulative mechanisms in this process is outlined. The identification of the attrition and recognition of its impact in dry eye pathophysiology could contribute to a better understanding of the disease and optimized treatment regimens.

Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system., (© 2019 Wiley Periodicals, Inc.)

Arterial thrombosis is in part contributed by excessive platelet aggregation, which can lead to blood clotting and subsequent heart attack and stroke. Platelets are sensitive to the haemodynamic environment. Rapid haemodynamcis and disturbed blood flow, which occur in vessels with growing thrombi and atherosclerotic plaques or is caused by medical device implantation and intervention, promotes platelet aggregation and thrombus formation. In such situations, conventional antiplatelet drugs often have suboptimal efficacy and a serious side effect of excessive bleeding. Investigating the mechanisms of platelet biomechanical activation provides insights distinct from the classic views of agonist-stimulated platelet thrombus formation. In this work, we review the recent discoveries underlying haemodynamic force-reinforced platelet binding and mechanosensing primarily mediated by three platelet receptors: glycoprotein Ib (GPIb), glycoprotein IIb/IIIa (GPIIb/IIIa) and glycoprotein VI (GPVI), and their implications for development of antithrombotic 'mechano-medicine' ., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The physico-chemical characteristics of the extracellular matrix (ECM) cause mechanical cues that could elicit responses in the survival rate of cortical neuronal cells. Efficient neurite outgrowth in vitro, is critical for successful cultivation of cortical neuronal cells and the potential for attempts at regeneration of the central nervous system (CNS) in vivo. Relatively soft and hydrophilic, microbially synthesized aromatic polyester, polyhydroxyphenylvalerate (PHPV) was blended 50:50 with the stiff and hydrophobic polycaprolactone (PCL) and electrospun in microfibers for use in a 3D (CellCrown™) configuration and in a 2D coverslip coated configuration. This blend allows a 2.3-fold increase in the life-span of human induced pluripotent stem derived cortical neuronal cells (hiPS) compared to pure PCL fibers. HiPS-derived cortical neuronal cells grown on PHPV/PCL fibers show a 3.8-fold higher cumulative neurite elaboration compared to neurites grown on PCL fibers only. 96% of cortical neuronal cells die after 8 days of growth when plated on PCL fibers alone while >83% and 55% are alive on PHPV/PCL fibers on day 8 and day 17, respectively. An increased migration rate of cortical neuronal cells is also promoted by the blend compared to the PCL fibers alone. The critical survival rate improvement of hiPS derived cortical neuronal cells on PHPV/PCL blend holds promise in using these biocompatible nanofibers as implantable materials for regenerative purposes of an active cortical neuronal population after full maturation in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)