α-Catenin and Piezo1 Mediate Cell Mechanical Communication via Cell Adhesions.

Cell-to-cell distant mechanical communication has been demonstrated using in vitro and in vivo models. However, the molecular mechanisms underlying long-range cell mechanoresponsive interactions remain to be fully elucidated. This study further examined the roles of α-Catenin and Piezo1 in traction force-induced rapid branch assembly of airway smooth muscle (ASM) cells on a Matrigel hydrogel containing type I collagen. Our findings demonstrated that siRNA-mediated downregulation of α-Catenin or Piezo1 expression or chemical inhibition of Piezo1 activity significantly reduced both directional cell movement and branch assembly. Regarding the role of N-cadherin in regulating branch assembly but not directional migration, our results further confirmed that siRNA-mediated downregulation of α-Catenin expression caused a marked reduction in focal adhesion formation, as assessed by focal Paxillin and Integrin α5 localization. These observations imply that mechanosensitive α-Catenin is involved in both cell-cell and cell-matrix adhesions. Additionally, Piezo1 partially localized in focal adhesions, which was inhibited by siRNA-mediated downregulation of α-Catenin expression. This result provides insights into the Piezo1-mediated mechanosensing of traction force on a hydrogel. Collectively, our findings highlight the significance of α-Catenin in the regulation of cell-matrix interactions and provide a possible interpretation of Piezo1-mediated mechanosensing activity at focal adhesions during cell-cell mechanical communication.

BitterDB database analysis plus cell stiffness screening identify flufenamic acid as the most potent TAS2R14-based relaxant of airway smooth muscle cells for therapeutic bronchodilation.

Rationale: Bitter taste receptors (TAS2Rs) are abundantly expressed in airway smooth muscle cells (ASMCs), which have been recognized as promising targets for bitter agonists to initiate relaxation and thereby prevent excessive airway constriction as the main characteristic of asthma. However, due to the current lack of tested safe and potent agonists functioning at low effective concentrations, there has been no clinically approved TAS2R-based drug for bronchodilation in asthma therapy. This study thus aimed at exploring TAS2R agonists with bronchodilator potential by BitterDB database analysis and cell stiffness screening. Methods: Bitter compounds in the BitterDB database were retrieved and analyzed for their working subtype of TAS2R and effective concentration. Compounds activating TAS2R5, 10, and 14 at < 100 µM effective concentration were identified and subsequently screened by cell stiffness assay using optical magnetic twisting cytometry (OMTC) to identify the most potent to relax ASMCs. Then the compound identified was further characterized for efficacy on various aspects related to relaxation of ASMCs, incl. but not limited to traction force by Fourier transform traction force microscopy (FTTFM), [Ca2+]i signaling by Fluo-4/AM intensity, cell migration by scratch wound healing, mRNA expression by qPCR, and protein expressing by ELISA. The compound identified was also compared to conventional ß-agonist (isoproterenol and salbutamol) for efficacy in reducing cell stiffness of cultured ASMCs and airway resistance of ovalbumin-treated mice. Results: BitterDB analysis found 18 compounds activating TAS2R5, 10, and 14 at < 100 µM effective concentration. Cell stiffness screening of these compounds eventually identified flufenamic acid (FFA) as the most potent compound to rapidly reduce cell stiffness at 1 µM. The efficacy of FFA to relax ASMCs in vitro and abrogate airway resistance in vivo was equivalent to that of conventional ß-agonists. The FFA-induced effect on ASMCs was mediated by TAS2R14 activation, endoplasmic reticulum Ca2+ release, and large-conductance Ca2+-activated K+ (BKCa) channel opening. FFA also attenuated lipopolysaccharide-induced inflammatory response in cultured ASMCs. Conclusions: FFA as a potent TAS2R14 agonist to relax ASMCs while suppressing cytokine release might be a favorite drug agent for further development of TAS2R-based novel dual functional medication for bronchodilation and anti-inflammation in asthma therapy.

High Stretch Associated with Mechanical Ventilation Promotes Piezo1-Mediated Migration of Airway Smooth Muscle Cells.

Ventilator-induced lung injury (VILI) during mechanical ventilation (MV) has been attributed to airway remodeling involving increased airway smooth muscle cells (ASMCs), but the underlying mechanism is not fully understood. Thus, we aimed to investigate whether MV-associated high stretch (>10% strain) could modulate mechanosensitive Piezo1 expression and thereby alter cell migration of ASMCs as a potential pathway to increased ASMCs in VILI. C57BL/6 mice and ASMCs were subjected to MV at high tidal volume (VT, 18 mL/kg, 3 h) and high stretch (13% strain, 0.5 Hz, 72 h), respectively. Subsequently, the mice or cells were evaluated for Piezo1 and integrin mRNA expression by immunohistochemical staining and quantitative PCR (qPCR), and cell migration and adhesion by transwell and cell adhesion assays. Cells were either treated or not with Piezo1 siRNA, Piezo1-eGFP, Piezo1 knockin, Y27632, or blebbistatin to regulate Piezo1 mRNA expression or inhibit Rho-associated kinase (ROCK) signaling prior to migration or adhesion assessment. We found that expression of Piezo1 in in situ lung tissue, mRNA expression of Piezo1 and integrin αVß1 and cell adhesion of ASMCs isolated from mice with MV were all reduced but the cell migration of primary ASMCs (pASMCs) isolated from mice with MV was greatly enhanced. Similarly, cell line mouse ASMCs (mASMCs) cultured in vitro with high stretch showed that mRNA expression of Piezo1 and integrin αVß1 and cell adhesion were all reduced but cell migration was greatly enhanced. Interestingly, such effects of MV or high stretch on ASMCs could be either induced or abolished/reversed by down/up-regulation of Piezo1 mRNA expression and inhibition of ROCK signaling. High stretch associated with MV appears to be a mechanical modulator of Piezo1 mRNA expression and can, thus, promote cell migration of ASMCs during therapeutic MV. This may be a novel mechanism of detrimental airway remodeling associated with MV, and, therefore, a potential intervention target to treat VILI.

High Stretch Modulates cAMP/ATP Level in Association with Purine Metabolism via miRNA-mRNA Interactions in Cultured Human Airway Smooth Muscle Cells.

High stretch (>10% strain) of airway smooth muscle cells (ASMCs) due to mechanical ventilation (MV) is postulated to contribute to ventilator-induced lung injury (VILI), but the underlying mechanisms remain largely unknown. We hypothesized that ASMCs may respond to high stretch via regulatory miRNA-mRNA interactions, and thus we aimed to identify high stretch-responsive cellular events and related regulating miRNA-mRNA interactions in cultured human ASMCs with/without high stretch. RNA-Seq analysis of whole genome-wide miRNAs revealed 12 miRNAs differentially expressed (DE) in response to high stretch (7 up and 5 down, fold change >2), which target 283 DE-mRNAs as identified by a parallel mRNA sequencing and bioinformatics analysis. The KEGG and GO analysis further indicated that purine metabolism was the first enriched event in the cells during high stretch, which was linked to miR-370-5p-PDE4D/AK7. Since PDE4D/AK7 have been previously linked to cAMP/ATP metabolism in lung diseases and now to miR-370-5p in ASMCs, we thus evaluated the effect of high stretch on the cAMP/ATP level inside ASMCs. The results demonstrated that high stretch modulated the cAMP/ATP levels inside ASMCs, which could be largely abolished by miR-370-5p mimics. Together, these findings indicate that miR-370-5p-PDE4D/AK7 mediated high stretch-induced modulation of cAMP and ATP synthesis inside ASMCs. Furthermore, such interactive miRNA-mRNA pairs may provide new insights for the discovery of effective biomarkers/therapeutic targets for the diagnosis and treatment of VILI and other MV-associated respiratory diseases.

Mechanical Ventilation-Related High Stretch Mainly Induces Endoplasmic Reticulum Stress and Thus Mediates Inflammation Response in Cultured Human Primary Airway Smooth Muscle Cells.

Ventilator-induced lung injury (VILI) occurs in mechanically ventilated patients of respiratory disease and is typically characterized by airway inflammation. However, recent studies increasingly indicate that a major cause of VILI may be the excessive mechanical loading such as high stretch (>10% strain) on airway smooth muscle cells (ASMCs) due to mechanical ventilation (MV). Although ASMCs are the primary mechanosensitive cells in airways and contribute to various airway inflammation diseases, it is still unclear how they respond to high stretch and what mediates such a response. Therefore, we used whole genome-wide mRNA-sequencing (mRNA-Seq), bioinformatics, and functional identification to systematically analyze the mRNA expression profiles and signaling pathway enrichment of cultured human ASMCs exposed to high stretch (13% strain), aiming to screen the susceptible signaling pathway through which cells respond to high stretch. The data revealed that in response to high stretch, 111 mRNAs with count ≥100 in ASMCs were significantly differentially expressed (defined as DE-mRNAs). These DE-mRNAs are mainly enriched in endoplasmic reticulum (ER) stress-related signaling pathways. ER stress inhibitor (TUDCA) abolished high-stretch-enhanced mRNA expression of genes associated with ER stress, downstream inflammation signaling, and major inflammatory cytokines. These results demonstrate in a data-driven approach that in ASMCs, high stretch mainly induced ER stress and activated ER stress-related signaling and downstream inflammation response. Therefore, it suggests that ER stress and related signaling pathways in ASMCs may be potential targets for timely diagnosis and intervention of MV-related pulmonary airway diseases such as VILI.

Chemical Activation of Piezo1 Alters Biomechanical Behaviors toward Relaxation of Cultured Airway Smooth Muscle Cells.

Inspired by the well-known phenomenon of stretch-induced airway dilation in normal lungs and the emerging stretch-responsive Piezo1 channels that can be chemically activated by specific agonists such as Yoda1, we attempted to investigate whether chemical activation of Piezo1 by Yoda1 can modulate the biomechanical behaviors of airway smooth muscle cells (ASMCs) so that it may be exploited as a novel approach for bronchodilation. Thus, we treated in vitro cultured rat ASMCs with Yoda1, and examined the cells for calcium signaling, cell stiffness, traction force, cell migration, and the mRNA expression and distribution of molecules relevant to cell biomechanics. The data show that ASMCs expressed abundant mRNA of Piezo1. ASMCs exposed to 1 µM Yoda1 exhibited a potent but transient Ca2+ signaling, and treatment with 1 µM Yoda1 for 24 h led to decreased cell stiffness and traction force, all of which were partially reversed by Piezo1 inhibitor GsMTx4 and Piezo1 knockdown, respectively. In addition, ASMCs treated with 1 µM Yoda1 for 24 h exhibited impaired horizontal but enhanced vertical cell migration, as well as significant changes in key components of cells' contractile machinery including the structure and distribution of stress fibers and alpha-smooth muscle actin (α-SMA) fibrils, the mRNA expression of molecules associated with cell biomechanics. These results provide the first evidence that chemical activation of Piezo1 by Yoda1 resulted in marked pro-relaxation alterations of biomechanical behaviors and contractile machinery of the ASMCs. These findings suggest that Piezo1-specific agonists may indeed have great potential as alternative drug agents for relaxing ASMCs.

Emerging toolset of three-dimensional pulmonary cell culture models for simulating lung pathophysiology towards mechanistic elucidation and therapeutic treatment of SARS-COV-2 infection.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a never before seen challenge to human health and the world economy. However, it is difficult to widely use conventional animal and cell culture models in understanding the underlying pathological mechanisms of COVID-19, which in turn hinders the development of relevant therapeutic treatments, including drugs. To overcome this challenge, various three-dimensional (3D) pulmonary cell culture models such as organoids are emerging as an innovative toolset for simulating the pathophysiology occurring in the respiratory system, including bronchial airways, alveoli, capillary network, and pulmonary interstitium, which provide a robust and powerful platform for studying the process and underlying mechanisms of SARS-CoV-2 infection among the potential primary targets in the lung. This review introduces the key features of some of these recently developed tools, including organoid, lung-on-a-chip, and 3D bioprinting, which can recapitulate different structural compartments of the lung and lung function, in particular, accurately resembling the human-relevant pathophysiology of SARS-CoV-2 infection in vivo. In addition, the recent progress in developing organoids for alveolar and airway disease modeling and their applications for discovering drugs against SARS-CoV-2 infection are highlighted. These innovative 3D cell culture models together may hold the promise to fully understand the pathogenesis and eventually eradicate the pandemic of COVID-19.

FRET Visualization of Cyclic Stretch-Activated ERK via Calcium Channels Mechanosensation While Not Integrin ß1 in Airway Smooth Muscle Cells.

Mechanical stretch is one type of common physiological activities such as during heart beating, lung breathing, blood flow through the vessels, and physical exercise. The mechanical stimulations regulate cellular functions and maintain body homeostasis. It still remains to further characterize the mechanical-biomechanical coupling mechanism. Here we applied fluorescence resonance energy transfer (FRET) technology to visualize ERK activity in airway smooth muscle (ASM) cells under cyclic stretch stimulation in airway smooth muscle (ASM) cells, and studied the mechanosensing pathway. FRET measurements showed apparent ERK activation by mechanical stretch, which was abolished by ERK inhibitor PD98059 pretreatment. Inhibition of extracellular Ca2+ influx reduced ERK activation, and selective inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel or SERCA Ca2+ pump on endoplasmic reticulum (ER) blocked the activation. Chemical inhibition of the L-type or store-operated Ca2+ channels on plasma membrane, or inhibition of integrin ß1 with siRNA had little effect on ERK activation. Disruption of actin cytoskeleton but not microtubule one inhibited the stretch-induced ERK activation. Furthermore, the ER IP3R-dependent ERK activation was not dependent on phospholipase C-IP3 signal, indicating possibly more mechanical mechanism for IP3R activation. It is concluded from our study that the mechanical stretch activated intracellular ERK signal in ASM cells through membrane Ca2+ channels mechanosensation but not integrin ß1, which was mediated by actin cytoskeleton.

MircroRNA Let-7a-5p in Airway Smooth Muscle Cells is Most Responsive to High Stretch in Association With Cell Mechanics Modulation.

Objective: High stretch (strain >10%) can alter the biomechanical behaviors of airway smooth muscle cells which may play important roles in diverse lung diseases such as asthma and ventilator-induced lung injury. However, the underlying modulation mechanisms for high stretch-induced mechanobiological responses in ASMCs are not fully understood. Here, we hypothesize that ASMCs respond to high stretch with increased expression of specific microRNAs (miRNAs) that may in turn modulate the biomechanical behaviors of the cells. Thus, this study aimed to identify the miRNA in cultured ASMCs that is most responsive to high stretch, and subsequently investigate in these cells whether the miRNA expression level is associated with the modulation of cell biomechanics. Methods: MiRNAs related to inflammatory airway diseases were obtained via bioinformatics data mining, and then tested with cultured ASMCs for their expression variations in response to a cyclic high stretch (13% strain) simulating in vivo ventilator-imposed strain on airways. Subsequently, we transfected cultured ASMCs with mimics and inhibitors of the miRNA that is most responsive to the high stretch, followed by evaluation of the cells in terms of morphology, stiffness, traction force, and mRNA expression of cytoskeleton/focal adhesion-related molecules. Results: 29 miRNAs were identified to be related to inflammatory airway diseases, among which let-7a-5p was the most responsive to high stretch. Transfection of cultured human ASMCs with let-7a-5p mimics or inhibitors led to an increase or decrease in aspect ratio, stiffness, traction force, migration, stress fiber distribution, mRNA expression of α-smooth muscle actin (SMA), myosin light chain kinase, some subfamily members of integrin and talin. Direct binding between let-7a-5p and ItgαV was also verified in classical model cell line by using dual-luciferase assays. Conclusion: We demonstrated that high stretch indeed enhanced the expression of let-7a-5p in ASMCs, which in turn led to changes in the cells' morphology and biomechanical behaviors together with modulation of molecules associated with cytoskeletal structure and focal adhesion. These findings suggest that let-7a-5p regulation is an alternative mechanism for high stretch-induced effect on mechanobiology of ASMCs, which may contribute to understanding the pathogenesis of high stretch-related lung diseases.

Compression enhances invasive phenotype and matrix degradation of breast Cancer cells via Piezo1 activation.

BACKGROUND: Uncontrolled growth in solid breast cancer generates mechanical compression that may drive the cancer cells into a more invasive phenotype, but little is known about how such compression affects the key events and corresponding regulatory mechanisms associated with invasion of breast cancer cells including cellular behaviors and matrix degradation. RESULTS: Here we show that compression enhanced invasion and matrix degradation of breast cancer cells. We also identified Piezo1 as the putative mechanosensitive cellular component that transmitted compression to not only enhance the invasive phenotype, but also induce calcium influx and downstream Src signaling. Furthermore, we demonstrated that Piezo1 was mainly localized in caveolae, and both Piezo1 expression and compression-enhanced invasive phenotype of the breast cancer cells were reduced when caveolar integrity was compromised by either knocking down caveolin1 expression or depleting cholesterol content. CONCLUSIONS: Taken together, our data indicate that mechanical compression activates Piezo1 channels to mediate enhanced breast cancer cell invasion, which involves both cellular events and matrix degradation. This may be a critical mechanotransduction pathway during breast cancer metastasis, and thus potentially a novel therapeutic target for the disease.

Emergent Differential Organization of Airway Smooth Muscle Cells on Concave and Convex Tubular Surface.

Airway smooth muscle cells (ASMCs) exist in a form of helical winding bundles within the bronchial airway wall. Such tubular tissue provides cells with considerable curvature as a physical constraint, which is widely thought as an important determinant of cell behaviors. However, this process is difficult to mimic in the conventional planar cell culture system. Here, we report a method to develop chips with cell-scale tubular (concave and convex) surfaces from fused deposition modeling 3D printing to explore how ASMCs adapt to the cylindrical curvature for morphogenesis and function. Results showed that ASMCs self-organized into two distinctively different patterns of orientation on the concave and convex surfaces, eventually aligning either invariably perpendicular to the cylinder axis on the concave surface or curvature-dependently angled on the convex surface. Such oriented alignments of the ASMCs were maintained even when the cells were in dynamic movement during migration and spreading along the tubular surfaces. Furthermore, the ASMCs underwent a phenotype transition on the tubular (both concave and convex) surfaces, significantly reducing contractility as compared to ASMCs cultured on a flat surface, which was reflected in the changes of proliferation, migration and gene expression of contractile biomarkers. Taken together, our study revealed a curvature-induced pattern formation and functional modulation of ASMCs in vitro, which is not only important to better understanding airway smooth muscle pathophysiology, but may also be useful in the development of new techniques for airway disease diagnosis and therapy such as engineering airway tissues or organoids.

Naringin as a plant-derived bitter tastant promotes proliferation of cultured human airway epithelial cells via activation of TAS2R signaling.

BACKGROUND: Bitter tastants can activate bitter taste receptors (TAS2Rs) and thus initiate relaxation of airway smooth muscle cells (ASMCs), which have great potential in the development of novel bronchodilator drugs for asthma therapy. However, the canonical bitter substance, denatonium is known to induce apoptosis of airway epithelial cells (AECs), indicating that other bitter tastants may also impair the epithelial integrity to prevent hazardous particulate matters such as coronaviruses. Therefore, any bitter tastants intended for treating airway disease should be carefully evaluated for potential toxicity to AECs. HYPOTHESIS/PURPOSE: Considering the vast diversity of bitter tastants in nature and different types of TAS2Rs expressed in airway cells, we hypothesized that there must be some natural bitter tastants to be not only potent in inducing relaxation of ASMCs but also unharmful to AECs. STUDY DESIGN AND METHODS: Here we evaluated a group of bitter flavonoids that are derived from fruits and commonly used in traditional herbal medicine, including apigenin, hesperetin, kaempferol, naringenin, quercetin, and naringin, for their effects on the proliferation of human airway epithelial-like (16HBE14o-, BEAS-2B, and A549) cells cultured in vitro. Cell proliferation and associated signaling pathways were assessed by cell counting, ATP assay, cell cycling assay, quantitative RT-PCR, Fluo-4 labeling, and fluorescence resonance energy transfer, respectively. RESULTS: The results show that five of the six tested bitter tastants inhibited, but only naringin promoted the proliferation of the 16HBE14o-, BEAS-2B, and A549 cells at the dose of a few hundred micromoles. Furthermore, the naringin-promoted proliferation of the 16HBE14o- cells was associated with enhanced cell cycle progression, mRNA expression of cyclin E, and evoked calcium signaling/ERK signaling, which were all attenuated by inhibition of the TAS2R signaling pathways with specific blockers. CONCLUSION: These findings indicate that although the majority of the bitter flavonoids may inhibit the proliferation of AECs, naringin emerged as one to promote the proliferation of AECs via cell cycle progression and TAS2R-activated intracellular signaling. It suggests that naringin and not a few other bitter tastants can be proven with nontoxicity to the airway epithelial structure and function, which provides further confidence in the development of safe and effective TAS2R-based bronchodilators for asthma therapy.

Evolution of international tourist flows from 1995 to 2018: A network analysis perspective.

Tourist arrivals and tourism revenues have been extensively studied to evaluate international tourist flows, whereas the structure and evolution of these flows have received less attention. Based on international tourist arrival data from 221 countries/regions during the period 1995-2018, this study applies network analysis to explore the structure and evolution of international tourist flows, and the roles and functions of countries/regions in the international tourist flow network. The results of this study reveal that the network density of international tourist flows is increasing. Countries/regions in Europe, East Asia and North America generally occupy a significantly important position within the international tourist flow network, especially Germany and China. Those geographically close countries/regions demonstrate the same or similar roles and positions in international tourism. This study has significant implications for tourist destination management and marketing.

Sanguinarine Rapidly Relaxes Rat Airway Smooth Muscle Cells Dependent on TAS2R Signaling.

Excessive contraction of airway smooth muscle cells (ASMCs) is a hallmark feature of asthma. Intriguing, the activation of bitter taste receptor (TAS2R) in ASMCs can relax ASMCs. However, there is a lack of potent TAS2R agonists that can be used in asthma therapies since those tested agonists cannot relax ASMCs at the dose below a few hundred micromolar. Considering that sanguinarine (SA) is a bitter substance often used in small doses for the treatment of asthma in folk medicine, the present study was to determine the rapid relaxation effect of SA on ASMCs and to reveal the underlying mechanisms associated with TAS2R signaling. Here, cell stiffness, traction force, calcium signaling, cAMP levels, and the mRNA expression were evaluated by using optical magnetic twisting cytometry, traction force microscopy, Fluo-4/AM labeling, enzyme-linked immunosorbent assay (ELISA), and quantitative (q)RT-PCR, respectively. We found that 0.5 µM SA immediately decreased cell stiffness and traction force, which is comparable with the effect of 5 µM isoproterenol. In addition, 0.5 µM SA immediately increased intracellular free calcium concentration ([Ca2+]i) and decreased the mRNA expression of contractile proteins such as calponin and α-smooth muscle actin after the treatment for 24 h. Furthermore, SA-mediated decrease in cell stiffness/traction force and increase in [Ca2+]i were significantly blunted by inhibiting the TAS2Rs signaling. These findings establish the rapid relaxation effect of SA at low concentration (<1 µM) on cultured ASMCs depending on TAS2R signaling, indicating that SA might be developed as a useful bronchodilator in asthma therapy.

Toward the Identification of Extra-Oral TAS2R Agonists as Drug Agents for Muscle Relaxation Therapies via Bioinformatics-Aided Screening of Bitter Compounds in Traditional Chinese Medicine.

Significant advances have been made in the past decade in mapping the distributions and the physiological functions of extra-oral bitter taste receptors (TAS2Rs) in non-gustatory tissues. In particular, it has been found that TAS2Rs are expressed in various muscle tissues and activation of TAS2Rs can lead to muscle cell relaxation, which suggests that TAS2Rs may be important new targets in muscle relaxation therapy for various muscle-related diseases. So far, however, there is a lack of potent extra-oral TAS2R agonists that can be used as novel drug agents in muscle relaxation therapies. Interestingly, traditional Chinese medicine (TCM) often characterizes a drug's property in terms of five distinct flavors (bitter, sweet, sour, salty, and pungent) according to its taste and function, and commonly regards "bitterness" as an intrinsic property of "good medicine." In addition, many bitter flavored TCM are known in practice to cause muscle relaxation after long term use, and in lab experiments the compounds identified from some bitter flavored TCM do activate TAS2Rs and thus relax muscle cells. Therefore, it is highly possible to discover very useful extra-oral TAS2R agonists for muscle relaxation therapies among the abundant bitter compounds used in bitter flavored TCM. With this perspective, we reviewed in literature the distribution of TAS2Rs in different muscle systems with a focus on the map of bitter flavored TCM which can regulate muscle contractility and related functional chemical components. We also reviewed the recently established databases of TCM chemical components and the bioinformatics software which can be used for high-throughput screening and data mining of the chemical components associated with bitter flavored TCM. All together, we aim to present a knowledge-based approach and technological platform for identification or discovery of extra-oral TAS2R agonists that can be used as novel drug agents for muscle relaxation therapies through screening and evaluation of chemical compounds used in bitter flavored TCM.

Clathrin-mediated endocytosis regulates fMLP-mediated neutrophil polarization.

A cell's ability to establish polarization is one of the key steps in directional migration. Upon the addition of a chemoattractant, N-formylmethionyl-leucyl-phenylalanine (fMLP), neutrophils rapidly develop a front end marked by a wide and dense actin network which is a feature of cell polarization. Despite a general understanding of bi-directional crosstalk between endocytosis and polarization, it remains unclear how clathrin-mediated endocytosis (CME) induced by chemoattractant binding to formyl peptide receptor (FPR) affects neutrophil polarization. In this work, we characterized the spatial organization of FPR and clathrin-coated pits (CCPs), the functional unit of CME, with and without fMLP and found that fMLP induced different distributions of FPR and CCPs. We further found that cells had impaired polarization induced by fMLP when CME is inhibited by small molecule inhibitors. Under these conditions, pERK, pAkt308, and pAkt473 were all severely blocked or had altered dynamics. The spatial organization between actin and two major clathrin-mediated endocytic proteins, clathrin and ß-arrestin, were distinct and supported clathrin and ß-arrestin's functional roles in mediating neutrophil polarization. Together these results suggest that CME plays a pivotal role in a complex process such as cell polarization.

[Effect of sanguinarine on biomechanics of rat airway smooth muscle cells].

This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured in vitro were treated with sanguinarine solution at different concentrations (0.005~5 µmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 µmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 µmol/L and 0.5 µmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.

Two New Shrinking-Circle Methods for Source Localization Based on TDoA Measurements.

Time difference of arrival (TDoA) measurement is a promising approach for target localization based on a set of nodes with known positions, with high accuracy and low complexity. Common localization algorithms include the maximum-likelihood, non-linear least-squares and weighted least-squares methods. These methods have shortcomings such as high computational complexity, requiring an initial guess position, or having difficulty in finding the optimal solution. From the point of view of geometrical analysis, this study proposes two new shrinking-circle methods (SC-1 and SC-2) to solve the TDoA-based localization problem in a two-dimensional (2-D) space. In both methods, an optimal radius is obtained by shrinking the radius with a dichotomy algorithm, and the position of the target is determined by the optimal radius. The difference of the two methods is that a distance parameter is defined in SC-1, while an error function is introduced in SC-2 to guide the localization procedure. Simulations and indoor-localization experiments based on acoustic transducers were conducted to compare the performance differences between the proposed methods, algorithms based on weighted least-squares as well as the conventional shrinking-circle method. The experimental results demonstrate that the proposed methods can realize high-precision target localization based on TDoA measurements using three nodes, and have the advantages of speed and high robustness.

[Influence of Fe 2O 3 nanoparticles on the rhological properties of simulated asthma airway mucus].

The properties of mucus in a person with asthma can alter with disease process so that it may lead to the airway embolism. Fe 2O 3 nanoparticles can be used for drug delivery. Up till now, however, little is known about how the Fe 2O 3 nanoparticles influence the properties of airway mucus. In this study, Fe 2O 3 nanoparticles were dispersed with ultrasound, and the morphological properties were measured with scanning electron microscope, atomic force microscope and nanometer laser particle size and zeta potential analyzer. Then the dispersed Fe 2O 3 nanoparticles were added to the simulated asthma airway mucus with different final concentration (0.03, 0.3, and 0.4 mg/mL). The measurements of flow curve, yield stress, large amplitude oscillatory shear (LAOS) and shock scanning were carried out with a rotational rheometer. Experimental results showed that the Fe 2O 3 nanoparticles reduced the zero shear viscosity of simulated asthma airway mucus. With increase of shear rate, the wind speed of mucus was reduced. The yield stress of simulated asthma airway mucus was 19.0 Pa, but the yield stresses of experimental group (0.03, 0.3 and 0.4 mg/mL) were 17.0, 0.99, and 0.7 Pa, respectively. The results showed that the viscoelastic modulus of asthma airway mucus treated with Fe 2O 3 nanoparticles were changed obviously as measured with large amplitude scanning and frequency scanning. By adopting the method of optical phase microscopy, we found that different structures of simulated airway mucus were absorbed. The results showed Fe 2O 3 nanoparticles distroyed mucus structure. The experimental results proved that Fe 2O 3 nanoparticles could change the rheological characteristics of simulated asthma airway mucus. This experimental result would lay a foundation for the further development of airway mucus sticky agent based on the function of Fe 2O 3 nanoparticles.

A novel peptide ADAM8 inhibitor attenuates bronchial hyperresponsiveness and Th2 cytokine mediated inflammation of murine asthmatic models.

A disintegrin and metalloproteinase 8 (ADAM8) has been identified as a signature gene associated with moderate and severe asthma. Studies in mice have demonstrated that the severity of asthma can be reduced by either transgenic knock-out or by antibodies blocking ADAM8 function, highlighting ADAM8 as potential drug target for asthma therapy. Here, we examined the therapeutic effect of an ADAM8 inhibitor peptide (BK-1361) that specifically blocks cellular ADAM8 activity in ovalbumin-sensitized and challenged Balb/c mice. We found that BK-1361 (25 µg/g body weight) attenuated airway responsiveness to methacholine stimulation by up to 42%, concomitantly reduced tissue remodeling by 50%, and decreased inflammatory cells (e.g. eosinophils down by 54%)/inflammatory factors (e.g. sCD23 down by 50%)/TH2 cytokines (e.g. IL-5 down by 70%)/ADAM8-positive eosinophils (down by 60%) in the lung. We further verified that BK-1361 specifically targets ADAM8 in vivo as the peptide caused significantly reduced levels of soluble CD23 in wild-type but not in ADAM8-deficient mice. These findings suggest that BK-1361 blocks ADAM8-dependent asthma effects in vivo by inhibiting infiltration of eosinophils and TH2 lymphocytes, thus leading to reduction of TH2-mediated inflammation, tissue remodeling and bronchial hyperresponsiveness. Taken together, pharmacological ADAM8 inhibition appears as promising novel therapeutic strategy for the treatment of asthma.