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Mitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+ signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+ oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+ oscillations by increasing the light frequency, intensity, and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy, and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue, but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1), via the activation of Ca2+-dependent kinases, CaMKII, ERK, and CDK1. However, optogenetically engineered Ca2+ signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 at Ser637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins, mitofusin (MFN)-1 and MFN2.Taken together, this study provides an effective and innovative approach to altering Ca2+ signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension.

This letter presents a vertically-stacked photovoltaic (PV)/bypass diode (BPD) module fabricated via localized substrate removal and flip-chip assembly with the aim of improving shadow performance under Gaussian laser illumination. The proposed scheme also prevents the direct illumination of BPDs to eliminate reverse current leakage. The proposed PV/BPD module achieved output voltage (electrical power) of 4.24V (1.6 mW) in a small form factor (4.84 mm2), as well as a linear shadow response with −0.118 mW electrical power loss per shaded PV cell. Under laser misalignment conditions ( $>0.4$ mm shift), the electrical power generated by the PV/BPD module was 2~4 times higher than that of a PV module without BPD. The fact that the PV and BPD chips can both be fabricated using foundry bulk CMOS processes facilitates the integration of the proposed module with sensor circuits and/or (super)capacitors to form a compact self-powered system.

Detached breakwaters are widely used for shore protection. The planforms of tombolos or salients behind structures have also been used to provide a recreational and sustainable coastal environment. In this study, the comprehensive XBeach model was used to numerically simulate the evolution of wave transformation, nearshore current, and morphological changes in tombolo planforms behind detached breakwaters. Given various gap spacings between consecutive breakwaters, the numerical results indicated that both equilibrium bay-shaped shorelines and bottom profiles form in the lee of detached breakwaters after long-term persistent wave action. These equilibrium shorelines and bottom profiles were verified using well-known empirical formulas. Post-wave-action retreat displacement to the initial shoreline was analyzed, and an empirical relationship was proposed for predicting the equilibrium bay-shaped shoreline. By associating the empirical formula with a parabolic bay-shape equation, some actual beaches were evaluated to validate the predictions of equilibrium shorelines behind detached breakwaters. In conclusion, to appropriately plan the layouts of breakwaters, bay-shaped shorelines of tombolo planforms in the lee of detached breakwaters can be predicted at the design stage by using the proposed relationship.

A series-connected, silicon-nanowire-array-decorated, and CMOS-enabled photovoltaic (PV) module is demonstrated for driving a smart dimmable glass. This miniaturized PV module provides a lifted voltage of 10.8 V and an electrical power of 1.25 mW under solar illumination.

We demonstrate vertically-stacked photovoltaic/bypass diode module to generate a lifted output voltage (4.24V) with linear shadow response in electrical power (around 0.2 mW per PV cell) in a compact manner (4.84 mm2). The proposed device configuration allows further integration of sensor circuits and super-capacitors to realize a compact self-powered system.

In recent years, classification techniques have been broadly adopted for a variety of smart manufacturing applications, including system health maintenance, defect detection and diagnosis, etc. However, most classification methods require a large set of training data that are accurately labeled by human experts in a specific domain. Collecting these training data is time-consuming and prohibitively expensive in practical applications. To overcome this challenge, we develop a classification framework using imperfectly labeled data. First, a statistical model is proposed to derive a set of probabilistic labels with consideration of labeling errors. Next, an accurate classifier is trained from these inaccurate labels. As demonstrated by the experimental results of two industrial examples, the proposed framework achieves superior classification accuracy over other conventional approaches.

In the past decade, there have been several disaster earthquakes occurred in Taiwan.From the observed data of the disaster earthquakes, the stations located in the sourcerupture direction have obvious directivity pulses, and the distribution of the earthquakedisaster is related to the peak ground velocity. Therefore, how to use a large and high-dense seismic database to develop a near-real-time detection system on the earthquakerupture directivity, which is a very important task in Taiwan. In this study, we determinethe earthquake rupture directivity using near-field velocity data from 1991 to 2018, whichwere collected under the Taiwan Strong Motion Instrument Program (TSMIP). The usedmethod is mainly constructed in the interpolation of the peak-ground-velocity map andthe directional attenuation regression analysis. Through the analysis of moderate-to-largemagnitude (M L > 5.5) seismic events, the source rupture directivity can be detectedeffectively and quickly by the applied method. The detection results are also comparablewith those from the previous source studies. We also find out a linear relationship betweenthe directivity effect and earthquake magnitude. Since the TSMIP station may providereal-time services in the future, the detection system proposed by this research can quicklyprovide disaster prediction information, which is of great importance for earthquakeemergency response and hazard mitigation.

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FceRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca2+ levels, respectively. Histamine release and Ca2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FceRI and GPCR-mediated Ca2+ increase. FceRI-induced Ca2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca2+ influx enhanced the process. Furthermore, cytosolic Ca2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca2+ signaling in regulating histamine degranulation.

A coupled wave-vegetation simulation is presented for the moving effect of the coastal vegetation on tsunami wave height damping. The problem is idealized by solitary wave propagating on a group of emergent cylinders. The numerical model is based on general Reynolds-averaged Navier-Stokes equations associated with renormalization group turbulent closure model by using volume of fluid technique. The general moving object (GMO) model developed in CFD code Flow-3D is applied to simulate the coupled motion of vegetation with wave dynamically. The damping of wave height and the turbulent kinetic energy dissipation as waves passed over both moving and stationary cylinders are discussed. As comparing with the stationary cylinders, it obtains markedly less wave height damping and turbulent kinetic energy dissipation by the moving cylinders. The result implies that the wave decay by the coastal vegetation might be overestimated if the mangrove vegetation was represented as stationary state.

Background Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca 2 + /calpain-dependent proteolysis and disassembly. Here, we investigated how Ca 2 + influx induces cascades of FA turnover in living cells. Methods Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca 2 + ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover. Results Vinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA. Conclusions These results suggest that Ca 2 + -mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca 2 + -mediated FA turnover. General significance This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca 2 + .

Additional file 4 : Fig. S4. SOCE inhibitors decrease migration ability of IGROV1-WT cells. In vitro (a, b) transwell migration assay and (c, d) wound healing assay were performed to evaluate the effect of SOCE inhibitors (2 μΜ SKF-96365, 10 μΜ YM-58483, 0.1 μM 2-APB) on IGROV1-WT cells. a Representative photomicrographs of cells that penetrated a filter of pore size 8 μm. Scale bars, 200 μm. b Migrated cells were counted in 15 random fields on the lower surface of the filters and expressed as a percentage (%) of SOCE inhibitor pretreated cells compared with DMSO control (Ctrl). c Cells were seeded into silicon inserts with 10% FBS medium. Following cell adhesion, inserts were removed and incubated for 96 h. Phase images were captured every 24 h and wound spaces were analyzed using ImageJ. d Cellular migratory ability is presented as the percentage of wound closure. Each bar represents mean ± SEM from three independent experiments. *: significant difference between cells treated with SOCE inhibitors and DMSO control (Ctrl). *: p

Additional file 2 : Fig. S2. Chemoresistant IGROV1 sublines exhibit high adhesive ability. Cell adhesion assay was performed 30 min after seeding to evaluate the adhesion ability of IGROV1 parental (WT) and chemoresistant cells (CP, SRT). a Nuclear staining by DAPI showed remaining cells after PBS washing. b Quantitative analyses of adherent cells per field. Each bar represents mean ± SEM from at least 300 cells of three independent experiments. *: significant difference between chemoresistant (CP, SRT) and parental (WT) cells. ***: p

Additional file 3 : Fig. S3. Effect of SOCE inhibitors on Ca2+ elevation in IGROV1-WT cells. a Pre-incubation of IGROV1-WT cells with 2 μM fura-2/AM and SOCE inhibitors (2 μΜ SKF-96365, 10 μΜ YM-58483, 0.1 μM 2-APB) at 37 °C for 30 min for cytosolic Ca2+ measurement using a single-cell fluorimeter. Depletion of ER lumen-resident Ca2+ was induced by treating cells in Ca2+-free buffer with 2 μM thapsigargin for 10 min. Representative tracings show the subsequent elevation of Ca2+, indicating that SOCE occurred during the exchange of Ca2+-free buffer to 2 mM Ca2+ buffer for 5 min. The data in representative curves for the measurement of SOCE from three independent experiments. b SOCE-mediated total Ca2+ elevation was calculated from area under the curve. a.u., arbitrary unit. Each bar represents mean ± SEM from at least 120 cells. *: significant difference between cells treated with SOCE inhibitors and DMSO control (Ctrl). ***: p

Additional file 1 : Fig. S1. Chemoresistant IGROV1 sublines exhibit characteristics of epithelial-to-mesenchymal transition (EMT). a Phase contrast images of parental (WT) and chemoresistant (CP, SRT) IGROV1 cells. Scale bars, 100 μm. b Epithelial markers (E-cadherin, ZO-1; blue rectangles) and mesenchymal marker (vimentin, fibronectin, and N-cadherin; red rectangles) were detected using immunoblotting in IGROV1 parental (WT) and chemoresistant (CP, SRT) cells. β-actin served as the internal control.

Shallow S-wave velocities have been widely used for earthquake ground-motion site characterization. Thus, the S-wave velocity structures of Taipei Basin, Taiwan were investigated using array records of microtremors at 15 sites (Huang et al., 2015). In this study, seven velocity structures are added to the database describing Taipei Basin. Validity of S-wave velocity structures are first examined using the 1D Haskell method and well-logging data at the Wuku Sewage Disposal Plant (WK) borehole site. Basically, the synthetic results match well with the observed data at different depths. Based on S-wave velocity structures at 22 sites, theoretical transfer functions at five different formations of the sedimentary basin are calculated. According to these results, predominant frequencies for these formations are estimated. If the S-wave velocity of the Tertiary basement is assumed to be 1000 m/s, the predominant frequencies of the Quaternary sediments are between 0.3 Hz (WUK) and 1.4 Hz (LEL) in Taipei Basin while the depths of sediments between 0 m (i.e. at the edge of the basin) and 616 m (i.e. site WUK) gradually increase from southeast to northwest. Our results show good agreement with available geological and geophysical information.

Objective: Cell migration is an essential process in skin wound healing. Photodynamic therapy (PDT) enhances wound healing by photoactivating a photosensitizer with a specific wavelength of light. Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel expressed in multiple layers of keratinocytes. Recent studies showed that the activation of CFTR-related downstream signaling affects skin wound healing. We examined whether indocyanine green (ICG)-mediated PDT-enhanced cell migration is related to CFTR activation. Approach: The spatial and temporal expression levels of CFTR and proteins involved in focal adhesion, including focal adhesion kinase (FAK) and paxillin, were evaluated during cell migration in vitro and in vivo for wound healing. Results: ICG-PDT-conditioned medium collected from cells exposed to 5 J/cm(2) near-infrared light in the presence of 100 μg/mL ICG activated CFTR and enhanced HaCaT cell migration. The expression of phosphorylated FAK Tyr861 and phosphorylated paxillin in focal adhesions was spatially and temporally regulated in parallel by ICG-PDT-conditioned medium. Curcumin, a nonspecific activator of CFTR, further increased PDT-enhanced cell migration, whereas inhibition of CFTR and FAK delayed cell migration. The involvement of CFTR in ICG-PDT-enhanced skin wound healing was confirmed in a mouse back skin wound model. Innovation: CFTR is a potential new therapeutic target in ICG-PDT to enhance wound healing. Conclusion: ICG-PDT-enhanced cell migration may be related to activation of the CFTR and FAK pathway. Conditioned medium collected from ICG-PDT may be useful for treating patients with chronic skin ulcer by regulating CFTR expression in keratinocytes.

Chemo-photothermal therapy, which exhibits synergistic effects, is more effective than either of the treatments administered alone because of its superior ability to target and destroy cancer cells. An anti-cancer compound (doxorubicin, DOX) was embedded in silica–carbon hollow spheres (SCHSs) using heat and vacuum to integrate multi-therapeutic effects onto one platform and subsequently improve the anti-cancer efficacy. SCHSs were synthesized via a surface activation method and its highly porous surface enhanced the loading content of the desired drug. SCHSs are an infrared photothermal material that can destroy targeted cells by heating under near-infrared (NIR) laser illumination at 808 nm. NIR laser illumination also enhances DOX release from SCHSs to increase the anti-cancer efficiency of DOX–loaded SCHSs (DOX–SCHSs) in both two-dimensional and three-dimensional multicellular tumor spheroid cultures. SCHSs exhibited high heat-generating ability and pH-responsive drug delivery. In conclusion, this study demonstrated that DOX–SCHSs represent a potential tool for chemo-photothermal therapy due to its photothermal effects. Thus, our findings imply that the high cancer cell killing efficiency of DOX–SCHSs induced by NIR illumination can be used for the treatment of tumors.

Obesity is a significant risk factor for various diseases. It is a clinical condition caused by the excessive accumulation of fat, which has a negative impact on human health. Galactin-12 is an adipocyte-expressed protein and possesses adipocyte-inducing activity. We investigated the expression level of candidate proteins involved in galactin-12-mediated adipocyte differentiation pathway. We performed a high-throughput screening assay to monitor galectin-12 promoter activity using 105 traditional Chinese herbs. Corn silk extract and [Formula: see text]-sitosterol reduced the expression of galactin-12 promoter in 3T3-L1 cells. In addition, corn silk extract and [Formula: see text]-sitosterol decreased the level of lipid droplets and downregulated the gene and protein expression level of C/EBP[Formula: see text], C/EBP[Formula: see text], PPAR[Formula: see text], Ap2, and adipsin in 3T3-L1 pre-adipocytes via AKT and ERK1/2 inhibition. In vivo study with the oral administration of corn silk extract and [Formula: see text]-sitosterol in a mouse model showed a significant weight reduction and decrease in adipocytes in several organs such as the liver and adipose tissue. Taken together, corn silk extract and [Formula: see text]-sitosterol may effectively reduce pre-adipocyte differentiation by inhibiting galectin-12 activity and exerting anti-obesity effects. These findings highlight the potential use of corn silk extract and [Formula: see text]-sitosterol as potential candidates for the prevention and treatment of obesity.

Galectin-12 is a member of an animal lectin family with affinity for β-galactosides and containing consensus amino acid sequences. Here, we found that galectin-12 was expressed in macrophages and thus aimed to determine how galectin-12 affects inflammation and macrophage polarization and activation. The ablation of galectin-12 did not affect bone marrow cells to differentiate into macrophages, but reduced phagocytic activity against Escherichia coli and lowered the secretion of nitric oxide. The ablation of galectin-12 also resulted in the polarization of macrophages into the M2 direction, as indicated by increases in the levels of M2 markers, namely, resistin-like β (FIZZ1) and chitinase 3-like 3 (Ym1), as well as a reduction in the expression levels of a number of M1 pro-inflammatory cytokines. We found that the diminished expression of pro-inflammatory cytokines in macrophages resulting from galectin-12 deletion was due to reduced activation of IKKα/β, Akt and ERK, which in turn caused decreased activation of NF-κB and activator protein 1. The activation of STAT3 was much higher in Gal12(-/-) macrophages activated by lipopolysaccharide, which was correlated with higher levels of IL-10. Adipocytes showed higher insulin sensitivity when treated with Gal12(-/-) macrophage-conditioned media than those treated with Gal12(+/+) macrophages. We conclude galectin-12 negatively regulates macrophage polarization into the M2 population, resulting in enhanced inflammatory responses and also in turn causing decreased insulin sensitivity in adipocytes. This has implications in the treatment of a wide spectrum of metabolic disorders.

The hydrodynamic performance of the wave power extraction of an innovative modified breakwater-integrated Oscillating Water Column (OWC) wave energy converter is both numerically and experimentally presented in this study. The modified OWC device comprises a non-conventional perforated wall in front of a typical OWC chamber. The air-water two-fluid model, considering the fluid compressibility involved in FLOW-3D solver, is employed for the numerical simulations of the hydrodynamic characteristics of the OWC devices. The numerical simulations of the hydrodynamic performance of the OWC device are first validated by the experimental measurements with a scaled model. The effects of the geometry of the OWC chamber on the hydrodynamic efficiency are then discussed by numerical simulations using a full-scale OWC device under regular waves. The numerical and experimental results show that the present modified device can form a U-type flow pattern by the oscillating water column, thus developing better performance in extracting pneumatic power, when compared with the typical OWC device which does not have the perforated front wall. The results indicate that the present modified OWC device can not only promote the efficiency of the pneumatic power extraction, but can also reduce the wave force acting on the structure.

Hepatocellular carcinoma (HCC) is one of the most common cancers and causes of death by cancer. Concanavalin A (ConA) lectin can specifically bind to the glycoprotein receptors of HCC, which are produced by the aberrant overexpression of liver cancer cells. ConA was used in the current study to conjugate on silica-carbon hollow spheres (SCHSs) and applied in the thermal ablation therapy of liver cancer cell lines under near-infrared (NIR) laser irradiation. We found that the amount of ConA-SCHS complex binding to hepatoma cells was significantly higher than that seen with normal hepatocytes, based on flow cytometric analysis and confocal imaging. Hepatoma cells incubated with ConA-SCHSs were thus more easily killed by the subsequent irradiation with a NIR laser. The results show that the ConA-SCHS complex may enhance the interaction with highly expressed ConA receptors on hepatoma cells, and thus serve as an effective photothermal therapy agent for liver cancer treatment.

Scope The aim of this study is to investigate the signaling pathways by which allyl isothiocyanate (AITC) reduces adipocyte differentiation and the efficacy of AITC in suppressing galectin-12 levels as a therapeutic for high fat diet (HFD)-induced obesity. Methods and results AITC presents anti-adipogenic effects on 3T3-L1 cells by decreasing lipid droplet accumulation in a dose-dependent manner. AITC suppresses 3T3-L1 differentiation into adipocytes by decreasing galectin-12 expression and by downregulating key adipogenic transcription factors. AITC influences the expression of 3T3-L1 pre-adipocytes by modulating adipokine expression (leptin and resistin) and by regulating the protein kinase B (PKB/Akt)/cAMP response element-binding protein (CREB) pathway. In HFD-fed mice, oral administration of AITC reduces the body weight, accumulation of lipid droplets in the liver, and white adipocyte size. Conclusion In summary, the results indicate that AITC inhibits adipocyte differentiation by suppressing galectin-12 levels in 3T3L1 cells and has antiobesity effects in HFD-fed mice.

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca

Local anesthetics are commonly used for pain relief by regional nerve blocking. In this study, we fabricated solvent-free biodegradable pellets to extend the duration of lidocaine release without any significant local or systemic toxicity levels. To manufacture the pellets, poly[(d,l)-lactide-co-glycolide] (PLGA) was first pre-mixed with lidocaine powder into different ratios. The powder mixture was then compressed with a mold (diameter of 1, 5, 8 or 10 mm) and sintered at 65 °C to form pellets. The in vitro release study showed that the lidocaine/PLGA pellets exhibited a tri-phase release behavior (a burst, a diffusion-controlled release and a degradation-dominated release) and reached completion around day 28. Scanning electron microscope (SEM) photos show that small channels could be found on the surfaces of the pellets on day 2. Furthermore, the polymer matrix swelled and fell apart on day 7, while the pellets became viscous after 10 days of in vitro elution. Perineural administration of the lidocaine/PLGA pellets produced anti-hypersensitivity effects lasting for at least 24 h in rats, significant when compared to the control group (a pure PLGA was pellet administered). In addition, no inflammation was detected within the nerve and in the neighboring muscle by histopathology.

Three-dimensional (3D) cell culture models have become powerful tools because they better simulate the in vivo pathophysiological microenvironment than traditional two-dimensional (2D) monolayer cultures. Tumor cells cultured in a 3D system as multicellular cancer aggregates (MCAs) recapitulate several critical in vivo characteristics that enable the study of biological functions and drug discovery. The microwell, in particular, has emerged as a revolutionary technology in the generation of MCAs as it provides geometrically defined microstructures for culturing size-controlled MCAs amenable for various downstream functional assays. This paper presents a simple and economical microwell fabrication methodology that can be conveniently incorporated into a conventional laboratory setting and used for the discovery of therapeutic interventions for liver cancer. The microwells were 400&ndash, 700 &micro, m in diameter, and hepatic MCAs (Huh-7 cells) were cultured in them for up to 5 days, over which time they grew to 250&ndash, 520 &micro, m with good viability and shape. The integrability of the microwell fabrication with a high-throughput workflow was demonstrated using a standard 96-well plate for proof-of-concept drug screening. The IC50 of doxorubicin was determined to be 9.3 &micro, M under 2D conditions and 42.8 &micro, M under 3D conditions. The application of photothermal treatment was demonstrated by optimizing concanavalin A-FITC conjugated silica-carbon hollow spheres (SCHSs) at a concentration of 500:200 &micro, g/mL after a 2 h incubation to best bind with MCAs. Based on this concentration, which was appropriate for further photothermal treatment, the relative cell viability was assessed through exposure to a 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of using photothermal treatment as a potential therapeutic intervention. The proposed microwell integration is envisioned to serve as a simple in-house technique for the generation of MCAs useful for discovering therapeutic modalities for liver cancer treatment.

Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by CaImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that CaVinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA.These results suggest that CaThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca

GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA-mediated gene silencing. Using lentiviral shRNA-induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA-mimic half-lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA-mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3'-5' exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute-bound miRNA.