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During differentiation, skeletal muscle develops mature multinucleated muscle fibers, which could contract to exert force on a substrate. Muscle dysfunction occurs progressively in patients with muscular dystrophy, leading to a loss of the ability to walk and eventually to death. The synthetic glucocorticoid dexamethasone (Dex) has been used therapeutically to treat muscular dystrophy by an inhibition of inflammation, followed by slowing muscle degeneration and stabilizing muscle strength. Here, in mice with muscle injury, we found that Dex significantly promotes muscle regeneration via promoting kinesin-1 motor activity. Nevertheless, how Dex promotes myogenesis through kinesin-1 motors remains unclear. We found that Dex directly increases kinesin-1 motor activity, which is required for the expression of a myogenic marker (muscle myosin heavy chain 1/2), and also for the process of myoblast fusion and the formation of polarized myotubes. Upon differentiation, kinesin-1 mediates the recruitment of integrin β1 onto microtubules allowing delivery of the protein into focal adhesions. Integrin β1-mediated focal adhesion signaling then guides myoblast fusion towards a polarized morphology. By imposing geometric constrains via micropatterns, we have proved that cell adhesion is able to rescue the defects caused by kinesin-1 inhibition during the process of myogenesis. These discoveries reveal a mechanism by which Dex is able to promote myogenesis, and lead us towards approaches that are more efficient in improving skeletal muscle regeneration.

Blood vessels are ubiquitous in the human body and play essential roles not only in the delivery of vital oxygen and nutrients but also in many disease implications and drug transportation. Although fabricating in vitro blood vessels has been greatly facilitated through various microfluidic organ-on-chip systems, most platforms that are used in the laboratories suffer from a series of laborious processes ranging from chip fabrication, optimization, and control of physiologic flows in micro-channels. These issues have thus limited the implementation of the technique to broader scientific communities that are not ready to fabricate microfluidic systems in-house. Therefore, we aimed to identify a commercially available microfluidic solution that supports user custom protocol developed for microvasculature-on-a-chip (MVOC). The custom protocol was validated to reliably form a smooth and functional blood vessel using a viscous fingering (VF) technique. Using VF technique, the unpolymerized collagen gel in the media channels was extruded by less viscous fluid through VF passive flow pumping, whereby the fluid volume at the inlet and outlet ports are different. The different diameters of hollow tubes produced by VF technique were carefully investigated by varying the ambient temperature, the pressure of the passive pump, the pre-polymerization time, and the concentration of collagen type I. Subsequently, culturing human umbilical vein endothelial cells inside the hollow structure to form blood vessels validated that the VF-created structure revealed a much greater permeability reduction than the vessel formed without VF patterns, highlighting that a more functional vessel tube can be formed in the proposed methodology. We believe the current protocol is timely and will offer new opportunities in the field of in vitro MVOC.

Cancer is one of the leading causes of death worldwide. There are only limited treatment strategies that can be applied to treat cancer, including surgical resection, chemotherapy, and radiotherapy, but these have only limited effectiveness. Developing a new drug for cancer therapy is protracted, costly, and inefficient. Recently, drug repurposing has become a rising research field to provide new meaning for an old drug. By searching a drug repurposing database ReDO_DB, a brief list of anesthetic/sedative drugs, such as haloperidol, ketamine, lidocaine, midazolam, propofol, and valproic acid, are shown to possess anti-cancer properties. Therefore, in the current review, we will provide a general overview of the anti-cancer mechanisms of these anesthetic/sedative drugs and explore the potential underlying signaling pathways and clinical application of these drugs applied individually or in combination with other anti-cancer agents.

Background Type 2 diabetes mellitus (T2DM) is a major cause of death with an increasing incidence at an epidemic rate. The existing treatments for T2DM lack long-term effective blood glucose control. In this study, the effects of miR-21 antagomir on T2DM and the related mechanism were investigated using streptozotocin (STZ)-induced T2DM rats. Methods 30 T2DM rats were randomly divided into 3 groups (n=10): T2DM group, T2DM rats with miR-21 antagomir group, T2DM rats with NC antagomir group. The expression of miR-21 in rats was detected by qRT-PCR. blood glucose, triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-Cho), insulin, adiponectin, ITT and GTT were detected. The expression of TIMP3 in si-TIMP3 rats and the expression of TIMP3 in T2DM rats with miR-21 antagomir and si-TIMP3 was detected by Western blotting. Results We found that miR-21 antagomir reduced blood glucose concentration in T2DM rats. MiR21 antagomir improved lipid metabolic disorder by decreasing the levels of triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-Cho) and increasing the level of high-density lipoprotein cholesterol (HDL-Cho). Also, miR-21 antagomir reduced the value of homeostasis model assessment of insulin resistance (HOMA-IR), hemoglobin A1c (HbAc1), plasma insulin, and up-regulated the plasma adiponectin. These results, combined with insulin tolerance tests (ITT) and glucose tolerance tests (GTT) results, showed that miR-21 improved insulin resistance in STZ-induced T2DM rats. Then the target relationship between miR-21 and tissue inhibitor of metalloproteinases 3 (TIMP3) was proved by luciferase reporter assay. More impressively, miR-21 significantly increased the expression level of TIMP3 in STZ-induced T2DM rats. Conclusions Our study taken together has shown that miR-21 antagomir improved insulin resistance and lipid metabolism disorder in STZ-induced T2DM rats by up-regulating the expression level of TIMP3. This study suggested that miR-21 antagomir could be used as an effective therapeutic strategy and the underlying mechanism was revealed.

For a number of years, oral cancer has remained in the top ten most common types of cancer, with an incidence rate that is steadily increasing. In total, ~75% oral cancer cases are associated with lifestyle factors, including uncontrolled alcohol consumption, betel and tobacco chewing, and the excessive use of tobacco. Notably, betel chewing is highly associated with oral cancer in Southeast Asia. Arsenic is a key environmental toxicant; however, arsenic trioxide has been used as a medicine for the treatment of acute promyelocytic leukemia, highlighting its anticancer properties. The present study aimed to investigate the role of arsenic compounds in the treatment of cancer, using FaDu oral squamous carcinoma cells treated with sodium arsenite (NaAsO

A better understanding of how streamflow interacts with climate change and human activities would contribute to the efficiency and effectiveness of water resources management. Specifically, quantifying the climate and human contributions has widely been used when attributing streamflow changes. However, only a few previous studies compared the results derived by different methods that are currently available, and even fewer studies have ever had a close look at the uncertainties induced by various estimations of evapotranspiration. This research first examined the streamflow changes for 12 catchments on the Loess Plateau in China during the period of 1961–2018 with Mann–Kendall test and relevant statistical measurements. Then, 8 Budyko-based climate elasticity methods, each with 13 estimations of evapotranspiration, were used to quantifying human and climate contributions to streamflow change in the study area (i.e., 104 pairs of values for human and climate contributions for one catchment). The results showed that significant declining trends could be found in 11 of the 12 catchments studied. In terms of contribution rates, human activity has been shown as the major contributor to the streamflow decrease (60–90%) compared to climate change (10–50%). By comparing the contribution results derived by possible combinations of attribution method and evapotranspiration estimation, the variability due to different Budyko-based methods being used seems to be related to geographical location and climate. Although the spatial pattern of variability due to different estimations of evapotranspiration is not obvious, it is necessary to consider the uncertainties induced when launching contribution analysis over specific regions.

Background: In vitro three-dimensional (3D) hepatic spheroid culture has shown great promise in toxicity testing because it better mimics the cell–cell and cell–matrix interactions found in in vivo conditions than that of the traditional two-dimensional (2D) culture. Despite embedding HepaRG spheroids with collagen type I (collagen I) extracellular matrix (ECM) revealed a much better differentiation capability, almost all the collagen utilized in in vitro hepatocytes cultures is animal-derived collagen that may limit its use in human toxicity testing. Method: Here, a preliminary investigation of HepaRG cells cultured in different dimensionalities and with the addition of ECM was performed. Comparisons of conventional 2D culture with 3D spheroid culture were performed based on their functional or structural differences over 7 days. Rat tail collagen (rtCollagen) I and recombinant human collagen (rhCollagen) I were investigated for their ability in promoting HepaRG spheroid differentiation. Results: An immunofluorescence analysis of the hepatocyte-specific functional protein albumin suggested that HepaRG spheroids demonstrated better hepatic function than spheroids from 2D culture, and the function of HepaRG spheroids improved in a time-dependent manner. The fluorescence intensities per unit area of spheroids formed by 1000 cells on days 7 and 10 were 25.41 and 45.38, respectively, whereas almost undetectable fluorescence was obtained with 2D cells. In addition, the embedding of HepaRG spheroids into rtCollagen and rhCollagen I showed that HepaRG differentiation can be accelerated relative to the differentiation of spheroids grown in suspension, demonstrating the great promise of HepaRG spheroids. Conclusions: The culture conditions established in this study provide a potentially novel alternative for promoting the differentiation of HepaRG spheroids into mature hepatocytes through a collagen-embedded in vitro liver spheroid model. This culture method is envisioned to provide insights for future drug toxicology.

BackgroundArctium lappa L.root (burdock root) has long been recommended for the treatment of different diseases in traditional Chinese medicine. Burdock root possesses anti-oxidative, anti-inflammatory, anti-cancer, and anti-microbial activities. The aim of the study was to elucidate whether aqueous extract of burdock root regulates mesenchymal stem cell proliferation and differentiation.MethodsHuman bone marrow-derived mesenchymal stem cells in 2D high density culture and in 3D micromass pellets were treated with chondrogenic induction medium and chondral basal medium in the absence or presence of aqueous extract of burdock root. The chondrogenic differentiation was accessed by staining glucosaminoglycans, immunostaining SOX9 and type II collagen and immuonblotting of SOX9, aggrecan and type II collagen.ResultsTreatment of aqueous extract of burdock root increased the cell proliferation of hMSCs. It did not have significant effect on osteogenic and adipogenic differentiation, but significantly enhanced chondrogenic induction medium-induced chondrogenesis. The increment was dose dependent, as examined by staining glucosaminoglycans, SOX9, and type II collagen and immunobloting of SOX9, aggrecan and type II collagen in 2D and 3D cultures. In the presence of supplemental materials, burdock root aqueous extract showed equivalent chondrogenic induction capability to that of TGF-β.ConclusionsThe results demonstrate that aqueous extract ofArctium lappa L.root promotes chondrogenic medium-induced chondrogenic differentiation. The aqueous extract of burdock root can even be used alone to stimulate chondrogenic differentiation. The study suggests that the aqueous extract of burdock root can be used as an alternative strategy for treatment purposes.

This empirical research examines the normalized difference vegetation index (NDVI) and the percent impervious surface area (%ISA) as indicators of urban heat island (UHI) effects, using the relationships between land surface temperature (LST), %ISA, and NDVI. Landsat 8 Operational Land Imager and Thermal Infrared Sensor data were used to estimate the LST in Connecticut at different times. A map of the percent impervious surface was generated using the Impervious Surface Analysis Tool developed by the Center for Land Use Education and Research and distributed through the National Oceanic and Atmospheric Administration. Strong linear relationships between LST and %ISA exist, as stated in previous studies, whereas the relationship between LST and NDVI is evidently affected by the seasons. As the patterns of LST in urban areas are influenced by the UHI, the results demonstrate that %ISA is a more reliable indicator of UHI effects than NDVI. Thus, %ISA could be a promising alternative for use in the quantitative analysis of LST when studying UHI effects.

Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high‐density culture performed with TGF‐β‐driven chondrogenic induction medium. Treatment of the Midazolam dose‐dependently inhibited chondrogenesis, examined using Alcian blue‐stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor‐β‐induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam‐induced congenital malformations of the musculoskeletal system through PBR.

Summary The physical environment critically affects cell shape, proliferation, differentiation, and survival by exerting mechanical forces on cells. These forces are sensed and transduced into intracellular signals and responses by cells. A number of different membrane and cytoplasmic proteins have been implicated in sensing mechanical forces, but the picture is far from complete, and the exact transduction pathways remain largely elusive. Furthermore, mechanosensation takes place alongside chemosensation, and cells need to integrate physical and chemical signals to respond appropriately and ensure normal tissue and organ development and function. Here, we report that ovarian cancer G protein coupled receptor 1 (OGR1) (aka GPR68) acts as coincidence detector of membrane stretch and its physiological ligand, extracellular H+. Using fluorescence imaging, substrates of different stiffness, microcontact printing methods, and cell-stretching techniques, we show that OGR1 only responds to extracellular acidification under conditions of membrane stretch and vice versa. The level of OGR1 activity mirrors the extent of membrane stretch and degree of extracellular acidification. Furthermore, actin polymerization in response to membrane stretch is critical for OGR1 activity, and its depolymerization limits how long OGR1 remains responsive following a stretch event, thus providing a “memory” for past stretch. Cells experience changes in membrane stretch and extracellular pH throughout their lifetime. Because OGR1 is a widely expressed receptor, it represents a unique yet widespread mechanism that enables cells to respond dynamically to mechanical and pH changes in their microenvironment by integrating these chemical and physical stimuli at the receptor level.

Mesenchymal stem cells (MSCs) are multipotent cells which can be easily isolated from different adult tissues and expanded in vitro. MSCs can be induced to differentiate into mesoderm lineages such as bone, cartilage, muscles and nonmesoderm lineage tissues such as cardiomyocytes and neurons. The maintenance of MSC self-renewal ability and multi-lineage differentiation potential are tightly associated with their residing microenvironment. Therefore, in this review, we summarized recent discoveries on how these microenvironmental cues, both from chemical and physical factors regulated MSC stemness. Understanding of how MSCs orchestrating these microenvironmental cues may provide us a better knowledge on how to manipulate and improve the in vitro expansion and induction of MSCs into certain lineage for the future tissue engineering and regenerative medicine.

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.

Neovascularization (NV) of the cornea disrupts vision which leads to blindness. Investigation of antiangiogenic, slow-release and biocompatible approaches for treating corneal NV is of great importance. We designed an eye drop formulation containing gelatin/epigallocatechin-3-gallate (EGCG) nanoparticles (NPs) for targeted therapy in corneal NV. Gelatin-EGCG self-assembled NPs with hyaluronic acid (HA) coating on its surface (named GEH) and hyaluronic acid conjugated with arginine-glycine-aspartic acid (RGD) (GEH-RGD) were synthesized. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the antiangiogenic effect of GEH-RGD NPs in vitro. Moreover, a mouse model of chemical corneal cauterization was employed to evaluate the antiangiogenic effects of GEH-RGD NPs in vivo. GEH-RGD NP treatment significantly reduced endothelial cell tube formation and inhibited metalloproteinase (MMP)-2 and MMP-9 activity in HUVECs in vitro. Topical application of GEH-RGD NPs (once daily for a week) significantly attenuated the formation of pathological vessels in the mouse cornea after chemical cauterization. Reduction in both vascular endothelial growth factor (VEGF) and MMP-9 protein in the GEH-RGD NP-treated cauterized corneas was observed. These results confirm the molecular mechanism of the antiangiogenic effect of GEH-RGD NPs in suppressing pathological corneal NV.

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

One of the key features of keloid is its fibroblasts migrating beyond the original wound border. During migration, cells not only undergo molecular changes but also mechanical modulation. This process is led by actin filaments serving as the backbone of intra-cellular force and transduces external mechanical signal via focal adhesion complex into the cell. Here, we focus on determining the mechanical changes of actin filaments and the spatial distribution of forces in response to changing chemical stimulations and during cell migration. Atomic force microscopy and micropost array detector are used to determine and compare the magnitude and distribution of filament elasticity and force generation in fibroblasts and keloid fibroblasts. We found both filament elasticity and force generation show spatial distribution in a polarized and migrating cell. Such spatial distribution is disrupted when mechano-signalling is perturbed by focal adhesion kinase inhibitor and in keloid fibroblasts. The demonstration of keloid pathology at the nanoscale highlights the coupling of cytoskeletal function with physical characters at the subcellular level and provides new research directions for migration-related disease such as keloid.

// Hsi-Hui Lin 1* , Hsiu-Kuan Lin 2* , I-Hsuan Lin 1 , Yu-Wei Chiou 3 , Horn-Wei Chen 1 , Ching-Yi Liu 2 , Hans I-Chen Harn 2 , Wen-Tai Chiu 3 , Yang-Kao Wang 4 , Meng-Ru Shen 5 and Ming-Jer Tang 1,2 1 Department of Physiology, National Cheng Kung University, Tainan, Taiwan 2 Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan 3 Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan 4 Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan, Taiwan 5 Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan * These authors have contributed equally to this work Correspondence to: Ming-Jer Tang, email: // Keywords : matrix stiffness, mechanical phenotype, stiffness sensing, cell stiffness, caveolin-1 Received : February 20, 2015 Accepted : May 02, 2015 Published : May 19, 2015 Abstract The stiffness sensing ability is required to respond to the stiffness of the matrix. Here we determined whether normal cells and cancer cells display distinct mechanical phenotypes. Cancer cells were softer than their normal counterparts, regardless of the type of cancer (breast, bladder, cervix, pancreas, or Ha-Ras V12 -transformed cells). When cultured on matrices of varying stiffness, low stiffness decreased proliferation in normal cells, while cancer cells and transformed cells lost this response. Thus, cancer cells undergo a change in their mechanical phenotype that includes cell softening and loss of stiffness sensing. Caveolin-1, which is suppressed in many tumor cells and in oncogene-transformed cells, regulates the mechanical phenotype. Caveolin-1-upregulated RhoA activity and Y397 FAK phosphorylation directed actin cap formation, which was positively correlated with cell elasticity and stiffness sensing in fibroblasts. Ha-Ras V12 -induced transformation and changes in the mechanical phenotypes were reversed by re-expression of caveolin-1 and mimicked by the suppression of caveolin-1 in normal fibroblasts. This is the first study to describe this novel role for caveolin-1, linking mechanical phenotype to cell transformation. Furthermore, mechanical characteristics may serve as biomarkers for cell transformation.

Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions (FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, β-PIX (PAK-interacting exchange factor-β). In H1299 cells, β-PIX's activity was found not to be down-regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, β-PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of β-PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of β-PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity.

Recently, a novel substrate known as an electrospun polylactic acid (PLLA) microtube array membrane (MTAM) was successfully developed as a cell coculture platform. Structurally, this substrate is made up of one-to-one connected, ultrathin, submicron scale fibers that are arranged in an arrayed formation. Its unique structure confers several key advantages which are beneficial in a cell coculture system. In this study, the interaction between rat fetal neural stem cells (NSC) and astrocytes was examined by comparing the outcome of a typical Transwell-based coculture system and that of an electrospun PLLA MTAM-based coculture system. Compared to tissue culture polystyrene (TCP) and Transwell coculture inserts, a superior cell viability of NSC was observed when cultured in lumens of electrospun PLLA MTAM (with supportive immunostaining images). Reverse transcription polymerase chain reaction revealed a strong interaction between astrocytes and NSC through a higher expression of doublecortin and a lower expression of nestin. These data demonstrate that MTAM is clearly a better coculture platform than the traditional Transwell system.

X-ray computed tomography (CT) is one of the most powerful non-invasive diagnostic techniques nowadays. The iodinated molecules used as CT contrast agents in the clinic have short circulation times in the body, which significantly restrict its applications. Furthermore, some patients are hypersensitive to iodine. So, researchers have made tremendous efforts to improve the property of iodine. Besides, cis-diammineplatinum (II) dichloride (cisplatin), a major chemo agent for cancer treatment, possess higher X-ray attenuation coefficient being a CT contrast agent. The incorporation of cisplatin with an iodinated agent could facilitate the quality of CT images and damage cancer cells simultaneously. To reduce toxicity of a contrast agent, polymer matrix, gelatin, was incorporated for avoiding contact with nontarget cells. In this study, we combined the iodine contrast agent, 1,3-N-bis (2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetamido]-2,4,6-triiodobenzene-1,3-dicarboxamide (iohexol), with cisplatin, and then examined them in a micro CT with different X-ray tube voltages (50 kV, 80 kV, 100 kV) to find optimal scanning conditions for imaging. As expected, iohexol combined with cisplatin enhanced X-ray attenuation and image contrast. The optimal CT image could be acquired at iohexol and cisplatin concentrations of 50 mg/ml and 3 mg/ml, respectively, under 80 kV irradiation. Finally, the iohexol-cisplatin-gelatin solution was then fabricated into nanoparticles of sizes about 240 nm, which may suitable for in vivo delivery.

Objective We evaluated the effect of three-dimensional static and dynamic culture on the proliferation, distribution, and differentiation of rabbit mesenchymal stem cells (MSCs) in a porous scaffold via autograft for bone regeneration. Methods Rabbit MSCs were seeded in a porous hydroxyapatite scaffold (MSCs/scaffold), and then cultured in petri dishes and a bidirectional flow reactor for 4 weeks for osteogenetic induction in vitro . Metabolic assay of the culture medium was carried out every 2 days; glucose, lactic acid, and calcium concentrations in the medium were also examined. Cell distribution in the scaffold was examined histologically. Cultured MSCs/scaffolds were implanted in rabbit condyles for the evaluation of bone regeneration in vivo . Results Histological sections showed that cells cultured in petri dishes grew only around the scaffolds and seldom in the inner part. However, the scaffolds cultured with MSCs in a bioreactor were almost fully occupied by cells. Metabolic results revealed that the average concentration of glucose in the medium was decreased as cells propagated. Glucose consumption was observed in both static and dynamic cultures, but higher lactic acid production was found in the static culture. Calcium ion concentration was reduced significantly in the dynamic culture after the addition of an osteogenetic induction medium, indicating progression of mineralization. The in vivo results showed that about 80% of the defect in the condyles with dynamically cultured MSCs/scaffold implants was filled with new bone tissue, a proportion much higher than that in the petri dish-cultured MSCs/scaffolds, in which only half of the bone regeneration occurred in the cavity. Conclusion This study provides evidence of the effectiveness of dynamic in vitro MSC culture for robust osteogenesis and indicates that it may impart superior potential for bone regeneration in vivo .

Focal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment.

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.

Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles and neurons. Although previous studies have focused intensively on the effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.

Summary Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that triggers the store-operated Ca2+ entry (SOCE). The clinical relevance of STIM1 has been highlighted in breast and cervical cancer, but the molecular mechanism by which STIM1 promotes cancer progression remains unclear. This study explores the regulatory mechanisms by which STIM1-dependent Ca2+ signaling controls cancer cell migration. Three different SOCE inhibitors, SKF96365, 2-APB and YM-58483, significantly inhibited cervical cancer cell migration to a similar extent to that of STIM1 silencing. In contrast, STIM1 overexpression significantly enhanced cervical cancer cell migration. Live cell confocal images and three-dimensional tomograms showed that STIM1 formed aggregates and translocated towards the plasma membranes of migratory cells, and this was accompanied by increasing cytosolic Ca2+ spikes. STIM1 silencing also inhibited the recruitment and association of active focal adhesion kinase (pTyr397-FAK) and talin at focal adhesions, indicating the blockade of force transduction from integrin signaling. Epidermal growth factor-induced phosphorylation of myosin II regulatory light chains was abolished by STIM1 knockdown and SOCE inhibition. Dual immunostaining of activated myosin II (pSer19-MLC) and actin revealed that actomyosin formation depended on STIM1-mediated Ca2+ entry. Most importantly, STIM1 expression levels as well as SOCE activity controlled the generation of cell contractile force, as measured by the microfabricated post-array-detector system. These results highlight the unique role of STIM1-dependent Ca2+ signaling in controlling cell migration by the regulation of actomyosin reorganization in conjunction with enhanced contractile forces.

Background Developing ultrasensitive and selective biological sensors has increased in interest for applications in disease detection, drug discovery, and biomedical diagnostics. This paper reports a CMOS-compatible technique for fabricating a silicon nanowire field-effect transistor. The nanowire sensor was applied to detect a fragment of the cancer-related interleukin-1β (IL-1β) gene, which is characterized as an indicator of breast, colon, lung, oral, head, and neck cancers. Methods We used the advanced integrated circuit (IC) technique to fabricate a back-gated nanowire field-effect transistor sensor. The dimensions of the nanowire were 60 nm in width and 20 μm in length. To enhance the sensitivity of the nanowire sensor, a plasma modification treatment with various parameters was employed on the surface of the device. Results The sensitivity of the silicon nanowire field-effect transistor could be improved after a 1-minute N2O plasma treatment, because the morphology of the detection region is rougher after the N2O plasma treatment. Hence, a more functional linker could be bound to the surface, thereby increasing the probability of DNA immobilization and hybridization. We used a specific sequence of the IL-1β gene to verify the sensitivity enhancement of the nanowire sensor by using plasma treatment. Conclusion The sensitivity and detection limit of the plasma-treated sensor can be extrapolated to 0.12/decade and 2.5fM, respectively. The detection results demonstrate that real-time monitoring of the expression of IL-1β using nanowire sensors could be useful for evaluating cancer treatment.

Many neurodegenerative diseases are accompanied by metabolic disorders. CCL5/RANTES, and its receptor CCR5 are known to contribute to neuronal function as well as to metabolic disorders such as type 2 diabetes mellitus, obesity, atherosclerosis and metabolic changes after HIV infection. Herein, we found that the lack of CCR5 or CCL5 in mice impaired regulation of energy metabolism in hypothalamus. Immunostaining and co-immunoprecipitation revealed the specific expression of CCR5, associated with insulin receptors, in the hypothalamic arcuate nucleus (ARC). Both ex vivo stimulation and in vitro tissue culture studies demonstrated that the activation of insulin, and PI3K-Akt pathways were impaired in CCR5 and CCL5 deficient hypothalamus. The inhibitory phosphorylation of insulin response substrate-1 at Ser302 (IRS-1S302) but not IRS-2, by insulin was markedly increased in CCR5 and CCL5 deficient animals. Elevating CCR5/CCL5 activity induced GLUT4 membrane translocation and reduced phospho-IRS-1S302 through AMPKα-S6 Kinase. Blocking CCR5 using the antagonist, MetCCL5, abolished the de-phosphorylation of IRS-1S302 and insulin signal activation. In addition, intracerebroventricular delivery of MetCCL5 interrupted hypothalamic insulin signaling and elicited peripheral insulin responsiveness and glucose intolerance. Taken together, our data suggest that CCR5 regulates insulin signaling in hypothalamus which contributes to systemic insulin sensitivity and glucose metabolism.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase of collagen, is primarily expressed in epithelial cells. Activation of DDR1 stabilises E-cadherin located on the cell membrane; however, the detailed mechanism of DDR1-stabilised E-cadherin remains unclear. We performed DDR1 knockdown (Sh-DDR1) on Mardin-Darby canine kidney cells to investigate the mechanism of DDR1-stabilised E-cadherin. Sh-DDR1 decreased junctional localisation, increased endocytosis of E-cadherin, and increased physical interactions between E-cadherin and clathrin. Treatment of the dynamin inhibitor Dyngo 4a suppressed Sh-DDR1-induced E-cadherin endocytosis. In addition, the phosphorylation level of Src tyrosine 418 was increased in Sh-DDR1 cell junctions, and inhibition of Src activity decreased Sh-DDR1-induced E-cadherin endocytosis. To characterise the molecular mechanisms, blocking integrin β1 decreased Src activity and E-cadherin junctional localisation in Sh-DDR1 cells. Photoconversion results showed that inhibition of Src activity rescued E-cadherin membrane stability and that inhibition of integrin β1-Src signalling decreased stress fibres and rescued E-cadherin membrane stability in Sh-DDR1 cells. Taken together, DDR1 stabilised membrane localisation of E-cadherin by inhibiting the integrin β1-Src-mediated clathrin-dependent endocytosis pathway.

// Cheng-Te Hsiao 1, 2, * , Hung-Wei Cheng 3, * , Chi-Ming Huang 3 , Hao-Ru Li 3 , Meng-Hsin Ou 3 , Jie-Rong Huang 3 , Kay-Hooi Khoo 1, 2 , Helen Wenshin Yu 4 , Yin-Quan Chen 4 , Yang-Kao Wang 5 , Arthur Chiou 4, 6 and Jean-Cheng Kuo 3, 4, 7 1 Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan 2 Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan 3 Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan 4 Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei 11221, Taiwan 5 Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan 70101, Taiwan 6 Institute of Biophotonics, National Yang-Ming University, Taipei 11221, Taiwan 7 Proteomics Research Center, National Yang-Ming University, Taipei 11221, Taiwan * These authors have contributed equally to this work Correspondence to: Jean-Cheng Kuo, email: jckuo@ym.edu.tw Keywords: fibronectin, glycoproteomics, N-glycans, integrin signals, cell migration Received: November 04, 2016 Accepted: July 18, 2017 Published: August 07, 2017 ABSTRACT Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair.

Keloids are pathological scars characterized by excessive extracellular matrix production that are prone to form in body sites with increased skin tension. Caveolin-1 (CAV1), the principal coat protein of caveolae, has been associated with the regulation of cell mechanics, including cell softening and loss of stiffness sensing ability in NIH3T3 fibroblasts. Although CAV1 is present in low amounts in keloid fibroblasts (KFs), the causal association between CAV1 downregulation and its aberrant responses to mechanical stimuli remain unclear. In this study, atomic force microscopy showed that KFs were softer than normal fibroblasts with a loss of stiffness sensing. The decrease of CAV1 contributed to the hyperactivation of fibrogenesis-associated RUNX2, a transcription factor germane to osteogenesis/chondrogenesis, and increased migratory ability in KFs. Treatment of KFs with trichostatin A (TSA), which increased the acetylation level of histone H3, increased CAV1 and decreased RUNX2 and fibronectin. TSA treatment also resulted in cell stiffening and decreased migratory ability in KFs. Collectively, these results suggest a novel role for CAV1 downregulation in linking the aberrant responsiveness to mechanical stimulation and extracellular matrix accumulation with the progression of keloids, findings that may lead to new developments in the prevention and treatment of keloid scarring.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.

Discoidin domain receptor 1 (DDR1) promotes cell differentiation through the increase of E-cadherin-mediated cell-cell contact. Life cell imaging with E-cadherin conjugated with Eos fluorescence protein showed that DDR1 stabilizes membrane-bound E-cadherin and the inactivation of Cdc42 mediates DDR1-regulated cell adhesion and differentiation., Discoidin domain receptor 1 (DDR1) promotes E-cadherin–mediated adhesion. The underlying mechanism and its significance, however, have not been elucidated. Here we show that DDR1 overexpression augmented, whereas dominant negative mutant (DN-DDR1) or knockdown of DDR1 inhibited E-cadherin localized in cell-cell junctions in epithelial cells. DDR1 changed the localization and abundance of E-cadherin, as well as epithelial plasticity, as manifested by enhancement of microvilli formation and alteration of cytoskeletal organization. DDR1 also reduced protein abundance of mesenchymal markers, whereas DN-DDR1 and sh-DDR1 showed opposite effects. These results suggest that expression of DDR1 increases epithelial plasticity. Expression of DDR1 augmented E-cadherin protein levels by decreasing its degradation rate. Photobleaching and photoconversion of E-cadherin conjugated with Eos fluorescence protein demonstrated that DDR1 increased the stability of E-cadherin on the cell membrane, whereas sh-DDR1 decreased it. Pull-down assay and expression of constitutively active or dominant-negative Cdc42 showed that DDR1 stabilized E-cadherin through inactivation of Cdc42. Altogether, our results show that DDR1 promotes cell-cell adhesion and differentiation through stabilization of E-cadherin, which is mediated by Cdc42 inactivation.

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.

In this study, we describe the development of surface-modified chitosan substrates to examine topographically related Neuro-2a cell behaviors. Different functional groups can be modified on chitosan surfaces to probe Neuro-2a cell morphology. To prepare chitosan substrates with micro/nano-scaled features, we demonstrated an easy-to-handle method that combined photolithography, inductively coupled plasma reactive ion etching, Ag nanoparticle-assisted etching, and solution casting. The results show that Neuro-2a cells preferred to adhere to a flat chitosan surface rather than a nanotextured chitosan surface as evidenced by greater immobilization and differentiation, suggesting that surface topography is crucial for neural patterning. In addition, we developed chitosan substrates with different geometric patterns and flat region depth; this allowed us to re-arrange or re-pattern Neuro-2a cell colonies at desired locations. We found that a polarity-induced micropattern provided the most suitable surface pattern for promoting neural network formation on a chitosan substrate. The cellular polarity of single Neuro-2a cell spreading correlated to a diamond-like geometry and neurite outgrowth was induced from the corners toward the grooves of the structures. This study provide greater insight into neurobiology, including neurotransmitter screening, electrophysiological stimulation platforms, and biomedical engineering.

The p38 MAPKs play important roles in the regulation of balance between cell survival and cell death on the development of various cancers. However, the roles of p38 MAPKs regulating apoptotic effects on Leydig tumor cells remain unclear. In the present study, we showed that cordycepin (3′-deoxyadenosine) selectively induced apoptosis in MA-10 mouse Leydig tumor cells through regulating the p38 MAPK and PI3K/AKT signaling pathways. Cordycepin reduced viability in MA-10, TM4 and NT2/D1 cells, but not cause cell death of primary mouse Leydig cells on moderate concentration. Cordycepin increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of p38 MAPKs activity by SB203580 significantly prevented cordycepin-induced apoptosis in MA-10 cells. Co-treatment with wortmannin or the autophagy inhibitor 3-methyladenine (3-MA) elevated levels of apoptosis in cordycepin-treated MA-10 cells. Moreover, cordycepin activated p53, p21 and TGFß; and downregulated CDK2. The antitumour activity of cordycepin-treated MA-10 cells was significantly distinct in severe combined immunodeficiency (SCID) mice in vivo. These results suggested that cordycein is a highly selective treatment to induce MA-10 cells apoptosis via p38 MAPKs signaling.