The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes.

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 µg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

RHOJ controls EMT-associated resistance to chemotherapy.

The resistance of cancer cells to therapy is responsible for the death of most patients with cancer1. Epithelial-to-mesenchymal transition (EMT) has been associated with resistance to therapy in different cancer cells2,3. However, the mechanisms by which EMT mediates resistance to therapy remain poorly understood. Here, using a mouse model of skin squamous cell carcinoma undergoing spontaneous EMT during tumorigenesis, we found that EMT tumour cells are highly resistant to a wide range of anti-cancer therapies both in vivo and in vitro. Using gain and loss of function studies in vitro and in vivo, we found that RHOJ-a small GTPase that is preferentially expressed in EMT cancer cells-controls resistance to therapy. Using genome-wide transcriptomic and proteomic profiling, we found that RHOJ regulates EMT-associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA-damage response, enabling tumour cells to rapidly repair DNA lesions induced by chemotherapy. RHOJ interacts with proteins that regulate nuclear actin, and inhibition of actin polymerization sensitizes EMT tumour cells to chemotherapy-induced cell death in a RHOJ-dependent manner. Together, our study uncovers the role and the mechanisms through which RHOJ acts as a key regulator of EMT-associated resistance to chemotherapy.

Alpha-terthienyl increases filamentous actin of Entamoeba histolytica.

This study aimed to know if alpha terthienyl (α-T) affects E. histolytica viability and to analyze its effect on the actin cytoskeleton. Trophozoites of E. histolytica HM1-IMSS were treated with α-T, then, cell viability and morphology were evaluated using tetrazolium salts and scanning electron microscopy, respectively; while actin filaments (F-actin) were stained with rhodamine-phalloidin, observed by confocal microscopy and quantified by fluorometry. Data showed that α-T inhibited cell viability of trophozoites (IC50, 19.43 µg / mL), affected the cell morphology, and increased the F-actin in a dose-dependent manner. Production of reactive oxygen species and RhoA-GTP levels remained normal in α-T-treated amebas. Two inhibitors that affect the organization of the trophozoites cytoskeleton, one that interacts directly with actin, Cytochalasin D (CD), and one that affects the Rho signaling pathway by inhibiting the downstream effector Rock, Y27632, were tested. Y27632 did not affect the increase of polymerized actin observed with α-T, this compound partially ameliorates the potent disrupting effects of CD on actin filaments. Docking results suggest that α-T could be an antagonist of CD for the same interaction zone in actin, however, more studies are needed to define the action mechanism of this compound.

Structure-activity relationship studies on an antitumor marine macrolide using aplyronine a-swinholide A hybrid.

An aplyronine A-swinholide A hybrid, consisting of the macrolactone part of aplyronine A and the side chain part of swinholide A, was designed, synthesized, and biologically evaluated. This hybrid induced protein-protein interactions between two major cytoskeletal proteins actin and tubulin in the same manner as aplyronine A, and exhibited potent cytotoxicity and actin-depolymerizing activity. The importance of the methoxy group in the N,N,O-trimethylserine ester was clarified by the structure-activity relationship studies of the amino acid moiety by using the hybrid analogs. Furthermore, the comparison of the actin-depolymerizing activities between the side chain analogs of aplyronine A and swinholide A showed that the side chain analog of swinholide A had much weaker actin-depolymerizing activity than that of aplyronine A.

Simple methods for quantifying super-resolved cortical actin.

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or 'corrals', in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by Super Resolved Radial Fluctuations (SRRF), Structured Illumination Microscopy (3D-SIM) and Expansion Microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 µm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus, this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

Bevacizumab and anexelekto inhibitor, TP-0903 inhibits TGF-ß1-induced epithelial-mesenchymal transition of colon cancer cells.

The incidence of colorectal cancer (CRC) is reported to be increasing nowadays, with a large proportion of newly diagnosed CRC patients being affected by metastasis. Epithelial-mesenchymal transition (EMT) is an important event in the development of metastasis of CRC. In this study, we investigated whether the anticancer drug bevacizumab and anexelekto inhibitor, TP-0903, regulate EMT of colon cancer cells induced by transforming growth factor-beta 1 (TGF-ß1). Using quantitative real-time PCR and western blot analysis, we found that bevacizumab and TP-0903 decreased the expression levels of fibronectin, alpha-smooth muscle actin, and vimentin, whereas they restored E-cadherin expression in TGF-ß1-exposed SW480 and HCT116 cells. In addition, we elucidated that bevacizumab and TP-0903 inhibited the migration and invasion of TGF-ß1-exposed colon cancer cells using scratched wound healing, transwell migration, and Matrigel-coated invasion assays. Finally, we discovered that bevacizumab and TP-0903 inactivated the Smad 2/3 signaling pathway in TGF-ß1-exposed SW480 and HCT116 cells. Therefore, we suggest that treatment of bevacizumab and TP-0903 inhibits TGF-ß1-induced EMT of colon cancer cells through inactivation of the Smad 2/3 signaling pathway.

MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway.

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.

Effect of docetaxel on mechanical properties of ovarian cancer cells.

Docetaxel could inhibit the proliferation of tumor cells by targeting microtubules. The extension of cellular microtubules plays an important role in the invasion and metastasis of tumor cells. This paper aims to study the distribution and mechanical properties of cytoskeletal proteins with low concentration of docetaxel. MTT assay was used to detect the minimum drug activity concentration of docetaxel on SKOV-3 cells, fluorescence staining was used to analyze the distribution of cytoskeleton proteins, scanning electron microscopy(SEM) was used to observe the morphology of single cells, and atomic force microscopy(AFM) was used to determine the microstructure and mechanical properties of cells. The results showed that the IC10 of docetaxel was 1 ng/ml. Docetaxel can effectively inhibit the formation of cell pseudopodia, hinder the indirectness between cells, reduce the cell extension area, and make the cells malformed. In addition, when AFM analyzes the effects of drugs on cell microstructure and mechanical properties, the average cell surface roughness and cell height are positively correlated with the concentration of docetaxel. Especially when the concentration was 100 ng/ml, the adhesion decreased by 37.04% and Young's modulus increased by 1.57 times compared with the control group. This may be because docetaxel leads to microtubule remodeling and membrane protein aggregation, which affects cell microstructure and increases cell strength, leading to significant changes in the mechanical properties of ovarian cells. This is of great significance to the study of the formation mechanism of tumor cell invasion and migration activities mediated by actin.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.

Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Homogentisic acid induces cytoskeleton and extracellular matrix alteration in alkaptonuric cartilage.

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease.

N-acetylcysteine maintains penile length and erectile function in bilateral cavernous nerve crush rat model by reducing penile fibrosis.

Penile length shortening and erectile dysfunction are common complications after radical prostatectomy. Various methods have been used to maintain erectile function, but less attention has been paid to preserving penis length. N-acetylcysteine (NAC) has the effect of antioxidation and antifibrotic, which may be beneficial to improve those postoperative complications. This study investigated the effect of NAC on maintaining the penile length and the erectile function after bilateral cavernous nerve crush (BCNC) and its underlying mechanism. Twenty-four male rats were randomly divided into three groups: control group, BCNC group, and BCNC + NAC group. NAC or equal volume of saline was daily administrated by intragastric gavage for 4 weeks. The initial and end penile lengths were measured. Intracavernosal pressure/mean arterial pressure (ICP/MAP) ratio was calculated to assess erectile function. Hematoxylin-eosin staining, Masson's trichrome staining, immunohistochemistry, and Western blot were performed to explore cellular and molecular changes of the penis. Compared to the BCNC group, the penile length, ICP/MAP ratio and smooth muscle/collagen ratio in the BCNC + NAC group were improved significantly (all P < 0.05), and the expressions of endothelial nitric oxide synthase, α-smooth muscle actin, glutathione, and glutathione peroxidase 1 were significantly increased after NAC treated (all P < 0.05), along with the decreased expressions of hypoxia-inducible factor-1α, transforming growth factor-ß1, collagen I, collagen III, collagen IV, malonaldehyde, and lysine oxidase (all P < 0.05). This study demonstrated that NAC could maintain penile length and partly improve erectile function. Possible mechanism is directly and/or indirectly related to antihypoxic and antifibrosis.

Melatonin suppresses Ti-particle-induced inflammatory osteolysis via activation of the Nrf2/Catalase signaling pathway.

Aseptic loosening induced by osteolysis is recognized as a late complication of joint replacement. Osteoclasts stimulated by Titanium (Ti) nanoparticles play a critical role in periprosthetic osteolysis. Emerging evidence indicates that melatonin, a hormone primarily synthesized by the pineal gland, has been shown an inhibitory effect on osteoclast formation. However, it is unclear whether melatonin could suppress Ti-particle-induced osteoclastogenesis and what the underlying mechanisms were involved in. Herein, we aimed to investigate the effect of melatonin on osteoclast differentiation and osteolysis stimulated by Ti particles. Our results showed that the in vitro osteoclastogenesis of mouse bone marrow monocytes (BMMs) stimulated by Ti particles was suppressed by melatonin treatments in a dose-dependent manner. Further experiments revealed that melatonin up-regulated the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and catalase (CAT) at both the mRNA and protein levels. The role of the Nrf2/CAT signaling pathway was confirmed by the fact that silencing the expression of NRF2 by small interfering RNA (siRNA) counteracted the anti-osteolysis effects of melatonin. Furthermore, in vivo intraperitoneal injection of melatonin successfully attenuated periprosthetic osteolysis induced by Ti particles in a murine calvarial model. Our findings demonstrate that melatonin is a promising therapeutic agent for treating periprosthetic osteolysis by inhibiting the Ti-particle-stimulated osteoclastogenesis via activation of the Nrf2/Catalase signaling pathway.

Emodin ameliorates ovalbumin-induced airway remodeling in mice by suppressing airway smooth muscle cells proliferation.

Increased number of airway smooth muscle cells (ASMCs) is a characteristic of airway remodeling in asthma. In this study we investigated whether emodin alleviated airway remodeling in a murine asthma model and reduced the proliferation of ASMCs in vitro. We provided in vivo evidence suggesting that intraperitoneal injection of emodin (20 mg/kg) 1 h prior to OVA challenge apparently alleviated the thickness of airway smooth muscle, the mass of alpha-smooth muscle actin (α-SMA), collagen deposition, epithelial damage, goblet cell hyperplasia, airway inflammation and airway hyperresponsiveness (AHR) in lung tissue. Meanwhile, we found that emodin suppressed the activation of the Akt pathway in lungtissue of allergic mouse models. Additionally, we found that emodin inhibited cellular proliferation and Akt activation in a dose-dependent manner in vitro. Furthermore, LY294002, an inhibitor for PI3K, abrogated serum-induced phosphorylation of Akt, and decreased the proliferation of ASMCs. These findings indicated that emodin alleviated ASMCs proliferation by inhibiting PI3K/Akt pathway in vivo and in vitro, which may provide a potential therapeutic option for airway smooth muscle remodeling in asthma.

VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

Purpose: A previous study reported that vasoactive intestinal peptide (VIP) can regulate the cytoskeleton of Schlemm's canal (SC) endothelium and expand the SC lumen in a rat glaucoma model. In this study, we aimed to investigate the molecular mechanism of VIP on cytoskeleton regulation. Methods: During in vivo experiments in rats, leucine-rich repeat kinase 2 (LRRK2) expression and the ratio of F-actin to G-actin (F-/G-actin) surrounding SC were examined by immunofluorescence after the application of VIP. For in vitro experiments in human umbilical vein endothelial cells, both quantitative PCR (qPCR) and western blotting were performed to evaluate Sp1 and LRRK2 expression after the application of VIP (and Sp1/LRRK2 inhibitor). In addition, the F-/G-actin ratio was examined by both immunofluorescence and western blotting after the application of VIP (and LRRK2 inhibitor). Results: VIP induced increases in the expression of LRRK2 both in vivo and in vitro and the nuclear translocation of Sp1 in vitro. The application of Sp1 inhibitor abolished the increase in LRRK2 expression induced by VIP in vitro. In addition, VIP changed the F-/G-actin ratio, and this effect was abolished by the LRRK2 inhibitor both in vivo and in vitro. Conclusions: VIP increased the expression of LRRK2, and this regulation was due to the nuclear translocation of Sp1. VIP further changed the F-/G-actin ratio and regulated the balance between the stabilization and destabilization of the F-actin architecture. This study elucidates a novel mechanism by which VIP regulates the actin cytoskeleton of SC endothelium via the Sp1-LRRK2 pathway, suggesting a potential novel treatment strategy for glaucoma.

CaMKII is activated in opioid induced conditioned place preference, but αCaMKII Thr286 autophosphorylation is not necessary for its establishment.

Activation of calcium/calmodulin-dependent protein kinase II (CaMKII), particularly its α isoform, is known to be important for neuronal processes central for learning and memory and has also been implicated in the maladaptive learning involved in drug addiction.Thr286 autophosphorylation of αCaMKII has been shown to be indispensable for establishment of cocaine-induced CPP (Easton et al., 2014). To study the contribution of CaMKII in opioid induced conditioned learning, we examined how establishment of conditioned place preference (CPP) induced by 10 or 30 µmol/kg morphine or its active metabolite morphine-6-glucuronide (M6G) affects the levels and Thr286 autophosphorylation of the α- and ß-isoforms of CaMKII, as well as ß-actin levels, in dorsal and ventral striatum and in hippocampus of mice. An acute and a sub-chronic treatment were used as controls. Whereas an acute single administration of morphine or M6G caused increases in CaMKII levels and phosphorylation at Thr286 and ß-actin in striatal areas, CPP induced by these opioids was accompanied primarily by an increase in the protein levels of both CaMKII isoforms and ß-actin in dorsal striatum and hippocampus. Decreases in CaMKII Thr286 phosphorylation were observed in dorsal striatum after the sub-chronic pharmacological treatment. Despite the changes observed in αCaMKII activity in wild type mice, morphine-induced CPP was not affected in αCaMKIIT286A autophosphorylation-deficient mice. These results indicate that opioid-induced CPP is accompanied by activation of α- and ßCaMKII in striatum and hippocampus, but, in opposition to what has been observed with cocaine, αCaMKII autophosphorylation is not essential for establishment of opioid-induced CPP.

Engineering blood outgrowth endothelial cells to optimize endothelial nitric oxide synthase and extracellular matrix production for coating of blood contacting surfaces.

Coverage of blood contacting surfaces by a functional endothelial layer is likely required to induce and maintain homeostasis. Blood outgrowth endothelial cells (BOECs), cultured from human peripheral blood monocytes, are readily available and functional autologous endothelial source that may represent a reasonable alternative to vascular derived cells. Endothelial nitric oxide synthase (eNOS) produces NO, an important factor that regulates homeostasis at the blood-contacting surface. We found that BOECs express markedly lower levels of eNOS protein (34% ± 13%, Western blot) and mRNA (29% ± 17%, qRT-PCR), as well as exhibiting reduced activity (49% ± 18%, Nitrite analysis) when compared to human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells. HUVECs grown on fibronectin, type I collagen, or laminin -coated surfaces exhibited significant reduction of eNOS mRNA and protein expression. However, no decrease in eNOS levels was observed in BOECs. Interestingly BOECs expressed significantly higher Collagen (Col) I compared to HUVECs, and blocking Col I synthesis significantly enhanced eNOS expression in BOECs. Inhibition of ß1 integrin, focal adhesion kinase (FAK), or actin polymerization increased eNOS in both BOECs and HUVECs suggesting involvement of a signaling pathway culminating in stabilization of the cytoskeleton. Finally, we demonstrated that a Rho-associated protein kinases (ROCK) inhibitor, as a disruptor of actin stabilization, enhanced both eNOS expression and bioactivity. Taken together, our findings demonstrate that cell-ECM interactions are fundamental to the regulation of eNOS in BOECs and suggest that disruption of key intracellular pathways (such as ROCK) may be necessary to enhance functional activity of an endothelialized surface. STATEMENT OF SIGNIFICANCE: Development of biocompatible blood-contacting biomaterial surfaces has not been possible to date, leading many investigators to believe that a complete autologous endothelial layer will be necessary. Blood outgrowth endothelial cells (BOECs), cultured from human peripheral blood monocytes, are readily available and functional autologous endothelial source. Endothelial nitric oxide synthase (eNOS) produces NO, an important factor that regulates homeostasis at the blood-contacting surface. In this study, we show that eNOS displays limited expression in cultured BOECs. We further demonstrate that a strong negative regulation of eNOS is mediated by collagen substrates and that treatment with ROCK inhibitor could enhance both eNOS expression and activity in BOECs and help to rapidly establish a functional autologous endothelial layer on cardiovascular biomaterials.

A comparison of microfluidic methods for high-throughput cell deformability measurements.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

Cofilin-1-induced actin reorganization in stored platelets.

BACKGROUND: During platelet storage, there are extensive changes in cytoskeleton and phosphatidylserine exposure. The intrinsic mitochondrial pathway of apoptosis, activated in stored platelets, is a major mediator these changes. Cofilin-1 is an effector of actin reorganization. We examined the effect of cofilin-1 deficiency on cytoskeleton and phosphatidylserine exposure during storage and following activation of apoptosis. METHODS AND RESULTS: We assessed actin filaments by Alexa-647-phalloidin and phosphatidylserine exposure by fluorescein isothiocyanate-lactadherin by fluorescence microscopy. In fresh platelets, actin filaments are distributed in the subcortical region, and they do not express phosphatidylserine in the outer surface. In stored platelets, there is retraction of actin filaments from the subcortical region with increased phosphatidylserine expression. These changes are seen in 20% of platelets of 6 days old and increases further with storage. Treatment with ABT-737, which activates the mitochondrial apoptosis, induces similar cytoskeletal changes in actin filaments with increased phosphatidylserine. Cofilin-1 is activated in stored platelets as well as in ABT-737 treated platelets by dephosphorylation. In cofilin-1 deficient murine platelets actin filaments are abnormal and ABT-737 induces less phosphatidylserine. Despite these changes in vitro, platelet survival of cofilin-1 deficient platelets in mice was not significantly different from their wild-type controls. CONCLUSION: These results show that cofilin-1 plays a role in apoptosis-induced actin rearrangement and phosphatidylserine exposure during storage. Despite the defects in platelet cytoskeleton and phosphatidylserine exposure in cofilin-1-deficient platelets, the in vivo life span of platelets is similar to littermate controls, indicating multiple redundant pathways for the clearance of platelets in vivo.

Effect of substrate topography on the regulation of human corneal stromal cells.

Optimal functionality of native corneal stroma depends on a well-ordered arrangement of extracellular matrix (ECM). To develop an in vitro corneal model, replication of the corneal in vivo microenvironment is needed. In this study, the impact of topographic cues on keratocyte phenotype is reported. Photolithography and polymer moulding were used to fabricate microgrooves on polydimethylsiloxane (PDMS) 2-2.5 µm deep and 5 µm, 10 µm, or 20 µm in width. Microgrooves constrained the cells body, compressed nuclei and led to cytoskeletal reorganization. It also influenced the concentration of actin filaments, condensation of chromatin and cell proliferation. Cells became more spread and actin filament concentration decreased as the microgroove width increased. Relationships were also demonstrated between microgroove width and cellular processes such as adhesion, migration and gene expression. Immunocytochemistry and gene expression (RT-PCR) analysis showed that microgroove width upregulated keratocyte specific genes. A microgroove with 5 µm width led to a pronounced alignment of cells along the edges of the microchannels and better supported cell polarization and migration compared with other microgroove widths or planar substrates. These findings provide important fundamental knowledge that could serve as a basis for better-controlled tissue growth and cell-engineering applications for corneal stroma regeneration through topographical patterns.

Blocking TG2 attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting EMT.

BACKGROUND: Epithelial-mesenchymal transformation (EMT) is a central mechanism for the occurrence and development of pulmonary fibrosis. Therefore, to identify the key target molecules regulating the EMT process is considered as an important direction for the prevention and treatment of pulmonary fibrosis. Transglutaminase 2 (TG2) has been recently found to play an important role in the regulation of inflammation and the generation of extracellular matrix. Here, our study focuses on the roles of TG2 in pulmonary fibrosis and EMT. METHODS: at first, the expression of TG2 and the EMT-related markers like E-cadherin, Vimentin, and α-SMA were detected with Western Blotting, immunohistochemistry and other methods in the mice with pulmonary fibrosis induced by bleomycin. Further, MLE 12 cells were used to study the effects on EMT of the inhibition of TG2 in vitro. Finally, GK921, an inhibitor against TG2, was used to show its function in both prevention and treatment of pulmonary fibrosis induced by bleomycin in mice. RESULTS: bleomycin succeeded to induce pulmonary fibrosis in mice, with increased TG2 expression, EMT and Akt activation. Knock-down of TG2 by siRNA technique in MLE 12 cell (a mouse alveolar epithelial cell line) and GK921 (an inhibitor of TG2) all inhibited the EMT process, however SC79, an activator of Akt rescued above inhibition. Finally, GK921 alleviated pulmonary fibrosis in mice induced by bleomycin. CONCLUSION: Blocking TG2 reduces bleomycin-induced pulmonary fibrosis in mice via inhibiting EMT.