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Network automation has evolved into a solution that emphasizes efficiency in all areas. Furthermore, communication and computer networks rely on a platform that provides the necessary technological infrastructure for packet transfer through the Internet using routing protocols. Border Gateway Protocol (BGP) is a standardized gateway protocol that exchanges routing information across autonomous systems (AS) on the Internet. The traditional technique to configure BGP is inefficient compared to the network automation concept. Network automation helps to assist network administrators in automating and verifying the BGP configuration using scripting. This paper implemented network automation using Ansible to configure BGP routing and advanced configuration in the live network environment. This study is focused on automated scripting to configure IP Addresses to the interfaces, BGP routing protocol, configuration backup. Ansible ran the scripting on Network Automation Docker and pushed the configurations to the routers. The network automation controller communicated with other routers via SSH. The findings show that Ansible has successfully deployed the configuration to the routers with no errors. Ansible can help network administrators minimized human mistakes, reduce time-consuming and enable device visibility across the network environment. This study can help network administrators minimized human mistakes, reduce time-consuming and enable device visibility across the network environment. Implementing different types of authentication and hardening process can enhance the network security level for future

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

Introduction High incidence of bleeding events remains a key risk for patients taking anticoagulants, especially those in need of long-term combination therapy with antiplatelet agents. As a consequence, patients may not receive clinically indicated combination antithrombotic therapy. Here, we report on VE-1902, a member of a novel class of precision oral anticoagulants (PROACs) that combines effective anticoagulation with reduced bleeding in preclinical testing. Methods and results Acting through covalent, reversible active-site modification of thrombin similar to a previously described molecule [ 1 ], VE-1902 shows potency and selectivity for thrombin inhibition in human plasma comparable to clinically relevant direct thrombin inhibitors (DTI) such as argatroban and dabigatran (thrombin generation assay ETP EC50 = 1.3 μM compared to 0.36 μM and 0.31 μM for argatroban and dabigatran; >100-fold selectivity against related serine proteases). Unlike the current anticoagulants, VE-1902 does not significantly inhibit thrombin-mediated platelet activation in in vivo models of thrombosis. In the thrombin generation assay, the compound inhibits thrombin formation without significantly delaying the initiation phase of the clotting cascade. These features are possibly responsible for the observed reduced bleeding in tail bleeding and saphenous vein bleeding models. Consistent with this novel pharmacological profile, VE-1902 shows efficacious anticoagulation in several fibrin-driven animal models of thrombosis (arteriovenous shunt, venous stasis thrombosis, and thrombin-induced thromboembolism models), whereas it does not significantly prevent arterial occlusion in the platelet dependent FeCl3 model. Conclusions By leaving platelet activation following vascular injury mostly unaffected, VE-1902, and the PROACs more generally, represent a new generation of precision anticoagulants with reduced bleeding risk.

Introduction: Anticoagulants, while an effective prophylactic for ischemic stroke increase a patient’s risk of major bleeding. There is currently an unmet need for new anticoagulants with the ideal combination of efficacy and low bleeding risk. Here we describe the in vitro and in vivo pharmacology of two development candidates VE-01902 and VE-02851, expected to enter phase I clinical trials in 2018 and 2019 respectively. These compounds belong to a new class of anti-coagulants, the PRecision Oral AntiCoagulants (PROACs), which inhibit fibrinogen cleavage at potencies comparable to the known Direct Thrombin Inhibitors (DTIs) while only weakly inhibiting thrombin induced platelet activation in plasma and whole blood. Methods: Enzyme activity assays by optical methods. Platelet status by expression of CD62P by flow cytometry. In vitro coagulation by Thrombin Generation Assay (TGA). In vivo efficacy and bleeding risk evaluated by rodent models of thrombosis and bleeding time tests. Results and Conclusions: The PROACs are a class of compounds that share a unique mechanism of action: reversible covalent inhibitors of thrombin with slow enzyme kinetics. We examined Thrombin Generation in platelet Poor Plasma (PPP) and Platelet Rich Plasma (PRP) of VE-01902 and VE-02851 and compared it to the known DTIs argatroban and dabigatran. In both plasma samples the PROACs potently inhibit the propagation phase (Endogenous Thrombin Potential (ETP)) while, unlike the known DTIs, only weakly affect the initiation phase of thrombin generation (10 to 20-fold weaker). In the PRP samples the PROACs do not significantly affect platelet activation even while suppressing ETP. To better understand the unique in vitro properties of the PROACs we studied the activity of multiple key factors of the coagulation cascade. Like argatroban and dabigatran, the PROACs protect mice from the effects of thrombin-induced pulmonary embolism and inhibit clot formation in the arteriovenous shunt rat model. However unlike these DTIs, they do not significantly inhibit platelet activation in either of these rodent models. Despite its strong anticoagulant properties, the PROACs show significantly lower bleeding than comparators.

Introduction In recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high. Methods We have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1. Results We demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay. Conclusion Compound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.

The main feature of this paper is to propose the reduction method for the pentagonal fuzzy number using the expected value criterion. In this regard, some theoretical development is proposed for the pentagonal fuzzy number. As an application to the proposed reduction method, a single objective carbon cost integrated solid transportation problem minimizing the transportation cost along with the emission cost is solved with the parameters are as the pentagonal fuzzy number. To validate the proposed reduction method an analysis is outlined with the existing expected value reduction methods (viz. triangular and trapezoidal fuzzy numbers). After defuzzified the pentagonal fuzzy parameters, the equivalent deterministic problem is solved using the LINGO optimization solver. Based on the obtained solution, some important managerial decisions are finalized.

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate β1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways.

The main investigation of the paper is to develop the defuzzification method of type-2 gamma fuzzy variables. For this purpose, we have proposed the deffuzification technique of type-2 gamma fuzzy variables with the help of CV based reduction method. The paper also focuses on the significance to use the type-2 fuzzy variables especially the type-2 gamma fuzzy variables. In this paper, a transportation problem (TP) has been used to perform the proposed defuzzification approach. Finally, some real life data collected from different parts of our state has been used to solve the transportation problem (TP). The proposed deffuzification method has been illustrated with two examples to enrich the method. To solve this model, Lingo 13.0 optimization software has been used. Finally some managerial views are discussed to illustrate the result along with future scope.

The main purpose of the paper is to present the reduction process of type-2 zigzag uncertain variables with an application to a two stage solid transportation problem (STP). The problem under consideration has been described with all its parameters are in type-2 zigzag uncertain variables. To solve the problem, we have used the proposed reduction method which is based on expected value of the type-2 zigzag uncertain variables. Whole the research will describe the superiority to use the uncertain variables in transportation problem. In connection with the real life situation, we have encountered a numerical example with the help of practical data to show the use of our proposed reduction method in two-stage STP. All the soft computations regarding the numerical problem solution are performed in Lingo 13.0 iterative platform. At the last, the overall contribution, iteration and managerial facts have been outlined following the result.

SummaryThe formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to downregulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell-cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)-enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3-enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells.

The asymmetric polarization of cells allows specialized functions to be performed at discrete subcellular locales. Spatiotemporal coordination of polarization between groups of cells allowed the evolution of metazoa. For instance, coordinated apical-basal polarization of epithelial and endothelial cells allows transport of nutrients and metabolites across cell barriers and tissue microenvironments. The defining feature of such tissues is the presence of a central, interconnected luminal network. Although tubular networks are present in seemingly different organ systems, such as the kidney, lung, and blood vessels, common underlying principles govern their formation. Recent studies using in vivo and in vitro models of lumen formation have shed new light on the molecular networks regulating this fundamental process. We here discuss progress in understanding common design principles underpinning de novo lumen formation and expansion.

The Bengal slow loris Nycticebus bengalensis is a nocturnal prosimian that inhabits the forests of northeastern India. As with many nocturnal prosimian species, little is known about its behavior or ecology; even less information is available on its distribution and population status in northeastern India. We conducted a survey of forest patches in the state of Meghalaya, in northeast India, in order to assess its distribution. A secondary aim of our study was to estimate the severity of threats that may affect the long-term survival of the slow loris in Meghalaya. We surveyed sixteen sites in six districts. Slow lorises were seen in only two sites; however, information obtained through secondary sources indicated that they were present in a number of other forest patches across the state. Many of the forests surveyed were severely affected by logging, poaching and forest fires; it is imperative that conservation measures, aimed at protecting existing forest patches, be implemented in order to ensure the long-term survival of the slow loris and other mammals in the state.

International audience; Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes-EGFR-ERK cascade, decreased pIgA-pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA-pIgR transcytosis. Our results reveal a novel Yes-EGFR-ERK-FIP5 signalling network for regulation of pIgA-pIgR transcytosis.

We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy. Citation Format: Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4851.

We present the most comprehensive list of birds for the upper Siang region based on surveys and opportunistic observations from 2010 to 2016. Of the 252 species recorded for this region, we report 66 for the first time, including six globally threatened species. The presence of migratory waterfowl indicates the importance of the Siang Valley as a migratory route. We also emphasise the importance of mixed-use shifting cultivation landscapes outside protected areas in sustaining bird diversity. Further focused surveys in remote parts of this landscape are required to fully understand the biodiversity significance of this region in the face of emerging large-scale threats.

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

Epithelial cells are polarized, with an apical surface facing a lumen or outer surface and a basolateral surface facing other cells and extracellular matrix (ECM). Hallmarks of epithelial carcinogenesis include loss of polarity, as well as uncontrolled proliferation and resistance to apoptosis. Are these features controlled by a common molecular mechanism? The partitioning-defective 3 (PAR3)-PAR6-atypical PKC (aPKC) complex is a master regulator that controls polarization in many animal cells. Here we show that PAR6 is involved in apoptosis by regulating aPKC and glycogen synthase kinase 3beta (GSK-3beta) activity. During epithelial morphogenesis in 3D culture of Madin-Darby canine kidney (MDCK) cells, expression of an N-terminally deleted PAR6 (PAR6DeltaN) leads to a significant increase in caspase-dependent cell death by downregulating aPKC activity. Accordingly, inhibition of aPKC in wild-type (WT) MDCK cells with either a cell-permeable PKCzeta pseudosubstrate or RNAi promotes apoptosis, which suggests that PAR6 regulates apoptosis via an aPKC-mediated pathway. GSK-3beta, a substrate of aPKC, is hyper-activated by expressing PAR6DeltaN. GSK-3beta inhibitors block PAR6DeltaN-induced apoptosis while expression of constitutively active GSK-3beta (S9A) promotes apoptosis, which is rescued by ectopic expression of aPKC. We conclude that a PAR6-aPKC-GSK-3beta mechanism links cell polarity and apoptosis.

The cytoplasmic domain of beta(3) integrin contains tyrosines at positions 747 and 759 in domains that have been implicated in regulation of alpha(v)beta(3) function and that serve as potential substrates for Src family kinases. The phosphorylation level of beta(3) integrin was modulated using a temperature-sensitive v-Src kinase. Increased beta(3) phosphorylation abolished alpha(v)beta(3)- but not alpha(5)beta(1)-mediated adhesion to fibronectin. alpha(v)beta(3)-Mediated cell adhesion was restored by the expression of beta(3) containing Y747F or Y759F mutations but not by wild type beta(3) integrin. Thus, phosphorylation of the cytoplasmic domain of beta(3) is a negative regulator of alpha(v)beta(3)-fibronectin binding strength.

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses. Fil: Tran, Cindy S.. University of California; Estados Unidos Fil: Eran, Yonatan. University of California; Estados Unidos Fil: Ruch, Travis. University of California; Estados Unidos Fil: Bryant, David. University of California; Estados Unidos Fil: Datta, Aniriban. University of California; Estados Unidos Fil: Brakeman, Paul. University of California; Estados Unidos Fil: Kierbel, Arlinet Verónica. Instituto Pasteur de Montevideo; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Wittman, Torsten. University of California; Estados Unidos Fil: Metzger, Ross. University of California; Estados Unidos Fil: Mostov, Keith. University of California; Estados Unidos Fil: Engel, Joanne. University of California; Estados Unidos

Wireless Sensor Network (WSN) is a collection of nodes, which are limited in energy content and interact with each other to complete a job assigned to them. Performance of WSN may degrade because of faulty nodes present in the network. The strategy in the paper discusses about a fault detection technique, which depends upon three components of the node. The strategy exploits a node to the fullest extent possible before declaring it as faulty. Moreover the faulty-node replacement technique replaces a faulty node with a healthy node,based on a regression plain model. Optimal use of sensor node is done by reallocation of jobs of the faulty nodesto the active nodes.

Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.

We have developed a new class of direct thrombin inhibitors (VE-DTIs) that act through reversible covalent enzyme inhibition. These compounds inhibit thrombin with high potency (IC50 < 10 nM) and with better than 100× selectivity against related serine proteases. In rodents, VE-DTIs show dose-dependent efficacy in preventing arterial and venous thromboses respectively in arteriovenous shunt thrombosis and venous stasis thrombosis models. They also prevent thrombin-induced pulmonary embolism in rodents. Despite their strong anticoagulation properties, VE-DTIs are associated with significantly lower bleeding times than other approved anticoagulants dabigatran, argatroban or apixaban in rodent bleeding models (tail bleeding time and saphenous vein bleeding time). We compared the effect of VE-DTIs and the DTIs argatroban and dabigatran on the coagulation cascade. In vitro, VE-DTIs inhibited thrombin-mediated fibrinogen cleavage with similar potency. Inhibition of thrombin/thrombomodulin complex-mediated activation of TAFI and protein C by VE-DTIs were also comparable. In contrast, VE-DTIs were more than 1000-fold less potent in inhibiting thrombin-mediated platelet activation than the other DTIs. Additionally, and also in contrast to argatroban and dabigatran, VE-DTIs protect rodents from the effects of thrombin-induced pulmonary embolism without significantly inhibiting platelet activation. The ability of VE-DTIs to prevent thrombosis without significantly impacting platelet function could lead to a new anticoagulant therapy with lower bleeding risk. Disclosures Sivaraja: Verseon Corporation: Employment, Equity Ownership. Sizikov:Verseon Corporation: Employment, Equity Ownership. Sandoval:Verseon Corporation: Employment, Equity Ownership. Lin:Verseon Corporation: Employment, Equity Ownership. Igoudin:Verseon Corporation: Employment, Equity Ownership. Dash:Verseon Corporation: Employment, Equity Ownership. Xu:Verseon Corporation: Employment, Equity Ownership. Clemens:Verseon Corporation: Employment, Equity Ownership. Estiarte:Verseon Corporation: Employment, Equity Ownership. Shiau:Verseon Corporation: Employment, Equity Ownership. Short:Verseon Corporation: Employment, Equity Ownership. Williams:Verseon Corporation: Employment, Equity Ownership. Datta:Verseon Corporation: Employment, Equity Ownership. Kita:Verseon Corporation: Employment, Equity Ownership.

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.

Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell–cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)- enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3- enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells.

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin–Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects., This work was supported by grants from the Human Frontier Science Program (HFSP-CDA 00011/2009) and Marie Curie (IRG-209382) to F. Martín- Belmonte, National Institutes of Health grants (R01 DK067153 and R01 DK074398) to K. Mostov, and grants from the Ministerio de Ciencia e Innovación to F. Martín-Belmonte (BFU2008-01916) and M.A. Alonso (BFU2006- 01925) and to F. Martín-Belmonte and M.A. Alonso (CONSOLIDER CSD2009-00016). An institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is also acknowledged.

To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation through the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing an interaction between the machineries of vesicular transport and polarization., Supported by a Susan G Komen Foundation Fellowship (D.M.B.), a DOD Breast Cancer Concept Award (A.D.), NIH grants R01DK074398, R01AI25144 and P01AI53194 (K.E.M.), grants of the Human Frontiers Science Program (HFSP-CDA00011/2009), Marie Curie (IRG-209382), MICINN (BFU2008-01916) and (CONSOLIDER CSD2009-00016) to F.M.-B.; and a JAE fellowship (MICINN) to A.E.R.F.

Epithelial cells are characterized by the ability to form sheets of cells that surround fluid-filled lumens. Cells in these sheets exhibit a characteristic subcellular polarity, with an apical pole that faces the lumen and a basolateral pole that is in contact with other cells and the extracellular matrix (ECM). To investigate the signaling events required for polarization and lumen formation, we have taken advantage of the ability of Madin-Darby canine kidney (MDCK) cells to dynamically remodel their polarity in response to changes in ECM cues. When MDCK cells are grown in suspension culture, they form multicellular “inside-out” cysts with apical proteins found on the peripheral surface and basolateral markers on the interior surface. When these inside-out cysts are embedded in ECM, they rapidly reorient their polarity: apical proteins become localized to the inside surface, and basolateral proteins are found on the surface that contacts ECM. Here we have characterized the signaling requirements for these early molecular reorientation events. Specifically, expression of a dominant-negative form of Rac1 (DN-Rac1) blocks the reorientation of polarity. Phosphoinositide 3′-kinase is required for apical membrane protein remodeling from the initial apical membrane surface. Cells expressing DN-Rac1 fail to detectably activate the PI 3-kinase/protein kinase B pathway. Last, we found that atypical protein kinase C (aPKC) is also required for reorientation of polarity, since an inhibitor of atypical PKC blocks reorientation. This effect cannot be overcome by constitutively active Rac1, demonstrating that both Rac1 and atypical PKC are required for reorientation of cellular polarity.