Platelet Secretion and Hemostasis Require Syntaxin-binding Protein STXBP5

Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion

Cognitive Profiles and Education of Female Cyber Defence Operators

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Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

Self-Regulated Learning in Web-Based Environments: Instructional Tools Designed to Facilitate Cognitive Strategy Use, Metacognitive Processing, and Motivational Beliefs

This study investigated strategies teachers can use to improve students' use of self-regulated learning strategies in a Web-based setting. SRL is defined as a learner's intentional efforts to manage and direct complex learning activities and is composed of three primary components including cognitive strategy use, metacognitive processing, and motivational beliefs. These three components are defined relative to note-taking methods (cognitive component), self-monitoring prompts (metacognitive component), and self-efficacy buildng feedback (motivation component). One hundred nineteen students were assigned randomly to one cell in a 2 × 2 × 2 design. Students took notes in a matrix or a free form method from a Web site about educational measurement and either received or did not receive self-monitoring prompts and self-efficacy building feedback. Results indicated note-taking method had the strongest influence on both the amount of information gathered and achievement. Additionally, both academic self-efficacy building feedback and self-monitoring prompts demonstrated modest effects on achievement. Results are discussed relative to SRL theory, classroom application, and Web-based instructional design.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline

Cellubrevin/Vesicle-Associated Membrane Protein-3–Mediated Endocytosis and Trafficking Regulate Platelet Functions

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbβ3, αvβ3), and purinergic receptors (P2Y1, P2Y12), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble N-ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function. Although not required for normal platelet exocytosis or hemostasis, VAMP-3−/− mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Other granule markers were unaffected. Direct experiments, both in vitro and in vivo, showed that loss of VAMP-3 led to a robust defect in uptake/storage of Fg in platelets and cultured megakaryocytes. Uptake of the fluid-phase marker, dextran, was only modestly affected. Time-dependent uptake and endocytic trafficking of Fg and dextran were followed using 3-dimensional–structured illumination microscopy. Dextran uptake was rapid compared with Fg, but both cargoes progressed through Rab4+, Rab11+, and von Willebrand factor (VWF)+ compartments in wild-type platelets in a time-dependent manner. In VAMP-3−/− platelets, the 2 cargoes showed limited colocalization with Rab4, Rab11, or VWF. Loss of VAMP-3 also affected some acute platelet functions, causing enhanced spreading on Fg and fibronectin and faster clot retraction compared with wild-type. In addition, the rate of Janus kinase 2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3−/− platelets. Collectively, our studies show that platelets are capable of a range of endocytosis steps, with VAMP-3 being pivotal in these processes

Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin

Improving PBL in Empowering Meta cognitive Skill of Students

Abstract Objective: Problem-Based Learning (PBL) is a potential constructivist learning strategy that empowers students’ Meta cognitive skill. PBL focuses on problem, involves thinking activity to solve problems, and correlates to cognitive function of students. Methods: The implementation of PBL reveals various benefits, but there are also some weaknesses in this learning strategy. Thus, it is necessary to implement a certain learning strategy that can cover the PBL weaknesses, such as Reading, Questioning, and Answering (RQA) learning strategy. RQA is a new learning strategy developed based on a fact that almost all students do not read the next lecture materials, causing failure of learning strategy planned and finally the students’ comprehension becomes low. RQA is also potential to empower students’ Meta cognitive skill. Findings: The integration of RQA and PBL learning strategy is called PBL-RQA learning strategy. This study was a quasi-experimental study designed to compare the effect of PBL, RQA, and PBL-RQA learning strategies on the students’ Meta cognitive skill of Faculty of Mathematics and Science, State University of Makassar. Application: The results of the study showed that the potency of PBL learning strategy in empowering the students’ Meta cognitive skill has been increased by integrating it to RQA learning strategy. The meta cognitive skill mean score of the students taught by PBL-RQA learning strategy was 21% higher than that of the students taught by PBL and 26.9% higher than that of the students taught by RQA learning strategy. Keywords: Answering, Meta Cognitive Skill, Problem-Based Learning, Questioning, Reading, PBL-RQ