Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future

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

Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

Peer reviewe

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Abstract 4335: Low dose 2 deoxy glucose induces ER stress and neuroblastoma cell death

Abstract Introduction. The prognosis for relapsed neuroblastoma (NB) is extremely poor. While standard therapies can produce brief periods of remission, many NB patients will eventually die due to recurrent disease. Therefore novel approaches are urgently needed for the treatment of NB. Cancer cells are characterized by increased rates of aerobic glycolysis, which correlate with increased tumor aggressiveness and a poor prognosis. 2-deoxy-D-glucose (2-DG) is a glucose analogue which blocks glycolysis and has recently been investigated in a phase I trial for advanced solid tumors. Here we examine the potential of 2-DG for neuroblastoma treatment. Methods. The effect of 2-DG was evaluated in a panel of neuroblastoma cell lines: N-Myc amplified: NB1691, SMS-KCNR, SK-N-BE2, and non N-Myc amplified: SK-N-SH, SH-SY5Y, and SVBM15, a newly developed cell line derived from a bone marrow aspirate of a stage IV relapsed NB patient. The effects of 2-DG were evaluated by MTS assay and LDH release. ATP levels were evaluated using an ATP colorimetric assay. Western blot analysis was used to determine protein levels of ER stress proteins and AKT. Results. To determine the effective dose (ED50) of 2-DG, we exposed NB cells to 0.1-8mM 2-DG for 72 hours and measured cell viability. The ED50 was found to be close to 2mM for most cell lines examined; SH-SY5Y: 1.86mM, SK-N-SH: 2.23mM, SVBM15: 2.4mM, SK-N-BE2: 1.87mM, SMSKNR: 1.98mM, and NB1691: 5.44mM. LDH analysis of SH-SY5Y and SK-N-BE2 cells indicated that this effect was primarily due to cell death and not cell cycle inhibition. Inhibiting glycolysis with 2mM 2-DG reduced ATP levels by ∼30% at 24 hours (SK-N-BE2: 70±6.3% and SH-SY5Y: 64±4.4% of non-treated controls). Examination of cell signaling pathways induced by 2-DG in SH-SY5Y and SK-N-BE2 cells revealed a dose-dependent induction of AKT and ER stress associated proteins; GRP94, GRP78, and GADD153. To determine if inhibiting GRP94 or AKT would potentiate 2-DG induced cell death, cells were exposed to 2mM 2-DG with 17-AAG (500nM), an inhibitor of GRP94, or AKT inhibitor × (10uM). Inhibiting the activity of both GRP94 and AKT further reduced viability by an additional ∼50% over 2-DG alone in SH-SY5Y cells (2-DG: 45±1.4%; 17-AAG: 62±1.6%; 2-DG+17-AAG: 25±0.61%; AKT X: 49±1.8%; 2-DG+AKT X: 23±1.93%) and SK-N-BE2 cells (2-DG: 41±3.3%; 17-AAG: 85±2.0%; 2-DG+17-AAG: 24±0.43%; AKT X: 75±7.3%; 2-DG+AKT X: 22±1.2%). Conclusions. These results demonstrate that clinically achievable levels of 2-DG reduce ATP concentrations and induce significant loss of NB cell viability. Furthermore, inhibiting cell survival signaling pathways activated by 2-DG exposure can increase the effectiveness of 2-DG treatment. Targeting NB cell metabolism is a novel approach to NB treatment, and when combined with additional chemotherapeutic agents, may be an effective treatment for NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2011-4335</jats:p

Identification of novel agonists of the integrin CD11b/CD18

We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC50 of 10.5 μM and in vitro selectivity for binding to the recombinant αA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding αA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound

High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique