Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. in this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrino-genolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate D-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca2+ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators. (C) 2013 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Inst Butantan, CAT Cepid, Lab Especial Toxinol Aplicada, BR-05503000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, São Paulo, BrazilFAPESP: 98/14307-9FAPESP: 11/08514-8FAPESP: 2011/11308-0Web of Scienc

Electroweak Baryogenesis and Dark Matter with an approximate R-symmetry

It is well known that R-symmetric models dramatically alleviate the SUSY flavor and CP problems. We study particular modifications of existing R-symmetric models which share the solution to the above problems, and have interesting consequences for electroweak baryogenesis and the Dark Matter (DM) content of the universe. In particular, we find that it is naturally possible to have a strongly first-order electroweak phase transition while simultaneously relaxing the tension with EDM experiments. The R-symmetry (and its small breaking) implies that the gauginos (and the neutralino LSP) are pseudo-Dirac fermions, which is relevant for both baryogenesis and DM. The singlet superpartner of the U(1)_Y pseudo-Dirac gaugino plays a prominent role in making the electroweak phase transition strongly first-order. The pseudo-Dirac nature of the LSP allows it to behave similarly to a Dirac particle during freeze-out, but like a Majorana particle for annihilation today and in scattering against nuclei, thus being consistent with current constraints. Assuming a standard cosmology, it is possible to simultaneously have a strongly first-order phase transition conducive to baryogenesis and have the LSP provide the full DM relic abundance, in part of the allowed parameter space. However, other possibilities for DM also exist, which are discussed. It is expected that upcoming direct DM searches as well as neutrino signals from DM annihilation in the Sun will be sensitive to this class of models. Interesting collider and Gravity-wave signals are also briefly discussed.Comment: 50 pages, 10 figure

Tokyo Guidelines 2018 management bundles for acute cholangitis and cholecystitis

Management bundles that define items or procedures strongly recommended in clinical practice have been used in many guidelines in recent years. Application of these bundles facilitates the adaptation of guidelines and helps improve the prognosis of target diseases. In Tokyo Guidelines 2013 (TG13), we proposed management bundles for acute cholangitis and cholecystitis. Here, in Tokyo Guidelines 2018 (TG18), we redefine the management bundles for acute cholangitis and cholecystitis. Critical parts of the bundles in TG18 include the diagnostic process, severity assessment, transfer of patients if necessary, and therapeutic approach at each time point. Observance of these items and procedures should improve the prognosis of acute cholangitis and cholecystitis. Studies are now needed to evaluate the dissemination of these TG18 bundles and their effectiveness. Free full articles and mobile app of TG18 are available at: . Related clinical questions and references are also include

Tokyo Guidelines 2018: initial management of acute biliary infection and flowchart for acute cholangitis

The initial management of patients with suspected acute biliary infection starts with the measurement of vital signs to assess whether or not the situation is urgent. If the case is judged to be urgent, initial medical treatment should be started immediately including respiratory/circulatory management if required, without waiting for a definitive diagnosis. The patient's medical history is then taken; an abdominal examination is performed; blood tests, urinalysis, and diagnostic imaging are carried out; and a diagnosis is made using the diagnostic criteria for cholangitis/cholecystitis. Once the diagnosis has been confirmed, initial medical treatment should be started immediately, severity should be assessed according to the severity grading criteria for acute cholangitis/cholecystitis, and the patient's general status should be evaluated. For mild acute cholangitis, in most cases initial treatment including antibiotics is sufficient, and most patients do not require biliary drainage. However, biliary drainage should be considered if a patient does not respond to initial treatment. For moderate acute cholangitis, early endoscopic or percutaneous transhepatic biliary drainage is indicated. If the underlying etiology requires treatment, this should be provided after the patient's general condition has improved; endoscopic sphincterotomy and subsequent choledocholithotomy may be performed together with biliary drainage. For severe acute cholangitis, appropriate respiratory/circulatory management is required. Biliary drainage should be performed as soon as possible after the patient's general condition has been improved by initial treatment and respiratory/circulatory management. Free full articles and mobile app of TG18 are available at: http://www.jshbps.jp/modules/en/index.php?content_id=47 . Related clinical questions and references are also include

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

International audienceSnake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues

Proteomic and Glycoproteomic Profilings Reveal That Post- translational Modifications of Toxins Contribute to Venom Phenotype in Snakes

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.Fundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Inst Butantan, Ctr Toxins Immune Response & Cell Signaling CeTIC, Lab Especial Toxinol Aplicada, BR-05503000 Sao Paulo, BrazilUniv Fed Sao Paulo ICT UNIFESP, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, BrazilUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, BR-04021001 Diadema, BrazilUniv Fed Sao Paulo ICT UNIFESP, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, BrazilUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, BR-04021001 Diadema, BrazilFAPESP: 2013/07467-1FAPESP: 2013/13548-4Web of Scienc

Echinococcus granulosus Antigen B binds to monocytes and macrophages modulating cell response to inflammation

Background: Antigen B (EgAgB) is an abundant lipoprotein released by the larva of the cestode Echinococcusgranulosus into the host tissues. Its protein moiety belongs to the cestode-specific family known as hydrophobicligand binding protein (HLBP), and is encoded by five gene subfamilies (EgAgB8/1-EgAgB8/5). The functions ofEgAgB in parasite biology remain unclear. It may play a role in the parasite?s lipid metabolism since it carries hostlipids that E. granulosus is unable to synthesise. On the other hand, there is evidence supporting immunomodulatingactivities in EgAgB, particularly on innate immune cells. Both hypothetical functions might involveEgAgB interactions with monocytes and macrophages, which have not been formally analysed yet.Methods: EgAgB binding to monocytes and macrophages was studied by flow cytometry using inflammationrecruitedperitoneal cells and the THP-1 cell line. Involvement of the protein and phospholipid moieties in EgAgBbinding to cells was analysed employing lipid-free recombinant EgAgB subunits and phospholipase D treatedEgAgB(lacking the polar head of phospholipids). Competition binding assays with plasma lipoproteins and ligandsfor lipoprotein receptors were performed to gain information about the putative EgAgB receptor(s) in these cells.Arginase-I induction and PMA/LPS-triggered IL-1β, TNF-α and IL-10 secretion were examined to investigate theoutcome of EgAgB binding on macrophage response.Results: Monocytes and macrophages bound native EgAgB specifically; this binding was also found with lipid-freerEgAgB8/1 and rEgAgB8/3, but not rEgAgB8/2 subunits. EgAgB phospholipase D-treatment, but not thecompetition with phospholipid vesicles, caused a strong inhibition of EgAgB binding activity, suggesting an indirectcontribution of phospholipids to EgAgB-cell interaction. Furthermore, competition binding assays indicated that thisinteraction may involve receptors with affinity for plasma lipoproteins. At functional level, the exposure ofmacrophages to EgAgB induced a very modest arginase-I response and inhibited PMA/LPS-mediated IL-1β andTNF-α secretion in an IL-10-independent manner.Conclusion: EgAgB and, particularly its predominant EgAgB8/1 apolipoprotein, are potential ligands for monocyteand macrophage receptors. These receptors may also be involved in plasma lipoprotein recognition and induce ananti-inflammatory phenotype in macrophages upon recognition of EgAgB.Fil: Silva Álvarez, Valeria. Universidad de la República; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Folle, Ana Maite. Universidad de la República; UruguayFil: Ramos, Ana Lía. Universidad de la República; UruguayFil: Kitano, Eduardo S.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Iwai, Leo K.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Corraliza, Ines. Universidad de Extremadura; EspañaFil: Córsico, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Ferreira, Ana María. Universidad de la República; Urugua