Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Abstract\ud \ud Background\ud Phospholipases A2 (PLA2s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA2 isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines.\ud \ud \ud Methods\ud The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I.\ud \ud \ud Results\ud It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %).\ud \ud \ud Conclusions\ud These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.The authors would like to thank the financial support provided by the State\ud of São Paulo Research Foundation (FAPESP, grants n. 2010/03243-43 and\ud 2011/23236-4), the Coordination for the Improvement of Higher Education\ud Personnel (CAPES) and the National Council for Scientific and Technological\ud Development (CNPq process n. 476932/2012-2). We are also grateful to\ud Fabiana Rosseto Morais, from FCFRP-USP, for the technical assistance in the\ud flow cytometry analyses. Thanks are also due to the Center for the Study of\ud Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication\ud of this special collection (CNPq process 469660/2014-7)

Beyond Sticks and Stones:Human capital enhancement efforts in response to violent crime in Latin America

Violent crime has been commonplace in Latin America over the past decades. While existing research has made progress in explaining the rationale and outcomes of government coercive strategies against crime, it has overlooked the non-coercive strategies implemented to improve public security. It is argued in this article that political authorities make human capital enhancement efforts to shape actors’ incentives about criminal activity and mitigate crime. Accordingly, it is hypothesised that violent crime increases human capital enhancement efforts, and that the effect of violent crime on human capital enhancement efforts is larger when left-oriented governments are in power because they stress actors’ motivations over windows of opportunities as the main drivers of crime. Support for these hypotheses is found in a sample of Latin American democracies in the period 1990–2007

Comparative analysis and biochemical and functional characterization of two serine proteases isolated from Bothrops pirajai snake venom

As peçonhas de serpentes possuem enzimas proteolíticas como metaloproteases e serinoproteases que afetam o sistema hemostático por diversos mecanismos. O presente trabalho teve como objetivo o isolamento e a caracterização bioquímica e funcional de duas serinoproteases da peçonha de Bothrops pirajai, bem como uma análise comparativa entre estas enzimas. O processo de isolamento foi realizado por três etapas cromatográficas consecutivas (exclusão molecular em Sephacryl S-200 seguida por afinidade em Benzamidine Sepharose e fase reversa em C2/C18), resultando em duas serinoproteases distintas, denominadas BpirSP27 e BpirSP41 de acordo com suas massas moleculares determinadas por espectrometria de massas (27 e 41 kDa, respectivamente). Conforme estimativas por SDS-PAGE, a BpirSP27 mostrou redução de sua massa molecular de 32 kDa para 27 kDa após deglicosilação com PNGase F em condições desnaturantes, enquanto que a BpirSP41 apresentou maior conteúdo de carboidratos N-ligados (~42%), tendo sua massa molecular reduzida de 49 kDa para 28 kDa após deglicosilação. Ambas são enzimas ácidas, sendo o pI da BpirSP27 de aproximadamente 4,7 e o da BpirSP41 de 3,7. As sequências parciais de aminoácidos das serinoproteases apresentaram identidade de 61% entre si, e o alinhamento múltiplo de ambas mostrou alta similaridade com as sequências de outras serinoproteases de peçonhas de serpentes. As enzimas mostraram ações distintas sobre o fibrinogênio bovino, com a BpirSP27 agindo preferencialmente sobre a cadeia Bβ e a BpirSP41 sobre as cadeias Aα e Bβ. As duas serinoproteases também foram capazes de degradar coágulos de fibrina e de sangue in vitro dependendo da dose e do tempo de incubação, com resultados superiores para a BpirSP41. Ambas as enzimas coagularam o plasma humano de forma dose-dependente, sendo que a BpirSP41 apresentou maior potencial coagulante, com dose coagulante mínima (DCM) de ~3,5 µg contra 20 µg para a BpirSP27. A perda da N-glicosilação por tratamento com PNGase F afetou a atividade coagulante das serinoproteases, sugerindo que os carboidratos N-ligados interferem na atividade catalítica destas enzimas. A atividade esterásica sobre o TAME foi avaliada em diferentes pHs e temperaturas, mostrando que as duas enzimas são bastante estáveis e perdem parte de suas atividades somente em pHs extremamente ácidos ou na temperatura extrema de 100°C. Além disso, as serino proteases foram capazes de hidrolisar substratos cromogênicos como o S-2238, específico para enzimas trombina-símile, e outros como S-2222, S-2266 e S-2302, sendo que somente a BpirSP27 agiu sobre o substrato S-2251 para plasmina. Na presença do íon Cu2+ e após tratamento com inibidores específicos de serinoproteases (PMSF, benzamidina, leupeptina e aprotinina), as atividades das duas enzimas foram significativamente reduzidas, sendo a BpirSP41 menos sensível ao PMSF do que a BpirSP27. O ensaio sobre plaquetas lavadas mostrou que a BpirSP27 induziu maior agregação plaquetária em relação à BpirSP41, tanto na presença quanto na ausência do íon cálcio. Os ensaios de inibição da atividade hemolítica promovida pelo sistema complemento mostraram uma maior ação da BpirSP41 sobre as vias clássica/das lectinas em relação à BpirSP27, e ambas apresentaram efeitos inibitórios similares sobre a via alternativa. As serinoproteases apresentaram efeitos discretos sobre processos inflamatórios como a indução de edema e dor, promovendo ainda um pronunciado recrutamento de neutrófilos sem induzir aumento significativo na quantidade total de leucócitos no exsudato inflamatório, o que sugere que estas enzimas devem apresentar um papel menor na inflamação causada pela peçonha de B. pirajai. Os resultados obtidos sugerem que as diferenças encontradas entre as atividades das enzimas BpirSP27 e BpirSP41 devem estar relacionadas principalmente às suas pronunciadas diferenças estruturais, incluindo diferentes sequências de aminoácidos e taxas de glicosilação.Snake venoms present proteolytic enzymes such as metalloproteases and serine proteases, which affect the hemostatic system through several mechanisms. This work aimed at the isolation and biochemical and functional characterization of two serine proteases from Bothrops pirajai snake venom, also providing a comparative analysis between these enzymes. The isolation procedure was performed by three consecutive chromatographic steps (molecular exclusion on Sephacryl S-200 followed by affinity on Benzamidine Sepharose and reverse phase on C2/C18) resulting in two distinct serine proteases, named BpirSP27 and BpirSP41 according to their molecular masses determined by mass spectrometry (27 and 41 kDa, respectively). As estimated by SDS-PAGE, BpirSP27 showed a reduction of its molecular mass from 32 kDa to 27 kDa after deglycosylation with PNGase F under denaturing conditions, while BpirSP41 presented a higher content of N-linked carbohydrates (~42%), having its molecular mass reduced from 49 kDa to 28 kDa after deglycosylation. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41. The partial amino acid sequences of these serine proteases showed 61% identity to each other, and multiple alignments of both revealed high homology with the sequences of other snake venom serine proteases. The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferably on the Bβ chain and BpirSP41 on both Aα and Bβ chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the dose and incubation period, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of ~3.5 µg versus 20 µg for BpirSP27. The loss of N-glycosylation by treatment with PNGase F affected the coagulant activity of B. pirajai serine proteases, suggesting that the N-linked carbohydrates interfere with the catalytic activity of these enzymes. Their esterase activity on TAME was assessed at different pHs and temperatures, showing that both enzymes are quite stable and lose some of their activities only at very acid pH values or at the extreme temperature of 100°C. Additionally, the serine proteases were able to hydrolyze chromogenic substrates including S-2238, specific for thrombin-like enzymes, S-2222, S-2266 and S-2302, with only BpirSP27 acting on the substrate S-2251 for plasmin. In the presence of Cu2+ ion and after treatment with specific serine protease inhibitors (PMSF, benzamidine, leupeptin and aprotinin), the activities of both enzymes were significantly reduced, and BpirSP41 showed to be less sensitive to PMSF than BpirSP27. The assay on washed platelets revealed that BpirSP27 induced greater platelet aggregation compared to BpirSP41 in the presence or absence of calcium ion. Tests of inhibition of the hemolytic activity promoted by the complement system showed a greater action of BpirSP41 on the classical/lectin pathways in comparison to BpirSP27, and both enzymes showed similar inhibitory effects on the alternative pathway. The serine proteases also presented mild effects on inflammatory processes such as the induction of edema and pain, promoting a pronounced neutrophil recruitment without inducing a significant increase in the total number of leukocytes present in the inflammatory exudate, which suggests that these enzymes should play a minor role in the inflammation caused by B. pirajai snake venom. The results suggest that the differences between the activities of BpirSP27 and BpirSP41 should be mainly related to their pronounced structural differences, including different amino acid sequences and rates of glycosylation

"THE DISCIPLINE OF CONFIDENTIAL INFORMATION IN EUROPEAN COMPETITION LAW"

reservedQuesta tesi tratta del concetto di informazione riservata all'interno del diritto europeo della concorrenza, fornendo definizioni tratte principalmente dalla “Direttiva 2016/943/UE”, dalla “Comunicazione della Commissione Europea 2020/C 242/01”, e servendosi degli Articoli 101 e 102 del TFEU. Partendo dall'introduzione, viene presentato il contesto del diritto europeo della concorrenza e viene spiegato il perché dell'importanza delle informazioni riservate nell'ambito trattato. Per quanto riguarda il primo capitolo, si inizia dando una definizione di segreto commerciale, per poi passare ai mezzi leciti e illeciti di acquisizione, utilizzo e divulgazione di suddetti segreti commerciali. Nel secondo capitolo viene trattata la disciplina di tutela delle informazioni riservata davanti alla Commissione Europea, con aggiunta degli argomenti del ring confidentiality e delle data rooms; quindi si inizia spiegando il ruolo delle informazioni riservate nella procedura della Commissione Europea e le modalità di protezione dell'informazione riservata (con limiti e controversie), per poi parlare appunto del ring confidentiality e delle data rooms. Successivamente nel terzo capitolo viene trattata la disciplina di tutela dell'informazione riservata davanti ai giudici nazionali; si inizia con una spiegazione del ruolo dei giudici nazionali nella gestione dei casi di concorrenza europea, con i loro obblighi e le loro responsabilità, parlando anche dei principi di trasparenza e di giustizia, per poi passare alla cooperazione e al coordinamento tra i giudici nazionali e la commissione europea presentando anche casi giuridici rilevanti. Infine, nella conclusione vengono riassunti i principali risultati raggiunti e vengono fatte delle riflessioni finali sulla ricerca svolta.This thesis deals with the concept of confidential information within European competition Law, providing definitions primarily drawn from “Direttiva 2016/943/UE”, from “Comunicazione della Commissione Europea 2020/C 242/01”, and making use of “Articolo 101” and “Articolo 102” of the TFUE. Starting from the introduction, we present the context of European competition law, and we explain the reason for the importance of confidential information in the treated area. As for the first chapter, it begins by providing a definition of trade secrets, followed by legal and illegal means of acquiring, using and disclosing such trade secrets. In the second chapter, we discuss the discipline for protecting confidential information before the European Commission, including topics such as confidentiality rings and data rooms; it begins by explaining the role of confidential information in the European Commission’s procedure and the methods of safeguarding confidential information (with limits and controversies), and then proceeds to discuss confidentiality rings and data rooms. Subsequently, in the third chapter, we address the discipline for protecting confidential information before national judges; it begins with an explanation of the role of national judges in handling European competition cases, along with their obligations and responsibilities, also discussing principles of transparency and justice, to then move on to the cooperation and coordination between national judges and the European Commission, presenting relevant legal cases. Finally, in the conclusion we summarise the main results achieved and we provide final reflections on the research conducted