Composição para inativação viral, processo de inativação viral, vacina e processo de produção de vacina

Em 19/05/2016: Anuidade de pedido de patente de invenção no prazo ordinário.DepositadaA presente invenção refere-se a uma composição para inativação viral que compreende solução aquosa de DEPC (dietilpirocarbonato); a referida composição é útil para a inativação do vírus VSV em um processo de inativação viral, que por sua vez é útil para a preparação de vacinas

Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein

© 2013 Freire et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.This work was supported by Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) [PTDC/QUI-BIQ/112929/2009], by the European Union [projects FP7-PEOPLE IRSES (MEMPEPACROSS) and FP7-HEALTH-F3-2008-223414 (LEISHDRUG)], by the Spanish Ministry of Economy and Competitiveness (SAF2011-24899), the Generalitat de Catalunya (2009 SGR 492), by the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnoloógico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the National Institute of Science and Technology in Dengue (INCT-Dengue). JMF also acknowledges FCT-MEC for Ph.D. fellowship SFRH/BD/70423/2010

Mechanisms of vesicular stomatitis virus inactivation by protoporphyrin ix, zinc- protoporphyrin ix, and mesoporphyrin ix

© 2017 American Society for Microbiology. All Rights Reserved.Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen (1O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1O2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ; Brazil; grant number E-26/201.167/2014), the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq; Brazil; grant number 306669/2013-7), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Brazil; grant number CsF 171/2012), the Fundacao para a Ciencia e Tecnologia-Ministério da Educação e Ciência (FCT-MEC; Portugal; project HIVERA/0002/2013), and Marie Skłodowska-Curie Actions (MSCA; European Commission project INPACT 644167). C.C.-O. acknowledges a Science without Borders postdoctoral fellowship from CAPES (171/2012), and J.M.F. acknowledges an FCT-MEC Ph.D. fellowship (SFRH/BD/70423/2010)info:eu-repo/semantics/publishedVersio

The ETNA mission concept: Assessing the habitability of an active ocean world

Enceladus is an icy world with potentially habitable conditions, as suggested by the coincident presence of a subsurface ocean, an active energy source due to water-rock interactions, and the basic chemical ingredients necessary for terrestrial life. Among all ocean worlds in our Solar System, Enceladus is the only active body that provides direct access to its ocean through the ongoing expulsion of subsurface material from erupting plumes. Here we present the Enceladus Touchdown aNalyzing Astrobiology (ETNA) mission, a concept designed during the 2019 Caltech Space Challenge. ETNA’s goals are to determine whether Enceladus provides habitable conditions and what (pre-) biotic signatures characterize Enceladus. ETNA would sample and analyze expelled plume materials at the South Polar Terrain (SPT) during plume fly-throughs and landed operations. An orbiter includes an ultraviolet imaging spectrometer, an optical camera, and radio science and a landed laboratory includes an ion microscope and mass spectrometer suite, temperature sensors, and an optical camera, plus three seismic geophones deployed during landing. The nominal mission timeline is 2 years in the Saturnian system and ∼1 year in Enceladus orbit with landed operations. The detailed exploration of Enceladus’ plumes and SPT would achieve broad and transformational Solar System science related to the building of habitable worlds and the presence of life elsewhere. The nature of such a mission is particularly timely and relevant given the recently released Origins, Worlds, and Life: A Decadal Strategy for Planetary Science and Astrobiology 2023–2032, which includes a priority recommendation for the dedicated exploration of Enceladus and its habitable potential

Mitochondrial Bioenergetic Alterations in Mouse Neuroblastoma Cells Infected with Sindbis Virus: Implications to Viral Replication and Neuronal Death

The metabolic resources crucial for viral replication are provided by the host. Details of the mechanisms by which viruses interact with host metabolism, altering and recruiting high free-energy molecules for their own replication, remain unknown. Sindbis virus, the prototype of and most widespread alphavirus, causes outbreaks of arthritis in humans and serves as a model for the study of the pathogenesis of neurological diseases induced by alphaviruses in mice. In this work, respirometric analysis was used to evaluate the effects of Sindbis virus infection on mitochondrial bioenergetics of a mouse neuroblastoma cell lineage, Neuro 2a. The modulation of mitochondrial functions affected cellular ATP content and this was synchronous with Sindbis virus replication cycle and cell death. At 15 h, irrespective of effects on cell viability, viral replication induced a decrease in oxygen consumption uncoupled to ATP synthesis and a 36% decrease in maximum uncoupled respiration, which led to an increase of 30% in the fraction of oxygen consumption used for ATP synthesis. Decreased proton leak associated to complex I respiration contributed to the apparent improvement of mitochondrial function. Cellular ATP content was not affected by infection. After 24 h, mitochondria dysfunction was clearly observed as maximum uncoupled respiration reduced 65%, along with a decrease in the fraction of oxygen consumption used for ATP synthesis. Suppressed respiration driven by complexes I- and II-related substrates seemed to play a role in mitochondrial dysfunction. Despite the increase in glucose uptake and glycolytic flux, these changes were followed by a 30% decrease in ATP content and neuronal death. Taken together, mitochondrial bioenergetics is modulated during Sindbis virus infection in such a way as to favor ATP synthesis required to support active viral replication. These early changes in metabolism of Neuro 2a cells may form the molecular basis of neuronal dysfunction and Sindbis virus-induced encephalitis

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Gourmet e glutão? Cérebro precisa de 'alimento' constante para manter seu funcionamento normal e esse 'combustível' é fornecido de diferentes formas

Submitted by Sandra Infurna ([email protected]) on 2018-12-11T10:13:15Z No. of bitstreams: 1 maurucio_luz_etal_IOC_2015.pdf.pdf: 179882 bytes, checksum: 9bbb9cb87cb2418d0f51f9300ffb160b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-12-11T10:17:08Z (GMT) No. of bitstreams: 1 maurucio_luz_etal_IOC_2015.pdf.pdf: 179882 bytes, checksum: 9bbb9cb87cb2418d0f51f9300ffb160b (MD5)Made available in DSpace on 2018-12-11T10:17:08Z (GMT). No. of bitstreams: 1 maurucio_luz_etal_IOC_2015.pdf.pdf: 179882 bytes, checksum: 9bbb9cb87cb2418d0f51f9300ffb160b (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.O cérebro representa cerca de 2 % da massa corporal, mas consome em torno de 20% do oxigênio usado pelo corpo humano. Esse alto consumo de oxigênio é necessário para manter o funcionamento cerebral, já que as células do cérebro gastam muita energia no constante transporte de íons através suas membranas, o que garante a transmissão do impulso nervoso. O consumo de energia desse glutão metabólico varia pouco ao longo do dia e se mantém quase inalterado durante o sono. Um gasto tão grande exige uma fonte de energia constante e eficiente. Em condições normais, ou seja, quando se segue uma dieta diversificada, quase toda a energia usada pelos neurônios vem da glicose transportada por meio da circulação sanguínea. Como o cérebro não dispõe de reservas de glicose, até pequenas quedas nas concentrações desse açúcar no sangue podem afetar seriamente seu funcionamento. Assim, além de ser glutão, o cérebro é também um gourmet que 'aceita' somente glicose para seu metabolismo. O aporte constante desse combustível para o cérebro parece uma coisa simples, mas, na verdade, é um desafio, vencido somente graças à ação de vários hormônios que permitem a sincronização do metabolismo de diversos tipos celulares

O ensino de nutrição e metabolismo energético a partir de um jogo de tabuleiro denominado Sobrevivência “Versão 1.0”

O estudo de nutrição e metabolismo energético no ensino médio depende de uma série de conceitos da química orgânica, que somente será trabalhada no último ano pelo professor de Química. A grande dificuldade dos professores de Biologia é introduzir esses conceitos químicos paralelamente a esse conteúdo. Cremos que apenas as aulas expositivas não sejam suficientes para despertar o interesse dos alunos. Partindo dessa premissa, acreditamos que seja muito relevante o desenvolvimento de novos materiais educacionais com o intuito de melhorar a aprendizagem. “Sobrevivência” é um jogo de tabuleiro de caráter investigativo/cooperativo sobre nutrição e metabolismo energético. Usado como uma estratégia lúdica, ele foi desenvolvido como material pedagógico para auxiliar os professores a revisar conceitos sobre o tema