Podcasts about dsrna

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.531699v1?rss=1 Authors: Hosseini, A., Lindholm, H. T., Chen, R., Mehdipour, P., Marhon, S. A., Ishak, C. A., De Carvalho, D. D. Abstract: Viral mimicry describes the immune response induced by endogenous stimuli such as dsRNA formed by endogenous retroelements. Activation of viral mimicry has the potential to kill cancer cells or augment anti-tumor immune response. Paradoxically, cancer cells frequently present a dysregulated epigenome, leading to increased expression of retroelements. We previously found that ADAR1 p150 upregulation is an adaptation mechanism to tolerate high retroelement-derived dsRNA levels, leading to a druggable dependency. Here, we systematically identified novel mechanisms of viral mimicry adaptation associated with cancer cell dependencies. We correlated the gene knockout sensitivity from the DepMap dataset and interferon stimulated gene (ISG) expression in the Cancer Cell Line Encyclopedia (CCLE) dataset of 1005 human cell lines and identified pathways such as RNA modification and nucleic acid metabolism. Among the top hits was the RNA decay protein XRN1 as an essential gene for the survival of a subset of cancer cell lines. XRN1-sensitive cancer cell lines have a high level of cytosolic dsRNA and high ISG expression. Furthermore, sensitivity to XRN1 knockout was mediated by MAVS and PKR activation, indicating that the cells die due to XRN1-dependent induction of viral mimicry. XRN1-resistant cell lines had low basal dsRNA levels, but became synthetically dependent on XRN1 upon treatment with viral mimicry inducing drugs such as 5-AZA-CdR or palbociclib. Finally, XRN1-dependency is partly independent of ADAR1 activity. These results confirm the potential for our ISG correlation analysis to discover novel regulators of viral mimicry and show that XRN1 activation is an adaptive mechanism to control high dsRNA stress induced by dysregulated retroelements in cancer cells and creates a dependency that can be explored for novel cancer therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.11.532206v1?rss=1 Authors: Fletcher, S. J., Tome-Poderti, L., Mongelli, V., Frangeul, L., Blanc, H., Verdier, Y., Vinh, J., Saleh, M.-C. Abstract: The siRNA pathway is the primary antiviral defense mechanism in invertebrates and plants. The systemic nature of this defense mechanism is one of its more fascinating characteristics and the recognition and transport of double-stranded RNA (dsRNA) of viral origin is required for the systemic activity of the siRNA pathway. Indeed, cellular internalization of dsRNA from the environment is a widespread phenomenon among insects. Here we aimed to identify cell surface proteins that bind to extracellular dsRNA and mediate its internalization. To this end, we developed a novel co-immunoprecipitation protocol that we followed with proteomics analysis. Among the hits from our screens was Hsc70-4, a constitutively expressed member of the heat shock protein family that has been implicated in clathrin-mediated endocytosis. We found that silencing Hsc70-4 impaired dsRNA internalization. Surprisingly, despite lacking a predicted transmembrane domain, Hsc70-4 localizes to the cell membrane and this localization was preserved when Hsc70-4 was expressed in mammalian cells, suggesting a conserved role at the cell surface. Furthermore, Hsc70-4 shows a previously undescribed dsRNA-specific binding capacity. Our results show that Hsc70-4 is a key element of the dsRNA internalization process and its detailed study may facilitate the development of RNA interference (RNAi)-based technologies for pest and vector borne disease control. ImportanceTo protect plants from pathogens or pests, the technology of "Host-induced gene silencing" has emerged as a powerful alternative to chemical treatments. This is an RNAi-based technology where small RNAs made in the plant silence the genes of the pests or pathogens that attack the plant. The small RNAs are generally derived from dsRNA expressed in transgenic plants. Alternatively, dsRNA can be sprayed onto the plant surface, where it can be taken up into the plant or ingested by pests. We have identified a cell surface protein that mediates the early steps of extracellular dsRNA internalization in insect cells. This could facilitate the development of new strategies for pest management. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.08.519618v1?rss=1 Authors: Knebel, E., Peleg, S., Dai, C., Cohen-Fultheim, R., Glaser, B., Levanon, E., Powers, A., Klochendler, A., Dor, Y. Abstract: A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response; however, a causal virus has not been identified. Here we use a mouse model, corroborated with human data, to demonstrate that endogenous dsRNA in beta-cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme ADAR in beta-cells triggers a massive interferon response, islet inflammation and beta-cell failure, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta-cell workload. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Hoje, um novo mundo encontra-se em testes nos laboratórios dos cientistas: e este mundo novo, que aliás não é tão novo assim, vamos combinar…. virá para aperfeiçoar o atual mundo, um tanto desajustado.  Cientista é um eterno inconformado, sempre persegue inovações - se assim não for não é cientista! E o mundo é movido pelo inconformismo da Ciência. Vamos ilustrar este ponto com tendências e pesquisas que se encontram em andamento neste momento, e que podem e irão impactar fortemente nossa agricultura, na próxima década. Bom aqui vou dar um pitaco sobre esta evolução, trazendo um texto bastante simplificado sobre o RNA mensageiro, de autoria do Décio Luiz Gazzoni, pesquisador da Embrapa.Todos os seres vivos possuem um código herdado dos progenitores, que são os genes localizados no núcleo de uma célula.  Os genes são compostos pelo DNA, e contêm instruções para a síntese de compostos químicos fundamentais para a vida - uma espécie de livro de receitas.  Além do DNA, existe uma estrutura chamada RNA mensageiro (mRNA), a qual gera uma cópia perfeita de um determinado gene, sendo sua função supervisionar a síntese das proteínas ligadas àquele gene. O mRNA é responsável por transferir o código de um gene do DNA para uma parte da célula chamada ribossomo, que podemos chamar de "fábrica de proteínas". Uma vez executada a tarefa, ou seja, depois que a proteína foi sintetizada, o mRNA cumpriu sua função e não é mais necessário. Surge, então, outro tipo de RNA, chamado de microRNA, que desliga ou altera o processo de produção de proteínas, degradando o RNA mensageiro. Se isto não ocorresse, a célula continuaria produzindo indefinidamente uma determinada proteína, provavelmente levando o organismo à morte. Bingo! E que tal transformar esta ameaça em oportunidade? Pois bem, para falarmos agora de cunho essencialmente prático, será necessário desenvolvermos métodos adequados para seu uso no campo, de forma extensiva, de maneira a controlar as pragas praticamente sem impacto sobre o ambiente ou à saude humana. Para isso trouxemos uma “fera” no assunto… Hugo Bruno Correa Molinari, atuou como pesquisador da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) durante 15 anos e atualmente é Diretor de P&D e Inovação na SEMPRE AgTech Agrônomo de formação, Mestre em Genética e Biologia Molecular, e Doutor em Agronomia.Em 2020 foi nomeado como membro titular da CTNBio, na vaga de Especialista em Biotecnologia indicado pelo Ministério da Agricultura, Pecuária e Abastecimento.  Na Embrapa foi Presidente do Portfólio de Biotecnologia Avançada aplicada ao Agronegócio durante 7 anos e coordenou projetos em edição genômica envolvendo as culturas de cana-de-açúcar, milho, feijão e soja e desenvolvimento de soluções via dsRNA de uso tópico para controle de ervas daninhas, insetos-praga e nematóides nas culturas de algodão, milho e soja.  Foi responsável pelo desenvolvimento das primeiras canas-de-açúcar editadas GM-free do mundo para produção de etanol de segunda geração e aumento na produção de açúcares. https://linktr.ee/academiadoagrohttps://podfollow.com/academia-do-agro ................Para assinar e ouvir o podcast:Spotify: https://spoti.fi/33WG1dwDeezer: https://bit.ly/2VOuubAApple: https://apple.co/3oyQzaxGoogle: https://bit.ly/3lWvpS7Amazon: https://bit.ly/34xCWReYouTube: https://bit.ly/3mZop7p................Participe da Academia: https://t.me/ADA_AcademiaDoAgro Links Citados no Episódiohttps://www.abms.org.brhttps://www.embrapa.br/xxxiii-congresso-nacional-de-milho-e-sorgo-debate-oportunidades-e-ameacas-para-commodities-agricolashttps://maissoja.com.br/interferencia-por-rna-rnai-para-o-controle-de-pragas-e-doencas/https://www.insper.edu.br/agenda-de-eventos/o-epicentro-da-informacao-genetica-e-as-perspectivas-de-uso-do-rna-mensageiro/https://agriculturasustentavel.org.br/artigo/como-surge-uma-inovacaohttps://www.paranacooperativo.coop.br/ppc/index.php/sistema-ocepar/comunicacao/2011-12-07-11-06-29/ ------------------------------------ Perfil do Entrevistado Hugo Bruno Correa MolinariHugo Bruno Correa Molinari, PhDR&D Director - SEMPRE Agtech INTERAJA COM Hugo Bruno Correa Molinari Email: hm@sempre.agr.br Linkedin: www.linkedin.com/in/hugomolinari Celular: (61) ‪9 83018656 Instagram: @sempre_agtech Twitter: @hbmolinariINTERAJA COM  ACADEMIA DO AGRO LinkedIn: https://www.linkedin.com/in/waldir.franzini Instagram:- www.instagram.com/academiadoagro Twitter: @academiadoagro Telegram: @academiadoagro Facebook: /academiadoagro Celular-Whatsapp: 062 9 9700 7049 E-mail: podcast.academiadoagro@gmail.com ACOMPANHE A REDE AGROCAST https://www.redeagrocast.com.br/ Somos da Agrocast. A primeira rede de podcasts do agronegócio brasileiro e tem o objetivo de aumentar o consumo da mídia pelo setor, bem como estimular a criação de novos podcasts do agro. Rede Agrocast: @redeagrocast Academia do Agro: @academiadoagro Agro Resenha: @agroresenha Bendito Agro: @benditoagro Bug Bites: @bugbitespodcast Cachaça, Prosa & Viola :@cpvpodcast Esalqast: @esalqast Mundo Agro Podcast: @mundoagropodcast Notícias do Front: @noticias_do_front Papo Agro: @papoagropodcast Rumen Cast: @rumencast Apoio: W. Franzini - Gestão do Agronegócio ME Ficha Técnica: Produção: Waldir Franzini Edição/masterização - A Fabrica de Podcast - https://www.afabricadepodcast.com.br/ Musica: CC BY  - CC BY SA http://creativecommons.org/licenses/by/3.0/us/ Se você tem alguma sugestão de pauta, reclamação ou dúvida envie um e-mail para podcast.academiadoagro@gmail.com  | waldir.franzini@gmail.com | Whatsapp: 062 99700-7049

Hoje, um novo mundo encontra-se em testes nos laboratórios dos cientistas: e este mundo novo, que aliás não é tão novo assim, vamos combinar…. virá para aperfeiçoar o atual mundo, um tanto desajustado.  Cientista é um eterno inconformado, sempre persegue inovações - se assim não for não é cientista! E o mundo é movido pelo inconformismo da Ciência. Vamos ilustrar este ponto com tendências e pesquisas que se encontram em andamento neste momento, e que podem e irão impactar fortemente nossa agricultura, na próxima década. Bom aqui vou dar um pitaco sobre esta evolução, trazendo um texto bastante simplificado sobre o RNA mensageiro, de autoria do Décio Luiz Gazzoni, pesquisador da Embrapa.Todos os seres vivos possuem um código herdado dos progenitores, que são os genes localizados no núcleo de uma célula.  Os genes são compostos pelo DNA, e contêm instruções para a síntese de compostos químicos fundamentais para a vida - uma espécie de livro de receitas.  Além do DNA, existe uma estrutura chamada RNA mensageiro (mRNA), a qual gera uma cópia perfeita de um determinado gene, sendo sua função supervisionar a síntese das proteínas ligadas àquele gene. O mRNA é responsável por transferir o código de um gene do DNA para uma parte da célula chamada ribossomo, que podemos chamar de "fábrica de proteínas". Uma vez executada a tarefa, ou seja, depois que a proteína foi sintetizada, o mRNA cumpriu sua função e não é mais necessário. Surge, então, outro tipo de RNA, chamado de microRNA, que desliga ou altera o processo de produção de proteínas, degradando o RNA mensageiro. Se isto não ocorresse, a célula continuaria produzindo indefinidamente uma determinada proteína, provavelmente levando o organismo à morte. Bingo! E que tal transformar esta ameaça em oportunidade? Pois bem, para falarmos agora de cunho essencialmente prático, será necessário desenvolvermos métodos adequados para seu uso no campo, de forma extensiva, de maneira a controlar as pragas praticamente sem impacto sobre o ambiente ou à saude humana. Para isso trouxemos uma “fera” no assunto… Hugo Bruno Correa Molinari, atuou como pesquisador da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) durante 15 anos e atualmente é Diretor de P&D e Inovação na SEMPRE AgTech Agrônomo de formação, Mestre em Genética e Biologia Molecular, e Doutor em Agronomia.Em 2020 foi nomeado como membro titular da CTNBio, na vaga de Especialista em Biotecnologia indicado pelo Ministério da Agricultura, Pecuária e Abastecimento.  Na Embrapa foi Presidente do Portfólio de Biotecnologia Avançada aplicada ao Agronegócio durante 7 anos e coordenou projetos em edição genômica envolvendo as culturas de cana-de-açúcar, milho, feijão e soja e desenvolvimento de soluções via dsRNA de uso tópico para controle de ervas daninhas, insetos-praga e nematóides nas culturas de algodão, milho e soja.  Foi responsável pelo desenvolvimento das primeiras canas-de-açúcar editadas GM-free do mundo para produção de etanol de segunda geração e aumento na produção de açúcares. https://linktr.ee/academiadoagrohttps://podfollow.com/academia-do-agro ................Para assinar e ouvir o podcast:Spotify: https://spoti.fi/33WG1dwDeezer: https://bit.ly/2VOuubAApple: https://apple.co/3oyQzaxGoogle: https://bit.ly/3lWvpS7Amazon: https://bit.ly/34xCWReYouTube: https://bit.ly/3mZop7p................Participe da Academia: https://t.me/ADA_AcademiaDoAgro Links Citados no Episódiohttps://www.abms.org.brhttps://www.embrapa.br/xxxiii-congresso-nacional-de-milho-e-sorgo-debate-oportunidades-e-ameacas-para-commodities-agricolashttps://maissoja.com.br/interferencia-por-rna-rnai-para-o-controle-de-pragas-e-doencas/https://www.insper.edu.br/agenda-de-eventos/o-epicentro-da-informacao-genetica-e-as-perspectivas-de-uso-do-rna-mensageiro/https://agriculturasustentavel.org.br/artigo/como-surge-uma-inovacaohttps://www.paranacooperativo.coop.br/ppc/index.php/sistema-ocepar/comunicacao/2011-12-07-11-06-29/ ------------------------------------ Perfil do Entrevistado Hugo Bruno Correa MolinariHugo Bruno Correa Molinari, PhDR&D Director - SEMPRE Agtech INTERAJA COM Hugo Bruno Correa Molinari Email: hm@sempre.agr.br Linkedin: www.linkedin.com/in/hugomolinari Celular: (61) ‪9 83018656 Instagram: @sempre_agtech Twitter: @hbmolinariINTERAJA COM  ACADEMIA DO AGRO LinkedIn: https://www.linkedin.com/in/waldir.franzini Instagram:- www.instagram.com/academiadoagro Twitter: @academiadoagro Telegram: @academiadoagro Facebook: /academiadoagro Celular-Whatsapp: 062 9 9700 7049 E-mail: podcast.academiadoagro@gmail.com ACOMPANHE A REDE AGROCAST https://www.redeagrocast.com.br/ Somos da Agrocast. A primeira rede de podcasts do agronegócio brasileiro e tem o objetivo de aumentar o consumo da mídia pelo setor, bem como estimular a criação de novos podcasts do agro. Rede Agrocast: @redeagrocast Academia do Agro: @academiadoagro Agro Resenha: @agroresenha Bendito Agro: @benditoagro Bug Bites: @bugbitespodcast Cachaça, Prosa & Viola :@cpvpodcast Esalqast: @esalqast Mundo Agro Podcast: @mundoagropodcast Notícias do Front: @noticias_do_front Papo Agro: @papoagropodcast Rumen Cast: @rumencast Apoio: W. Franzini - Gestão do Agronegócio ME Ficha Técnica: Produção: Waldir Franzini Edição/masterização - A Fabrica de Podcast - https://www.afabricadepodcast.com.br/ Musica: CC BY  - CC BY SA http://creativecommons.org/licenses/by/3.0/us/ Se você tem alguma sugestão de pauta, reclamação ou dúvida envie um e-mail para podcast.academiadoagro@gmail.com  | waldir.franzini@gmail.com | Whatsapp: 062 99700-7049

Hoje, um novo mundo encontra-se em testes nos laboratórios dos cientistas: e este mundo novo, que aliás não é tão novo assim, vamos combinar…. virá para aperfeiçoar o atual mundo, um tanto desajustado.  Cientista é um eterno inconformado, sempre persegue inovações - se assim não for não é cientista! E o mundo é movido pelo inconformismo da Ciência. Vamos ilustrar este ponto com tendências e pesquisas que se encontram em andamento neste momento, e que podem e irão impactar fortemente nossa agricultura, na próxima década. Bom aqui vou dar um pitaco sobre esta evolução, trazendo um texto bastante simplificado sobre o RNA mensageiro, de autoria do Décio Luiz Gazzoni, pesquisador da Embrapa.Todos os seres vivos possuem um código herdado dos progenitores, que são os genes localizados no núcleo de uma célula.  Os genes são compostos pelo DNA, e contêm instruções para a síntese de compostos químicos fundamentais para a vida - uma espécie de livro de receitas.  Além do DNA, existe uma estrutura chamada RNA mensageiro (mRNA), a qual gera uma cópia perfeita de um determinado gene, sendo sua função supervisionar a síntese das proteínas ligadas àquele gene. O mRNA é responsável por transferir o código de um gene do DNA para uma parte da célula chamada ribossomo, que podemos chamar de "fábrica de proteínas". Uma vez executada a tarefa, ou seja, depois que a proteína foi sintetizada, o mRNA cumpriu sua função e não é mais necessário. Surge, então, outro tipo de RNA, chamado de microRNA, que desliga ou altera o processo de produção de proteínas, degradando o RNA mensageiro. Se isto não ocorresse, a célula continuaria produzindo indefinidamente uma determinada proteína, provavelmente levando o organismo à morte. Bingo! E que tal transformar esta ameaça em oportunidade? Pois bem, para falarmos agora de cunho essencialmente prático, será necessário desenvolvermos métodos adequados para seu uso no campo, de forma extensiva, de maneira a controlar as pragas praticamente sem impacto sobre o ambiente ou à saude humana. Para isso trouxemos uma “fera” no assunto… Hugo Bruno Correa Molinari, atuou como pesquisador da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) durante 15 anos e atualmente é Diretor de P&D e Inovação na SEMPRE AgTech Agrônomo de formação, Mestre em Genética e Biologia Molecular, e Doutor em Agronomia.Em 2020 foi nomeado como membro titular da CTNBio, na vaga de Especialista em Biotecnologia indicado pelo Ministério da Agricultura, Pecuária e Abastecimento.  Na Embrapa foi Presidente do Portfólio de Biotecnologia Avançada aplicada ao Agronegócio durante 7 anos e coordenou projetos em edição genômica envolvendo as culturas de cana-de-açúcar, milho, feijão e soja e desenvolvimento de soluções via dsRNA de uso tópico para controle de ervas daninhas, insetos-praga e nematóides nas culturas de algodão, milho e soja.  Foi responsável pelo desenvolvimento das primeiras canas-de-açúcar editadas GM-free do mundo para produção de etanol de segunda geração e aumento na produção de açúcares. https://linktr.ee/academiadoagrohttps://podfollow.com/academia-do-agro ................Para assinar e ouvir o podcast:Spotify: https://spoti.fi/33WG1dwDeezer: https://bit.ly/2VOuubAApple: https://apple.co/3oyQzaxGoogle: https://bit.ly/3lWvpS7Amazon: https://bit.ly/34xCWReYouTube: https://bit.ly/3mZop7p................Participe da Academia: https://t.me/ADA_AcademiaDoAgro Links Citados no Episódiohttps://www.abms.org.brhttps://www.embrapa.br/xxxiii-congresso-nacional-de-milho-e-sorgo-debate-oportunidades-e-ameacas-para-commodities-agricolashttps://maissoja.com.br/interferencia-por-rna-rnai-para-o-controle-de-pragas-e-doencas/https://www.insper.edu.br/agenda-de-eventos/o-epicentro-da-informacao-genetica-e-as-perspectivas-de-uso-do-rna-mensageiro/https://agriculturasustentavel.org.br/artigo/como-surge-uma-inovacaohttps://www.paranacooperativo.coop.br/ppc/index.php/sistema-ocepar/comunicacao/2011-12-07-11-06-29/ ------------------------------------ Perfil do Entrevistado Hugo Bruno Correa MolinariHugo Bruno Correa Molinari, PhDR&D Director - SEMPRE Agtech INTERAJA COM Hugo Bruno Correa Molinari Email: hm@sempre.agr.br Linkedin: www.linkedin.com/in/hugomolinari Celular: (61) ‪9 83018656 Instagram: @sempre_agtech Twitter: @hbmolinariINTERAJA COM  ACADEMIA DO AGRO LinkedIn: https://www.linkedin.com/in/waldir.franzini Instagram:- www.instagram.com/academiadoagro Twitter: @academiadoagro Telegram: @academiadoagro Facebook: /academiadoagro Celular-Whatsapp: 062 9 9700 7049 E-mail: podcast.academiadoagro@gmail.com ACOMPANHE A REDE AGROCAST https://www.redeagrocast.com.br/ Somos da Agrocast. A primeira rede de podcasts do agronegócio brasileiro e tem o objetivo de aumentar o consumo da mídia pelo setor, bem como estimular a criação de novos podcasts do agro. Rede Agrocast: @redeagrocast Academia do Agro: @academiadoagro Agro Resenha: @agroresenha Bendito Agro: @benditoagro Bug Bites: @bugbitespodcast Cachaça, Prosa & Viola :@cpvpodcast Esalqast: @esalqast Mundo Agro Podcast: @mundoagropodcast Notícias do Front: @noticias_do_front Papo Agro: @papoagropodcast Rumen Cast: @rumencast Apoio: W. Franzini - Gestão do Agronegócio ME Ficha Técnica: Produção: Waldir Franzini Edição/masterização - A Fabrica de Podcast - https://www.afabricadepodcast.com.br/ Musica: CC BY  - CC BY SA http://creativecommons.org/licenses/by/3.0/us/ Se você tem alguma sugestão de pauta, reclamação ou dúvida envie um e-mail para podcast.academiadoagro@gmail.com  | waldir.franzini@gmail.com | Whatsapp: 062 99700-7049

Hoje, um novo mundo encontra-se em testes nos laboratórios dos cientistas: e este mundo novo, que aliás não é tão novo assim, vamos combinar…. virá para aperfeiçoar o atual mundo, um tanto desajustado.  Cientista é um eterno inconformado, sempre persegue inovações - se assim não for não é cientista! E o mundo é movido pelo inconformismo da Ciência. Vamos ilustrar este ponto com tendências e pesquisas que se encontram em andamento neste momento, e que podem e irão impactar fortemente nossa agricultura, na próxima década. Bom aqui vou dar um pitaco sobre esta evolução, trazendo um texto bastante simplificado sobre o RNA mensageiro, de autoria do Décio Luiz Gazzoni, pesquisador da Embrapa.Todos os seres vivos possuem um código herdado dos progenitores, que são os genes localizados no núcleo de uma célula.  Os genes são compostos pelo DNA, e contêm instruções para a síntese de compostos químicos fundamentais para a vida - uma espécie de livro de receitas.  Além do DNA, existe uma estrutura chamada RNA mensageiro (mRNA), a qual gera uma cópia perfeita de um determinado gene, sendo sua função supervisionar a síntese das proteínas ligadas àquele gene. O mRNA é responsável por transferir o código de um gene do DNA para uma parte da célula chamada ribossomo, que podemos chamar de "fábrica de proteínas". Uma vez executada a tarefa, ou seja, depois que a proteína foi sintetizada, o mRNA cumpriu sua função e não é mais necessário. Surge, então, outro tipo de RNA, chamado de microRNA, que desliga ou altera o processo de produção de proteínas, degradando o RNA mensageiro. Se isto não ocorresse, a célula continuaria produzindo indefinidamente uma determinada proteína, provavelmente levando o organismo à morte. Bingo! E que tal transformar esta ameaça em oportunidade? Pois bem, para falarmos agora de cunho essencialmente prático, será necessário desenvolvermos métodos adequados para seu uso no campo, de forma extensiva, de maneira a controlar as pragas praticamente sem impacto sobre o ambiente ou à saude humana. Para isso trouxemos uma “fera” no assunto… Hugo Bruno Correa Molinari, atuou como pesquisador da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) durante 15 anos e atualmente é Diretor de P&D e Inovação na SEMPRE AgTech Agrônomo de formação, Mestre em Genética e Biologia Molecular, e Doutor em Agronomia.Em 2020 foi nomeado como membro titular da CTNBio, na vaga de Especialista em Biotecnologia indicado pelo Ministério da Agricultura, Pecuária e Abastecimento.  Na Embrapa foi Presidente do Portfólio de Biotecnologia Avançada aplicada ao Agronegócio durante 7 anos e coordenou projetos em edição genômica envolvendo as culturas de cana-de-açúcar, milho, feijão e soja e desenvolvimento de soluções via dsRNA de uso tópico para controle de ervas daninhas, insetos-praga e nematóides nas culturas de algodão, milho e soja.  Foi responsável pelo desenvolvimento das primeiras canas-de-açúcar editadas GM-free do mundo para produção de etanol de segunda geração e aumento na produção de açúcares. https://linktr.ee/academiadoagrohttps://podfollow.com/academia-do-agro ................Para assinar e ouvir o podcast:Spotify: https://spoti.fi/33WG1dwDeezer: https://bit.ly/2VOuubAApple: https://apple.co/3oyQzaxGoogle: https://bit.ly/3lWvpS7Amazon: https://bit.ly/34xCWReYouTube: https://bit.ly/3mZop7p................Participe da Academia: https://t.me/ADA_AcademiaDoAgro Links Citados no Episódiohttps://www.abms.org.brhttps://www.embrapa.br/xxxiii-congresso-nacional-de-milho-e-sorgo-debate-oportunidades-e-ameacas-para-commodities-agricolashttps://maissoja.com.br/interferencia-por-rna-rnai-para-o-controle-de-pragas-e-doencas/https://www.insper.edu.br/agenda-de-eventos/o-epicentro-da-informacao-genetica-e-as-perspectivas-de-uso-do-rna-mensageiro/https://agriculturasustentavel.org.br/artigo/como-surge-uma-inovacaohttps://www.paranacooperativo.coop.br/ppc/index.php/sistema-ocepar/comunicacao/2011-12-07-11-06-29/ ------------------------------------ Perfil do Entrevistado Hugo Bruno Correa MolinariHugo Bruno Correa Molinari, PhDR&D Director - SEMPRE Agtech INTERAJA COM Hugo Bruno Correa Molinari Email: hm@sempre.agr.br Linkedin: www.linkedin.com/in/hugomolinari Celular: (61) ‪9 83018656 Instagram: @sempre_agtech Twitter: @hbmolinariINTERAJA COM  ACADEMIA DO AGRO LinkedIn: https://www.linkedin.com/in/waldir.franzini Instagram:- www.instagram.com/academiadoagro Twitter: @academiadoagro Telegram: @academiadoagro Facebook: /academiadoagro Celular-Whatsapp: 062 9 9700 7049 E-mail: podcast.academiadoagro@gmail.com ACOMPANHE A REDE AGROCAST https://www.redeagrocast.com.br/ Somos da Agrocast. A primeira rede de podcasts do agronegócio brasileiro e tem o objetivo de aumentar o consumo da mídia pelo setor, bem como estimular a criação de novos podcasts do agro. Rede Agrocast: @redeagrocast Academia do Agro: @academiadoagro Agro Resenha: @agroresenha Bendito Agro: @benditoagro Bug Bites: @bugbitespodcast Cachaça, Prosa & Viola :@cpvpodcast Esalqast: @esalqast Mundo Agro Podcast: @mundoagropodcast Notícias do Front: @noticias_do_front Papo Agro: @papoagropodcast Rumen Cast: @rumencast Apoio: W. Franzini - Gestão do Agronegócio ME Ficha Técnica: Produção: Waldir Franzini Edição/masterização - A Fabrica de Podcast - https://www.afabricadepodcast.com.br/ Musica: CC BY  - CC BY SA http://creativecommons.org/licenses/by/3.0/us/ Se você tem alguma sugestão de pauta, reclamação ou dúvida envie um e-mail para podcast.academiadoagro@gmail.com  | waldir.franzini@gmail.com | Whatsapp: 062 99700-7049

AIM ImmunoTech Inc CEO Tom Equels provided a summary of positive clinical data supporting the synergistic potential of its flagship drug Ampligen (rintatolimod) with checkpoint blockade therapies. Ampligen is the company's dsRNA drug being developed to fight cancers. It has shown synergy with checkpoint inhibitors, including increasing survival rates and efficacy, in the treatment of animal tumors when used with checkpoint blockade therapies.

最先端の研究やゴキブリだけではない神経科学全般、今後の研究などについて語っていただきました。 【ゲスト】 アザラシさん (https://twitter.com/olfneuron) ゴキブリについて神経科学の研究中。 ▶RNAi: RNA interference、広範囲な細胞タイプにおけるタンパク質機能を解析するために遺伝子発現をノックダウンする手法 RNA干渉(RNAi)に関する基礎知識のページ ▶若虫と幼虫 ▶チャネルロドプシン: 単細胞緑藻の感覚光受容体として働き、光に向かって構造する運動を制御する。シーズン5の海洋微生物の世界も参照。 ▶dsRNA: double stranded RNA、二本鎖RNA ▶Dicer: 二本鎖RNA前駆体を21塩基対からなる短い二本鎖RNAへと切断する酵素。 ▶RISC: RNA誘導サイレンシング複合体。タンパク質とsiRNAから構成される複合体で、これに取り込まれたsiRNAは相補的なmRNAを標的として切断を起こす。 ▶サーカディアンリズム：概日リズム、体内時計ともいう。約25時間周期で変動する生理現象で、動物、植物、菌類、藻類などほとんどの生物に存在している。 ▶Twitterスペース: Twitter上で複数人で会話できる機能。レンもよく使用しており、本番組のゲストと出会う場でもある。 質問、感想、ゲスト出演の問い合わせなどは以下Twitter・お便りフォームまでお願いします。 Twitter　＃サイエンマニア https://twitter.com/REN_SciEnTALK おたよりフォーム https://forms.gle/H4vg7MuN77VqbHV79 研究者レンのもう１つの番組　SciEnTALK/サイエントークはこちら→ https://lit.link/scientalk 1シーズン1人のゲスト形式で、各エピソードは一区切りのチャプターとしてご利用いただけます。 流し聞きする場合は、エピソードを古い順に並べ替えていただくとシームレスに聞くことができます。 BGM Future Sky/ SAKURA BEATZ.JP Somehow/Khaim https://www.khaimmusic.com/

Nesse episódio eu conversei com o Luis Antônio de Avila, Engenheiro Agrônomo, Ph.D em Agronomia e Professor da Universidade Federal de Pelotas. Nós batemos um papo sobre RNA de interferência, também conhecido como RNAi. O RNAi é um mecanismo desencadeado pela presença de RNA de fita dupla (dsRNA, double-stranded RNA) que resulta na redução da expressão e silenciamento de genes. Essa técnica tem potencial de uso no manejo de plantas daninhas, em especial no silenciamento de genes responsáveis por resistência de plantas daninhas a herbicidas. Por essa técnica, por exemplo poderia “desligar” a expressão de genes que conferem resistência e a planta daninha voltaria a ser sensível a um determinado herbicida. Quer saber mais sobre o RNAi e sobre as possibilidades no manejo de plantas daninhas? Fique com a gente e ouça esse episódio do MIPD47. . . . Weed.Con Entre os dias 22 a 24 de novembro de 2021 ocorrerá o Weed.Con, segundo evento 100% digital, realizado pela SBCPD. Acesse o site https://weedcon.com.br/ e saiba mais informações sobre o evento.

Susan Weiss returns to TWiV to discuss coronavirus entry into cells and the role of spike protein cleavage by proteases, why lab escaped hypotheses for SARS-CoV-2 are unlikely, and modulation of innate immune responses during infection. Hosts: Vincent Racaniello, Rich Condit, Alan Dove, Brianne Barker, and Amy Rosenfeld Guest: Susan Weiss Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode CoV pathogenesis in CNS (J Neuro Pharm) SARS-CoV-2 induces dsRNA-mediated innate responses (bioRxiv) Screen of FDA-approved drugs (J Viral) Letters read on TWiV 735 Timestamps by Jolene. Thanks! Weekly Picks Brianne – Tiktok of How mRNA Vaccines Work Alan – Dark Rich – Unmixing Color Machine (Ultra Laminar Reversible Flow) – Smarter Every Day 217 Amy – MOMA Calder exhibit, reviewed in NYTimes Vincent – Ripple Training (free YouTube channel) Listener Picks Ruth – 100 million year old seafloor bacteria resuscitated Iradis – Linda Zall, CIA spy for science Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv

Susan Weiss returns to TWiV to discuss coronavirus entry into cells and the role of spike protein cleavage by proteases, why lab escaped hypotheses for SARS-CoV-2 are unlikely, and modulation of innate immune responses during infection. Hosts: Vincent Racaniello, Rich Condit, Alan Dove, Brianne Barker, and Amy Rosenfeld Guest: Susan Weiss Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode CoV pathogenesis in CNS (J Neuro Pharm) SARS-CoV-2 induces dsRNA-mediated innate responses (bioRxiv) Screen of FDA-approved drugs (J Viral) Letters read on TWiV 735 Timestamps by Jolene. Thanks! Weekly Picks Brianne – Tiktok of How mRNA Vaccines Work Alan – Dark Rich – Unmixing Color Machine (Ultra Laminar Reversible Flow) – Smarter Every Day 217 Amy – MOMA Calder exhibit, reviewed in NYTimes Vincent – Ripple Training (free YouTube channel) Listener Picks Ruth – 100 million year old seafloor bacteria resuscitated Iradis – Linda Zall, CIA spy for science Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390161v1?rss=1 Authors: Jonely, M., Singh, R. K., Bass, B., Noriega, R. Abstract: Drosophila melanogaster Dicer-2 is a large, multidomain protein that cleaves double-stranded RNA (dsRNA) into small interfering RNAs in a terminus-dependent manner as part of the RNA interference pathway. We characterize the local binding environment involved in this substrate-selective molecular recognition event by monitoring the time-resolved photophysics of a cyanine dye linked to the dsRNA terminus. We observe substantial changes in the molecular rigidity and local freedom of motion of the probe as a function of distinct conformations of the biomolecular complex between Dicer-2 and dsRNA as a function of dsRNA termini, the presence of regulatory proteins, and the addition of a biochemical energy source (ATP) or a non-hydrolysable equivalent (ATP-{gamma}S). With a clustering analysis based solely on these molecular-scale measures of the local binding environment at the dsRNA terminus, we identify sub-populations of similar conformations that define distinct modes of molecular recognition which are correlated with biochemical activity. These observations reveal the important role of substrate-selective molecular recognition properties for proteins with multiple domains that can bind RNA, regulatory proteins, and cofactors. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.319475v1?rss=1 Authors: SINGH, R. K., Jonely, M., Leslie, E., Rejali, N. A., Noriega, R., Bass, B. L. Abstract: Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3{per thousand}-overhanging termini. We investigated the transient kinetic mechanism of these reactions using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. We found that ATP binding to dmDcr-2{per thousand}s helicase domain impacts the kinetics of dsRNA binding and dissociation in a termini-dependent manner, emphasizing the termini-dependent discrimination of dsRNA on a biologically-relevant time-scale. ATP-hydrolysis mediates local unwinding of dsRNA, and directional translocation on unwound single-stranded RNA, which is concurrent with a slow rewinding prior to dsRNA cleavage. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent change in conformational dynamics of the helicase and Platform.PAZ domains in the nanosecond timescale that is correlated with termini-dependent dsRNA cleavage. Our study delineates kinetic events and transient intermediates for a Dicer-catalyzed reaction, thus establishing a framework for understanding other Dicers and how accessory factors modulate the reaction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.24.264465v1?rss=1 Authors: Caruso, I. P., Sanches, K., Da Poian, A., Pinheiro, A., Almeida, F. C. L. Abstract: The N protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs' Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. N-NTD dsRNA destabilizing activity was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of {beta}2-{beta}3 and 2-{beta}5 loops, played a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTDs of dimeric N would act independently, increasing efficiency; (ii) two N-NTDs of dimeric N would bind to two different RNA sites, bridging distant regions of the genome; and (iii) monomeric N would be active, opening up the possibility of a regulatory dissociation event. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.31.231274v1?rss=1 Authors: Mickolajczyk, K. J., Shelton, P. M. M., Grasso, M., Cao, X., Warrington, S. R., Aher, A., Liu, S., Kapoor, T. M. Abstract: The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication. Copy rights belong to original authors. Visit the link for more info

Dr. Luis Garza MD-PhD is an Associate Professor of Dermatology with secondary appointments in Cell Biology and Oncology at the Johns Hopkins University School of Medicine. He did his undergraduate work at Cornell University, followed by an MD-PhD at the University of Pennsylvania. He completed his dermatology residency at the University of Michigan, followed by a postdoctoral fellowship in the lab of George Cotsarelis at the University of Pennsylvania. Dr. Garza’s current research focuses on wound healing and regenerative medicine. His lab is funded by the NIH/NIAMS, the DoD, and Maryland State Stem Cell Fund. His lab explores regeneration using the skin as their model system. They are inspired by the limb regeneration of a salamander as a clue to therapeutic interventions for fibrosis, one of the larges sources of human morbidity. They focus on two areas. In the first, if mammals cannot regrow an entire appendage, can they regrow a hair follicle mini-appendage? On this they focus on the mechanisms of how dsRNA sensing is a damage-repair response and robustly stimulates regeneration. For the second, they ask-- if they cannot regrow an entire limb in mammals-- can they regrow the pressure-responsive type of skin found at the palms and soles at the stump site of an amputee? For this project they focus on how cell-cell interactions modify tissue identity, and the mechanisms of how cell therapy might modify skin identity in mouse models and FDA approved human trials.”

Dickson, Daniel and Vincent solve the case of the Two Volunteers With Watery Diarrhea, discuss diagnosis of SARS-CoV-2 in the New York area, and reveal an approach to preventing honeybee colony collapse by imbuing their gut bacteria with the ability to produce antiviral and antiparasite double-stranded RNAs. Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Subscribe (free): iTunes, Google Podcasts, RSS, email Links for this episode PWB social media: Facebook, Instagram, Twitter Daniel on TWiV Special Engineered symbionts limit honeybee pathogens (Science) Letters read on TWiP 181 Become a patron of TWiP. Case Study for TWiP 181 Risks of being helpful. Volunteer from previous case. Diarrhea is gone, now has second issue. Since coming to Uganda noticing on waking in AM often has series of 1 cm red raised lesions in a line, 3-4, on torso. Swollen and itchy. New ones in the AM. Manager says welcome to Uganda. They move out of room, leave bedding behind. Lesions stop. What is going on and when can they move back into room? Send your case diagnosis, questions and comments to twip@microbe.tv Music by Ronald Jenkees

Agradece a este podcast tantas horas de entretenimiento y disfruta de episodios exclusivos como éste. ¡Apóyale en iVoox! El presidente de la Cámara, #JohnnyMéndez, dijo hoy que le había advertido a Ricardo Rosselló Nevárez los alegados líos de #Taniua Vázquez, secretaria del #DSRNA con el Superintendente del #Capitolio, José #erón Muñiz Lasallle.Escucha este episodio completo y accede a todo el contenido exclusivo de Bonita Radio. Descubre antes que nadie los nuevos episodios, y participa en la comunidad exclusiva de oyentes en https://go.ivoox.com/sq/90599

El presidente de la Cámara, #JohnnyMéndez, dijo hoy que le había advertido a Ricardo Rosselló Nevárez los alegados líos de #Taniua Vázquez, secretaria del #DSRNA con el Superintendente del #Capitolio, José #erón Muñiz Lasallle.

Michelle Flenniken joins TWiV to talk about honeybees and the work of her laboratory on honeybee viruses. Hosts: Vincent Racaniello, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Guest: Michelle Flenniken Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode MSU Pollinator Health Center Honeybee antiviral defenses (Sci Rep) The buzz about honeybee viruses (PLoS Path) Recently identified bee viruses (Curr Op Insect Sci) Videos on honey bee researchat MSU, honey bee pathogens, honey bee pathogen detection (YouTube) Timestamps by Jolene. Thanks! Weekly Science Picks Michelle - First 21 days of a bee's life; NPR's Hidden Brain; Varroa destructor triggers deformed wing virus in bees Brianne - Flu Near You Alan - Skype a Scientist program Rich- NASA's Voyager 2 Probe Enters Interstellar Space Kathy- Which ones are bees? And What it feels like to be a bee Vincent - Fecal sequencing and Vaccines did not cause Rachel's autism by Peter Hotez Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Michelle Flenniken joins TWiV to talk about honeybees and the work of her laboratory on honeybee viruses. Hosts: Vincent Racaniello, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Guest: Michelle Flenniken Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode MSU Pollinator Health Center Honeybee antiviral defenses (Sci Rep) The buzz about honeybee viruses (PLoS Path) Recently identified bee viruses (Curr Op Insect Sci) Videos on honey bee researchat MSU, honey bee pathogens, honey bee pathogen detection (YouTube) Timestamps by Jolene. Thanks! Weekly Science Picks Michelle - First 21 days of a bee's life; NPR's Hidden Brain; Varroa destructor triggers deformed wing virus in bees Brianne - Flu Near You Alan - Skype a Scientist program Rich- NASA's Voyager 2 Probe Enters Interstellar Space Kathy- Which ones are bees? And What it feels like to be a bee Vincent - Fecal sequencing and Vaccines did not cause Rachel's autism by Peter Hotez Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiVumvirate reviews this years crop of Nobel Prizes, and how cells prevent leakage of mitochondrial double-stranded RNA into the cytoplasm, which would otherwise lead to the production of interferon. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Sea Phages program and application materials Plant biologists penalized by CNRS (The Scientist) 2018 Nobel Prize in Medicine (pdf) 2018 Nobel Prize in Chemistry (pdf) 2018 Nobel Prize in Physics (pdf) 2018 Nobel Peace Prize (Nobel) Mitochondrial dsRNA triggers IFN (Nature) Letters read on TWiV 514 Timestamps by Jolene. Thanks! Weekly Science Picks 1:28:33 Alan- Compound Interest chemistry graphics Kathy- Writing letters of recommendation Guidelines Trix & Psenka Schmader et al. Madera et al. Dickson- Japan Fireworks Vincent - The Game of T-Cells and Apple Park Lego Listener Picks Steve - Shomu's Biology Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

From the 13th International Symposium on dsRNA viruses in Belgium, Vincent speaks with Harry Greenberg about his career and his work on rotaviruses, noroviruses, hepatitis B virus, and influenza virus. Host: Vincent Racaniello Guest: Harry Greenberg Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode 13th dsRNA Virus Symposium Hepatitisin blood donors (Lancet) Interferon treatmentof hepatitis B (NEJM) Mycoplasmamutantsin volunteers (PNAS) Norwalk virus infectionof chimpanzees (J Med Virol) Rotavirus rescueby reassortment (PNAS) Cold-adaptedinfluenza vaccine (Virol) Rotavirus correlates of protection(Curr Opin Virol) Rotavirus vaccine and intussusception(NEJM) Timestamps by Jolene. Thanks! Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiM team notes the passing of Allan Campbell, and explains how aminoglycoside antibiotics like neomycin enhance host resistance to viral infection. Hosts:  Vincent Racaniello, Michael Schmidt, and Michele Swanson. Subscribe to TWiM (free) on iPhone, Android, RSS, or by email. You can also listen on your mobile device with the Microbeworld app. Become a patron of TWiM. Links for this episode: Happy Birthday, Elio! (STC) Allan Campbell, 88 (Stanford News) Life in Science by Allan Campbell (Bacteriophage) Aminoglycosides inhibit viral infection (Nat Micro) Image credit Letters read on TWiM 175 Send your microbiology questions and comments (email or recorded audio) to twim@microbe.tv

The TWiV hosts discuss a plant virus that infects a fungus, and whether you need to work insane hours to succeed in science. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Become a patron of TWiV! Links for this episode Phytopathogenic fungus hosts a plant virus (PNAS) Franklin column (PNAS) Potato famine mystery solved (History) Protein Data Bank You do not need to work 80 hours per week (THE) How I Learned To Stop Worrying (Sci Am) Image credit: Truth Wins by Jon Yewdell Letters read on TWiV 469 This episode is brought to you by the Department of Microbiology at the Icahn School of Medicine at Mount Sinai. Composed of over 20 virology labs, all centralized in one building in the heart of New York City, this department is a perfect fit for anyone with an interest in pursuing virus research. For more information about the Department, visit http://bit.ly/micromssm This episode is brought to you by  the Virology and Gene Therapy PhD program at the Mayo Clinic Graduate School of Biomedical Sciences in Rochester, Minnesota. The renowned Mayo Clinic Virology and Gene Therapy program is currently accepting applications until December first. If you want to be trained in the exciting fields of oncolytic virotherapy, gene therapy, vaccine creation, or basic virology, visit mayoclinic.org to apply to the Virology and Gene Therapy program at the Mayo Clinic Graduate School of Biomedical Sciences. Weekly Science Picks Kathy - Patterns Video (WonderLab) Dickson - Nikon 2017 Photomicrography Contest Rich - Leonardo Da Vinci by Walter Isaacson Alan - Sail Magazine Best Boats 2018 Vincent - Science Is Dry, Obscure, Complex? Listener Picks Ben - Is the staggeringly profitable business of scientific publishing bad for science? Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Theodora Hatziioannou joins the TWiV team to discuss a macaque model for AIDS, and how a cell protein that blocks HIV-1 infection interacts with double-stranded RNA. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Guest: Theodora Hatziioannou Become a patron of TWiV! Links for this episode ASU-UofA Joint Virology Symposium Laboratory of Retrovirology, Rockefeller University HIV-1-induced AIDS in monkeys (Science) APOBEC3H bound to duplex RNA (Nat Comm) Center for HIV RNA studies Image: Two molecules of APOBEC3H bound to dsRNA This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. Get $30 off your first delivery and FREE SHIPPING by going to blueapron.com/twiv. This episode is brought to you by the Defense Threat Reduction Agency. Part of the U.S. Department of Defense, the Agency’s Chemical and Biological Technologies Department hosts the 2017 Chemical and Biological Defense Science & Technology Conference to exchange information on the latest and most dynamic developments for countering chemical and biological weapons of mass destruction. Find out more at http://www.cbdstconference.com Weekly Science Picks Theodora - To Kill A Mockingbird by Harper Lee Kathy - 25 MILLION Orbeez in a pool Dickson - Fluid Dynamics of Paint Rich - The Hitchhiker's Guide to the Galaxy by Douglas Adams Alan - 30 days at sea: timelapse Vincent - TWiEVO 24 and Every Time Zone Listener Picks Fernando - Lego Women of NASA Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Nels and Vincent reveal how the protein DHX9 suppresses RNA processing defects caused by invasion of the Alu retroelement into the human genome. Hosts: Nels Elde and Vincent Racaniello Become a patron of TWiEVO DHX9 suppresses Alu defects (Nature) Asifa Akhtar on The Latest Thinking This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twie. Science Picks Nels - Saving Hellbender Salamanders Vincent - The Living River Music on TWiEVO is performed by Trampled by Turtles Send your evolution questions and comments to twievo@microbe.tv

The TWiV scientists reveal that mosquitoes transmit different West Nile virus populations with each blood meal, only to have the diversity purged in a bird host. Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Kathy Spindler   Become a patron of TWiV! Links for this episode Driving past a milestone (TWiP 100) Debug Fresno (Verily blog) What could possibly go wrong? (TWiV 388) Chestnut blight (ProMedMail) Hypovirus molecular biology (Adv Virus Res) Mosquitoes transmit unique WNV populations (Cell Rep) Teaching science using pseudoscience Image credit Letters read on TWiV 451 Weekly Science Picks Kathy - Approaching Jupiter and Michigan parents about vaccination Rich - Math Limericks from Futility Closet Alan - NASA flyover of Pluto Vincent - Talk show host nominated as USDA chief scientist Listener Pick Neil - These smart girls are here to debunk anti-vaxxer nonsense John - John Sever AMA Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin The TWiP Trinity solve the case of the Peace Corps volunteer with diarrhea, and reveal how immunizing against a virus ameliorates exacerbated leishmaniasis. Become a patron of TWiP. Links for this episode: Viral vaccine prevents exacerbated leishmaniasis (PLoS NTD) For whom the trich tolls (TWiP 47) A virus in a parasite in a human (virology blog) Virologists in the mist (TWiV 128) Letters read on TWiP 126 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free - WITH FREE SHIPPING - by going to blueapron.com/twip. Case Study for TWiP 126 Another Peace Corps volunteer in Fiji. 24 yo male, several days of fever, headache, dry cough, rash. Feels poorly, starts diarrhea. No blood or mucus, no vomiting but abdominal discomfort. Heart rate over 100. At private nearby hospital for evaluation: no prior med probs or surgeries. Social history: MSM, not always protected, drinks every weekend. Home blown away by cyclone. Alcohol: drinks beer, a lot. White rice, split peas, bread diet. Fan of cava, also drank unfiltered water. He is admitted, continues to feel poorly. Continued fevers, localized abdominal pain RUQ. On exam he has tender palpable liver, elevated WBC 17.8, eosinopenia, 0 cells. AST/ALT slightly above normal. Dengue, chick, lepto, blood all negative. Ultrasound of liver: shows 8x8 cm mixed echogenic lesion in right lobe. HIV negative.  Send your case diagnosis, questions and comments to twip@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Guest: Jared Rice The TWiV academia discuss induction of diarrhea by the capsid protein of an astrovirus, and association of a fungal RNA virus with white-nose syndrome of North American bats.   Become a patron of TWiV! Links for this episode Register for ASV 2017 ASM-ASV Conference on Interplay of Viral and Bacterial Pathogens Astrovirus capsid protein induces diarrhea (mBio) Fungal virus associated with white-nose syndrome (PLoS Path) Image credit Letters read on TWiV 423 Weekly Science Picks Alan - GPS visualizer Dickson - James Webb Space Telescope and The Vertical Farm Kathy - First Snowflake Photos (One and Two) Rich - Red Mars by Kim Stanley Robinson Vincent - Carrie Fisher Fought for the Rebel Alliance and Mental Health Awareness Listener Pick Ken - The Abate Lab Sean - The Death of Expertise Send your virology questions and comments to twiv@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin The parasite prophets solve the case of the Thai Man with Abdominal Distention, and discuss the finding that metastatic leishmaniasis dependent on a virus can be prevented by blocking IL-17A. Links for this episode: TWiP 27: Trematodes Leishmaniavirus and IL-17A dependent leishmaniasis (PLoS Path) Image credit Letters read on TWiP 117 This episode is sponsored by CuriosityStream, a subscription streaming service that offers over 1,400 documentaries and non­fiction series from the world's best filmmakers. Get unlimited access starting at just $2.99 a month, and for our audience, the first two months are completely free if you sign up at curiositystream.com/microbe and use the promo code MICROBE. This episode is also sponsored by Drobo, a family of safe, expandable, yet simple to use storage arrays. Drobos are designed to protect your important data forever. Visit www.drobo.com to learn more. Become a patron of TWiP. Case Study for TWiP 117 Woman 66 yo born in Guinea, grew up the moved to US past 10 years, just retired. Lives in Washington Heights. Mother having issues in Guinea, so went back for 3 months in 2016. Just came back a week ago, reporting headache, fever, feeling poorly. Staying in big city, with Mom (80s). Has own private toilet in nice home. No screens or bednets. Married for 40 years, recently divorces. Has had 10 children. Not sexually active. Starts with high fever, breaks, then 2 days later another for several hours, goes to ER. Given Ebola screening questions, negative, do some blood work, send her back out. 2-3 days later high fever, double vision, headache, comes to Columbia ER. No diarrhea, no urination discomfort. Has backache, feels that mouth is dry. Was admitted. Past med history: high bp, cholesterol, diabetes; not overweight; appendix out; has unknown reaction to novocaine. No smoking, drinking. Physical: 39.4 temp, 14-16 breath rate, heart rate over 100, rapid heartbeat, 2/6 systolic murmur with radiation to left carotid (flow murmur). No jugular venous distention. Abdomen right upper quadrant: slight enlargement of liver, not tender, can palpate spleen tip in left upper quadrant, slightly enlarged spleen. Normal bowel sounds, no rash. Blood: elevated white count, bands 9%. 0.1 eosinophils, platelets 79, hemoglobin 11. Bilirubin 1.5, bicarb 20, chest xray clear. Red cells: small, 79.4 mcv. Animals: don’t like animals! In middle of rainy season. Likes to walk outside in rain during day. No cats to keep out rodents. Food: all food is prepared in home. Eats all favorite foods: rice. No sick contacts, no exposure to health care setting, no bug bites.  Send your case diagnosis, questions and comments to twip@microbe.tv

Bovine neonatal pancytopenia (BNP) is a recently emerged alloimmune disease affecting neonatal calves and is strongly associated with the vaccination of dams with a specific inactivated BVDV (Bovine Viral Diarrhoea Virus) vaccine (PregSure® BVD, Pfizer Animal Health). The vaccine comprises a unique adjuvant based on nanoparticles and has been shown to be highly contaminated with cellular proteins of the vaccine producer cell line MDBK (Madin-Darby bovine kidney). The disease is induced by ingestion of colostral alloantibodies of certain PregSure® BVD-vaccinated dams. The hypothesis that BNP-associated alloantibodies might be directed against MHC class I has been proposed. Currently, there are published observations, which contradict this hypothesis and which suggest that the aetiopathogenesis of BNP has not been fully elucidated yet. The aims of this study were to gain more insights into selected factors and underlying mechanisms involved in BNP. The hypothesis of a genetic predisposition for clinical BNP had been proposed in a previous study, but the incidence and potential genetic predisposition for subclinical cases had not been investigated thoroughly before. Therefore, a well-defined cattle resource population was monitored for the incidence, vaccination-associated epidemiological factors and a possible genetic predisposition involved in subclinical BNP. Prominent immune responses to PregSure® BVD vaccination had been reported. This study characterised the immune response to a booster immunisation with PregSure® BVD to obtain insights into the structural and quantitative regulation of the blood transcriptome after vaccination by means of deep sequencing transcriptome analysis. Furthermore, this approach should facilitate insights into the composition of the vaccine. The previously proposed hypothesis of a genetic predisposition required for giving birth to clinical BNP-calves could be confirmed in this study. Our results suggest that a genetic predisposition is potentially also involved in subclinical BNP and additionally required for a significant decrease of thrombocytes and leucocytes in healthy calves without manifestation of clinical or subclinical BNP. In the monitored cattle resource population, the frequency of subclinical BNP did not exceed the frequency of clinical BNP-cases. Further vaccination-associated epidemiological factors, e.g. frequency of vaccination or time point of vaccination relative to parturition, could be excluded. Transcriptome analysis 14 days after booster vaccination with PregSure® BVD highlighted a very coordinated immune response to double-stranded (ds) RNA, although the inactivated vaccine was directed against a single-stranded (ss) RNA BVD virus. The source of the dsRNA is unknown and could have originated from the MDBK cell line, which would underline the contamination of the vaccine with host cells from vaccine production. Alternatively, a dsRNA analogue was potentially added to improve the efficacy of the vaccine. Finally, this study identified a cytokine-like gene, which was highly upregulated across all animals after booster vaccination. This gene has not been described before in any other species. It has to be evaluated if upregulation of this gene is specific for ruminant immune response and linked to PregSure® BVD vaccination. In conclusion, this study confirmed that a genetic predisposition of PregSure® BVD-vaccinated dams is required for producing BNP-colostrum. Therefore, prospective studies will have to consider genetically determined differential immune responses between BNP- and non-BNP-dams towards exogenous proteins combined with very potent adjuvants. Regarding the MHC class I hypothesis, published observations raise the question, if MHC class I should be considered as single causal agent for BNP-associated alloantibodies. This requires further research. Potential allogeneic effects of a whole panel of contaminating proteins contained in the vaccine still have to be validated.

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Vincent, Dickson, Alan, Rich, and Kathy review two papers that present evidence for RNA interference as an antiviral immunity mechanism in mammals. Links for this episode: World Polio Day Mole Day John Holland's publications RNAi is antiviral in mammals (Science) Antiviral RNA interference in mammals (Science) RNAi, antiviral after all (Science) Is RNA interference antiviral in mammals? (Cell Host Microbe) Nodamura virus (Nature) Ebolavirus proteins suppress RNAi (J Virol) Illustrated is an siRNA (orange), dicer (top right), and argonaute (bottom) Letters read on TWiV 256 Weekly Science Picks Dickson - Wildlife Photographers of the Year 2013Kathy - John Holland's Emerging Infectious Disease lecture (YouTube)Alan - The worst part is notRich - The Universe in a Single Atom by Dalai Lama (Mind and Life Institute)Vincent - The Truth about T. Rex by Brian Switek Listener Pick of the Week Stephen - International Institute for Species Exploration (Top 10 species choice)  Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello and Dickson Despommier Vincent and Dickson review evidence that a virus of the protozoan parasite Trichomonas vaginalis may exacerbate disease. Links for this episode: Endobiont viruses sensed by the human host (PLoS One) Letters read on TWiP 47 Contact Send your questions and comments (email or mp3 file) to twip@twiv.tv

Background: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from `knocking down' expression of the actin gene. Conclusion: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Es ist bereits bekannt, dass akute Infektionen zur Verschlechterung einer vorbestehenden Glomerulonephritis führen können. Dies ist zum Beispiel bei der Lupusnephritis, der IgA-Nephropathie oder der renalen Vaskulitis der Fall (mesangioproliferative Glomerulonephritiden). Chronische virale Infektionen (z.B. Hepatitis C) können sogar eine de novo Glomerulonephritis auslösen. Hierbei handelt es sich um eine Immunkomplex-Glomerulonephritis, die durch die Bildung von Immunkomplexen aus eindringendem Antigen und daraufhin gebildetem Antikörper entsteht. Die Immunkomplexe werden über den Fc-Teil des Antikörpers erkannt. Ob die viralen Komponenten dieser Komplexe auch direkt erkannt werden und von welchen Zellen ist unbekannt. Neben der Bildung von Immunkomplexen spielt die Produktion von Interferonen eine wichtige Rolle bei der Entstehung von virusassoziierten Glomerulonephritiden. Die Aktivierung der systemischen antiviralen Immunität führt zur systemischen Interferonfreisetzung. Es wird angenommen, dass die meisten Zellen Typ I-Interferone produzieren können, wenn sie viral infiziert werden. So kann beispielsweise die TLR-3- vermittelte Erkennung viraler dsRNA in den Inselzellen des Pankreas über lokale IFN-a-Produktion eine autoimmune Inselzellzerstörung auslösen. Dahingegen ist unklar, ob lokal produzierte Typ I-Interferone zur Entstehung von irusassoziierten Glomerulonephritiden beitragen können. Wurde Mäusen virale dsRNA injiziert, gelangte diese zu den glomerulären Mesangialzellen. Mesangialzellen besitzen als einzigen nukleinsäurespezifischen Toll-like Rezeptor den TLR-3 und produzieren auf Stimulation durch virale dsRNA hin Interleukin-6 und CCL2. Ob dieser Effekt über den endosomalen TLR-3 oder über zytosolische dsRNA-Rezeptoren abläuft und ob Mesangialzellen überhaupt Interferone produzieren können, ist allerdings noch unklar. Unsere Hypothese ist, dass Mesangialzellen virale RNA sowohl TLR-abhängig als auch TLR-unabhängig erkennen können und dass die virale RNA daraufhin ein angeborenes antivirales Antwortprogramm, einschließlich der Produktion von Typ I-Interferonen, in glomerulären Mesangialzellen aktiviert.

The cytosolic helicases RIG-I and MDA5 are primary sensors for viral RNA during infection. Although their overall role as key players in the antiviral response and the induced signaling pathways have been elucidated in great detail over the past years, a structural and functional understanding of virus recognition by these sensors is missing. On the basis of an X-ray structure of RIG-I RD the 5’-triphosphate interaction site could be mapped to a previously identified positively charged groove. Structural modeling of the homologous RD of MDA5 gave a rational for its lower affinity to RNA. Based on a comparison of enzymatic activities of several RIG-I truncation variants, a model for the transition from the inactive to the active state was postulated. In contrast to RIG-I, the molecular patterns which lead to MDA5-dependent anti-viral signaling are still insufficiently understood. Here it is shown that the dsRNA-mimic poly I:C is a potent activator of MDA5 ATPase activity in vitro. The ATPase activity is inhibited by V-protein, which stably binds to the N-terminal RecA domain of the MDA5 helicase domain. In summary, the results presented here broaden our understanding of virus sensing and pinpoint several intriguing avenues for future research. Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP)are reported. One nanobody could reversibly reduce GFP fluorescence about fivefold, whereas its displacement by a second nanobody caused a ten-fold increase. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment leading to altered absorption properties. Unlike conventional antibodies, the small and stable nanobodies are functional in living cells. Nanobody induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization, and translocation events like the Tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

Background/Aims: Viral infections are a major problem worldwide and many of them are complicated by virally induced glomerulonephritides. Progression of kidney disease to renal failure is mainly attributed to the development of renal fibrosis characterized by the accumulation of extracellular matrix components in the mesangial cell compartment and the glomerular basement membrane. Plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA) are major regulators of plasmin generation and play an important role in generation and degradation of glomerular extracellular matrix components. Viral receptors expressed by mesangial cells (MC) are known to be key mediators in immune-mediated glomerulonephritis. We investigated the effect of stimulation of the viral receptors toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I) on the expression of PAI-1 and t-PA. Methods: Expression of PAI-1 and t-PA in immortalized human MC stimulated with polyriboinosinic: polyribocytidylic acid {[}poly(I:C)] RNA and cytokines were analyzed by real-time RT-PCR and ELISA. Results: Incubation of MC with poly(I:C) RNA to activate the viral receptors TLR3 and RIG-I upregulates the expression of PAI-1 and t-PA. Knockdown of viral receptors with specific siRNA abolishes the induction of PAI-1 and t-PA. Conclusion: For the first time a link between the activation of viral receptors on MC and potentially causative agents in the development of glomerulosclerosis and tubulointerstitial fibrosis is shown. The progression of inflammatory processes to glomerulosclerosis can be postulated to be directly enhanced by viral infection. Copyright (C) 2009 S. Karger AG, Basel

Hintergrund und Ziele der Arbeit: Bakterien und DNA Viren werden anhand unmethylierter CpG-Motive innerhalb ihrer DNA von den TLR 9 tragenden PDCs und den B Zellen des humanen Immunsystems als Gefahrensignale erkannt. Mittels synthetischer, CpG-enthaltender ODN nutzt man diese Grundsatzmechanismen, um vergleichbare Immunantworten auszulösen. Auf Grundlage eines unterschiedlichen immunologischen Aktivierungsprofils wurden bislang drei CpG-Klassen definiert: CpG-A, CpG-B und CpG-C. Mit Hilfe von CpG-A war es erstmals möglich, IFN-α in PDCs (den endogenen Hauptproduzenten dieses Zytokins) in Mengen zu induzieren, wie es bislang nur mit Viren selbst möglich war. Auch CpG-C stimuliert PDCs zur Sekretion von IFN α und aktiviert darüber hinaus B Zellen - eine Eigenschaft, die CpG-A nicht besitzt. Die sequenzspezifischen und strukturellen Voraussetzungen für diese differenziellen Wirkprofile waren bislang unzureichend verstanden, auch weil die Struktur-Analysen nur begrenzt auf die tatsächlichen Vorgänge im physiologischen Milieu übertragbar waren. Um CpG-ODN für die therapeutische Anwendung zu optimieren, sind die genauen Kenntnisse der Struktur-Wirkungsbeziehungen jedoch unverzichtbar. Ein zweiter Ansatzpunkt zur Optimierung der Anwendung liegt in der Verbesserung der systemischen Stabilität von CpG-ODN. Die Bindung von CpG-ODN an partikuläre Trägersysteme (z.B. Gelatine-Nanopartikel) wurde bereits in unserer Abteiliung als mögliches drug-delivery-System etabliert. Eine Weiterentwicklung dieses Prinzips wären partikuläre Strukturen, die aus immunstimulatorischen Nukleinsäuren aufgebaut keiner weiteren Trägermaterialien bedürfen. Beide Ansatzpunkte führen zu den Zielen dieser Arbeit: 1) Die Aufklärung der Struktur-Wirkungsbeziehungen der CpG-Klassen A und C durch Etablierung geeigneter Methoden zur Untersuchung im physiologischen Milieu. 2) Die Entwicklung immunstimulatorischer partikulärer Strukturen auf Basis der in Teil 1) identifizierten wirksamen Strukturelemente beider CpG-Klassen. Ergebnisse: 1) Struktur-Wirkungsbeziehungen von ODN 2216 (CpG-A) und ODN M362 (CpG-C): CpG-A bildet im physiologischen Milieu spontan multimolekulare Strukturen, deren mittlere Durchmesser mit 24 40 nm im Größenbereich von Viren liegen. Es zeigte sich, dass für diese Multimerisierungen das Zusammenspiel aus flankierenden Poly-G-Motiven, palindromischem Zentrum und eingelagerten Natrium- oder Kaliumionen entscheidend ist. Physiologisches Milieu wirkt sich sowohl den Umgebungs-pH und die Na+/K+-Konzentrationen als auch die Temperatur (37 °C) betreffend optimal förderlich auf die Strukturbildung aus. Die Identifizierung dieser maßgeblichen Faktoren machte es möglich, den Strukturaufbau von CpG-A experimentell zu kontrollieren und die immunologischen Wirkungen der verschiedenen Strukturen direkt zu vergleichen. Für die rasche und hohe Induktion von IFN-α und anderen inflammatorischen Zytokinen durch PDCs sind große Partikel verantwortlich. Die Multimerisierungen von ODN 2216 werden bei pH < 6 zunehmend aufgehoben. Unterbindet man die Multimerisierungen durch Präinkubation der ODN bei Temperaturen > 60 °C oder durch Entzug der stabilisierenden Natriumionen (indem man sie zuvor in Aqua ad inj. löst), so verliert ODN 2216 seine immunstimulatorische Aktivität in Bezug auf PDCs. Die schwache Wirkung der CpG-A-Monomere kann jedoch durch Präinkubation von PDCs mit IFN β deutlich gesteigert werden. Im Gegensatz zu den ebenfalls einzelsträngig vorliegenden ODN 2006 (CpG-B) haben auch Monomere von ODN 2216 keine aktivierende Wirkung auf B Zellen. CpG-C hat durch die palindromische Sequenz die Möglichkeit, Hairpins und Duplices zu bilden. ODN M362 zeigt jedoch keine Hairpinstrukturen. Die Duplexformationen sind bei 37 °C in vitro nicht stabil und spielen keine Rolle bei der durch diese ODN initiierten B-Zell-Aktivierung. Duplices haben jedoch Anteil an der Induktion von IFN-α in PDCs. Die in dieser Arbeit etablierten Protokolle der Temperatur-Präinkubation ermöglichen erstmalig eine experimentelle Kontrolle der Strukturbildungen von CpG-A und CpG-C und dadurch den Vergleich von Struktur und Wirkung. Das Standardprotokoll für Gelelektrophorese wurde dahingehend modifiziert, dass ein physiologisches Milieu sowohl durch die anwesenden Ionen als auch durch die Umgebungstemperatur (37°C) simuliert werden konnte. 2)Design Nukleinsäure-basierter Nanopartikel: Zentrale Elemente von CpG-A und CpG-C (palindromische Sequenz, gerüstartige Verbindung mehrerer Nukleinsäuren) wurden eingesetzt, indem ODN M362-Sequenzen (CpG-C) an bi- und trivalenten Grundgerüsten (Linkern) für den Strukturaufbau optimiert wurden. Trivalente Linker ermöglichen die variierende Zusammenlagerung der palindromischen Nukleinsäuren in drei Richtungen des Raumes und dadurch die Bildung großer Partikel. Diese sind den bisher bekannten Maximalstimuli CpG-B und CpG-C hinsichtlich der Aktivierung von B-Zellen gleichwertig. Erstmalig konnten auf diese Weise B-Zellen durch partikuläre Strukturen stark aktiviert werden. Nach Vor-Komplexierung der Partikel mit Poly-L-Arginin wird die Aktivität bei B-Zellen nochmals verstärkt. Kurze, nicht-palindromische CpG-DNA-Sequenzen an trivalenten Grundgerüsten induzieren nach Vor-Komplexierung mit Poly-L-Arginin deutlich mehr IFN-α in PBMCs als CpG-A, obwohl sie selbst nicht multimerisieren. Wird die (palindromische) RNA-Sequenz von CpG-C an einem trivalenten Linker verwendet, so können ebenfalls große Strukturen generiert werden, die nach Transfektion vergleichbare Mengen IFN-α in PBMCs induzieren wie CpG-A. Ausblick: Die vorliegende Dissertation verbindet Fragestellungen der Immunologie und der pharmazeutischen Technologie mit den Möglichkeiten der Biochemie. Es werden nicht nur verschiedene Methoden zur strukturellen Untersuchung von CpG-ODN im physiologischen Milieu etabliert, sondern auch die experimentelle Kontrolle der Strukturbildung von CpG-A ermöglicht. Die entwickelte Technik der Generierung dreidimensionaler, über palindromische Nukleinsäuren aufgebauter Partikel ist nicht auf CpG-Motive in DNA begrenzt, sondern kann auf eine andere für Viren charakteristische Nukleinsäure (Einzelstrang-RNA) übertragen werden. Dadurch würde zusätzlich möglich, die immunologischen Profile von ssRNA, dsRNA und CpG in einem Partikel zu kombinieren und die Art der Immunantwort je nach Zusammensetzung der Partikel gezielt zu bestimmen. Die klinische Relevanz dieser Arbeit ergibt sich aus den neuen Erkenntnissen über die Multimerisierungen von CpG-A, welche dessen therapeutischen Einsatz optimieren und besser standardisierbar machen sollen. Außerdem werden neue Hinweise auf die unterschiedlichen Aufnahme- und Erkennungsmechanismen beider CpG-Klassen und deren Aktivierung der Synthese von IFN-α gewonnen. Darüber hinaus wurde durch die Entwicklung der Polyvalenten Linker eine grundsätzlich neue Technik im Stil eines Baukastensystems etabliert, welche als Grundstein einer neuen Generation von immunstimulatorischen Multimeren dienen soll. Die Koadministration von Adjuvans und Antigen in direkter räumlicher Nähe bietet neue Gestaltungsmöglichkeiten in der Vakzineentwicklung. Zudem ist zu erwarten, dass unter Einbeziehung der RNA basierten immunologischen Wirkprofile innerhalb eines Partikels der Einsatz von CpG-ODN zur Therapie von Virusinfektionen und Tumoren weiter verbessert werden kann.

Nucleic acids that occur free or as immune complexes may trigger immune activation leading to aggravation of diseases with autoimmune predisposition. TLR3 and TLR 9 represent receptors that signal for viral and bacterial nucleic acids respectively. pI:C RNA, a synthetic double stranded RNA with identical properties to that viral origin activates TLR3 led to aggravation of lupus nephritis, a form of immune complex glomerulonephritis, in pre-existing lupus in MRLlpr/lpr mice. Exposure to pI:C RNA (a structural analogue of viral dsRNA) can aggravate lupus nephritis through TLR3 on antigen-presenting cells and glomerular mesangial cells. pI:C RNA–induced cytokine and chemokine production represents a major mechanism in this context. Likewise, CpG DNA a classical activator for TLR-9 led to disease aggravation in this mouse model albeit, through mechanisms that shared some commonality as well as differences to that observed with pI:C RNA. Apparently, pathogen associated immunomodulation relates to the cell-type-specific expression pattern of the respective pattern-recognition receptor. dsRNA-induced disease activity is independent of B cell activation and humoral antichromatin immunity in experimental SLE and therefore differs from CpG-DNA–induced autoimmunity. These findings contribute to the understanding of pathogen-associated modulation of autoimmunity but may also be involved in the pathogenesis of other types of inflammatory kidney diseases, e.g., flares of IgA nephropathy, renal manifestations of chronic hepatitis C virus infection, and renal vasculitis. Further, besides signifying the role of foreign and self-DNA as a pathogenic factor in autoimmune disease activity in lupus, this detailed study reveals, that certain synthetic G-rich nucleic acids may potentially block nucleic acid specific TLR functions and thus prove beneficial in arresting disease activity during progressive systemic lupus. One such G-rich DNA employed in this study has proven to be beneficial and suppressed systemic lupus in MRLlpr/lpr mouse model. Thus, modulating the CpG-DNA - TLR9 pathway may offer new opportunities for the understanding and treatment of lupus.

2. Summary In Drosophila melanogaster the transcriptional activity of the male X chromosome is upregulated to compensate for the reduced dosage of X-linked genes as compared to the two X chromosomes in females. This process is mediated by the Dosage Compensation Complex (DCC), a ribonucleoprotein complex consisting of five proteins (MSL1, MSL2, MSL3, MOF and MLE) and two non-coding RNAs (roX1 and roX2). The DCC preferentially localizes on the X chromosomes in males where it doubles its transcription rate. Two enzymes are associated with the DCC: the acetyltransferase MOF, specific for the lysine 16 of H4 (H4-K16), and the DNA/RNA helicase MLE. Genetic experiments demonstrated that both activities are required for dosage compensation in male flies. However, the weak association of MLE to the DCC has complicated its biochemical analysis and, so far, the involvement of MLE RNA helicase in dosage compensation has only been demonstrated genetically. Using different in vivo and in vitro approaches the physical and functional interactions of MLE with the other MSL proteins and with the roX RNAs was addressed. Monoclonal antibodies, specifically recognizing MLE, were raised in rats, offering a new tool for MLE characterization. By coexpression of the DCC subunits in SF9 cells, a recombinant complex containing MSL1-2-3, MOF, MLE and the roX2 RNA was reconstituted and purified. A specific integration of roX2 into the DCC could be observed only in the absence of MLE. Non specific RNA binding properties seemed instead associated to MLE RNA helicase. Moreover, the purified MSL complex did not affect the ATPase activity of MLE in the presence or absence of roX2 RNA. In vitro, MLE showed a preferential association with MSL1 and MSL2 and MLE interaction with both MSL proteins were not RNA mediated. In view of these results we suggest that binding to roX2 is not the main determinant for MLE integration into the DCC complex and protein-protein interactions might instead contribute to its proper recruitment to the X chromosome. MLE is a member of the DEAD-box RNA helicase family and it shares with the other members the same domain organization. In addition to a central ATPase/helicase domain, two predicted N-terminal double strand (ds) RNA-binding motifs (dsRBM1 and dsRBM2) and a predicted C-terminal single strand (ss) RNA/DNA-binding domain (RGG-box) are also present in MLE protein. These domains have been extensively characterized in RHA, human ortholog of MLE, and their RNA binding properties confirmed. However, it is not known how MLE binds RNA and how the different RNA binding modules contribute to stimulate its enzymatic activities. A preferential binding of MLE to dsRNA compared to ssRNA was shown by binding assays. In addition, changes in the affinity of MLE for both ssRNA and dsRNA were observed in the presence of different nucleotides. Deletion mutants of MLE were produced and purified from insect cells in order to address the contribution of the different RNA binding domains to MLE enzymatic activities. By transient expression in Drosophila cells of the same deletion mutants fused to GFP, the effects of individual domains on MLE recruitment to the X chromosome were also determined. The results show that unlike RHA, the dsRB1 and the RGG domains are dispensable for MLE RNA binding and unwinding, whereas dsRB2 seems to play the major role in coordinating both activities. However, the enzymatic activities alone are not sufficient to properly target MLE to the X chromosome. These results provide new data on the functional properties of MLE RNA helicase that may help to elucidate its molecular mechanisms of action.

Background Glioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine poly IC]), a strong activator of apoptosis, selectively to cancer cells. Methods and Findings Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEl(25)-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and ``bystander killing'' of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors. Conclusion The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

Ein Ziel der vorliegenden Arbeit war es, die biologische Aktivität des kürzlich von Schneider et al. klonierten, rekombinanten Hühner IL-6 (rChIL-6), zu charakterisieren. In vitro wurde der Nachweis der biologischen Aktivität von rChIL-6 durch die Induktion der Proliferation der streng IL-6 abhängigen murinen Zelllinie 7TD1 erbracht. Diese Tatsache wurde für die Etablierung eines quantitativen rChIL-6 Nachweissystems genutzt. Für weiterführende Studien wurde ein neutralisierendes, polyklonales ChIL-6 Antiserum entwickelt. Durch wiederholte Vakzinierung von Kaninchen mit rChIL-6 (E.coli) war es möglich, ein ChIL-6 Antiserum zu gewinnen, welches auch die biologische Aktivität von natürlichem ChIL-6 neutralisiert. In vivo induzierte die intravenöse Applikation von rChIL-6 (E.coli) einen deutlichen Anstieg der Kortikosteronkonzentration im Serum von Hühnern. Somit standen mit biologisch aktivem rChIL-6, einem ChIL-6 Nachweistest und einem neutralisierenden Antiserum geeignete Werkzeuge zur Verfügung, die für erste Untersuchungen zur funktionellen Charakterisierung des aviären Toll-like Rezeptor (TLR)-Systems genutzt wurden. Diese, unter anderem auf Makrophagen exprimierten Rezeptoren, sind für eine adäquate Reaktion des Immunsystems von essentieller Bedeutung, denn sie erkennen die Anwesenheit pathogener Mikroorgansimen anhand so genannter „pathogen-associated molecular pattern“ (PAMP). Eine Konsequenz der Rezeptoraktivierung ist die Transkription von Genen pro-inflammatorischer Cytokine, unter anderem von IL-6. Primäre Hühnermakrophagen wurden durch Inkubation mit verschiedenen, aus dem Säuger-System bekannten TLR-Agonisten stimuliert; als Nachweissystem der erfolgreichen Aktivierung wurde die quantitative Analyse der ChIL-6 Sekretion genutzt. Zunächst wurden primäre Hühnermakrophagen mit LPS stimuliert worauf sie mit einer starken ChIL-6 Sekretion reagierten. Durch die gleichzeitige Inkubation der Makrophagen mit LPS und IFN- ließ sich das Ausmaß der ChIL-6 Sekretion deutlich steigern. Auch die Inkubation mit bakterieller DNA induzierte die Sekretion von ChIL-6. Dieser Makrophagen-aktivierende Effekt war DNA-spezifisch, denn zum einen beendete der DNA Verdau mit DNase I diesen Effekt. Zum anderen induzierten CpG-ODNs nicht aber GpC-ODNs eine starke ChIL-6 Sekretion. Weiterhin führte die Stimulation der Makrophagen mit synthetischem bakteriellem Lipopeptid und synthetischer dsRNA zu einer deutlichen Sekretion von ChIL-6. Bei in vivo Studien wurde eine deutliche Induktion von ChIL-6 durch LPS (TLR 4) beobachtet. Im Rahmen dieser Arbeit konnte also gezeigt werden, dass rekombinantes ChIL-6 sowohl in vitro als auch in vivo biologisch aktiv ist. Auf der Basis der dabei etablierten Methoden konnte bei ersten Untersuchungen zur funktionellen Charakterisierung des TLR-Systems des Huhnes Hinweise dafür gewonnen werden, dass beim Huhn funktionelle Homologe zu den, aus dem Säuger bekannten TLR 2, 3, 4, und 9 vorhanden sind