Development of Si-based electrical biosensors: Simulations and first experimental results

In this work, we simulated and experimentally assessed the possibility to detect, through electrical transduction, hybridization of DNA molecules on MOS-like devices, having different dielectrics: SiO2, Si3N4 and SiO2/Si3N4/SiO2 (ONO). The electrical characterization was performed after the various functionalization steps, consisting of dielectric activation, silanization, DNA spotting and anchoring, and after the hybridization process, to test the devices effectiveness as DNA recognition biosensors. The experimental results were used to validate device simulations. The comparison shows the ability to determine a priori the DNA probe density needed to maximize the response. The results confirm that the structures analyzed are sensitive to the immobilization of DNA and its hybridization

Diversidade Morfológica de Rizobactérias Obtidas de Solos SoB Distintos Manejos de Cultivo da Região Oeste do Paraná

As bactérias diazotróficas encontradas na rizosfera apresentam características fenotípicas distintas e mutáveis dependendo do meio ambiente em que estão inseridas. Neste contexto, o presente trabalho tem como objetivo isolar e caracterizar a morfologia de colônias  de bactérias presentes em amostras de solo coletadas de diferentes manejos de cultivo da região oeste do estado do Paraná. Os manejos contemplados para o estudo foram Sistema Agropastoril (M1), Horticultura (M2), Pastagem (M3), Soja 1 (M4), Mata nativa (M5) e Soja 2 (M6). As amostras de solo foram analisadas em laboratório diluindo-as em solução salina 0,85% até a diluição de trabalho 10-3 a qual foi plaqueada em meio de cultura sólido. Após três dias de crescimento em B.O.D á 27ºC as colônias foram classificadas morfologicamente de acordo com o protocolo estabelecido por Fonseca et al. (2000). Os dados processados após a classificação revela que foram obtidos 136 isolados bacterianos, que foram selecionados para a construção dos agrupamentos obtendo o total de 29 grandes grupos (GG), sendo o manejo M5 (mata nativa) o que teve um maior número de representantes.

Diversidade Morfológica de Rizobactérias Obtidas de Solos SoB Distintos Manejos de Cultivo da Região Oeste do Paraná

As bactérias diazotróficas encontradas na rizosfera apresentam características fenotípicas distintas e mutáveis dependendo do meio ambiente em que estão inseridas. Neste contexto, o presente trabalho tem como objetivo isolar e caracterizar a morfologia de colônias  de bactérias presentes em amostras de solo coletadas de diferentes manejos de cultivo da região oeste do estado do Paraná. Os manejos contemplados para o estudo foram Sistema Agropastoril (M1), Horticultura (M2), Pastagem (M3), Soja 1 (M4), Mata nativa (M5) e Soja 2 (M6). As amostras de solo foram analisadas em laboratório diluindo-as em solução salina 0,85% até a diluição de trabalho 10-3 a qual foi plaqueada em meio de cultura sólido. Após três dias de crescimento em B.O.D á 27ºC as colônias foram classificadas morfologicamente de acordo com o protocolo estabelecido por Fonseca et al. (2000). Os dados processados após a classificação revela que foram obtidos 136 isolados bacterianos, que foram selecionados para a construção dos agrupamentos obtendo o total de 29 grandes grupos (GG), sendo o manejo M5 (mata nativa) o que teve um maior número de representantes.

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.</p

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.</p

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson’s disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2’s phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation