Use of medication and associated factors in adults living in Rio Branco, Acre. Use of medication in adults

Abstract We analyzed use of medication and associated factors in adults aged 18-59 years living in Rio Branco, Acre. This is a cross-sectional and population-based study that used a probabilistic sample of the population from rural and urban areas of the city of Rio Branco, Acre. The Prevalence Ratio (PR) was calculated with 95% confidence intervals and associations were estimated by Poisson regression. This study found a 29.4% prevalence ratio of use of medication among individuals aged from 18 to 59 years (685 adults: 473 women and 212 men; producing estimates for 211,902 adults: 110,769 women and 101,133 men). After adjusted analysis, their use was associated with: age (50-59 years, PR: 2.36; 95%CI: 2.29-2.43); women (PR: 1.25; 95%CI: 1.23-1.27); up to elementary school (PR: 1.13; 95%CI: 1.11-1.15); and poor or very poor self-rated health (PR: 1.47; 95%CI: 1.43-1.51). The health conditions associated with use of medication were: number of comorbidities, hypertension, diabetes, insomnia, depression, number of health complaints and use of health services. The most frequently used drugs were those belonging to the following ATC categories: alimentary tract and metabolism, cardiovascular system, nervous system, and the musculoskeletal system.

pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.

The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.

Caracterização termodinâmica e funcional da proteína não estrutural 3 (NS3) do vírus da dengue do tipo 2

O propósito deste trabalho foi a clonagem, expressão e purificação da proteína NS3 íntegra do DENV-2, seus domínios e complexos isoladamente, com objetivo de avaliar i) a estabilidade da NS3 íntegra e dos domínios protease e helicase; ii) o efeito do domínio protease sobre a atividade NTPásica; e iii) o papel do cofator NS2B na atividade e estrutura da NS3 protease. Utilizamos técnicas espectroscópicas para o monitoramento da fluorescência intrínseca do Trp e ensaios de atividade helicásica, ATPásica e proteásica. Nossos dados termodinâmicos demonstram a importância do domínio protease na estabilidade da NS3 íntegra, e que o interdomínio E169-K185 (que conecta os domínios protease e helicase) é importante na modulação da atividade proteolítica, interação da protease com o cofator NS2B, e estabilidade do domínio protease. Observamos que o cofator NS2B fornece maior estabilidade e cooperatividade ao domínio protease em pH básico, além de promover modificações conformacionais que reduzem a exposição de resíduos hidrofóbicos que parecem prejudicar na interação de resíduos dibásicos com a tríade catalítica. Ao avaliar se a baixa atividade ATPásica da NS3 íntegra em comparação com a NS3 helicase estaria associada com a oclusão do motivo Walker A pelo domínio protease, demonstramos que a NS3 helicase apresenta maior atividade ATPase, em pH 6,0, devido à exposição de resíduos hidrofóbicos que favorecem a ligação do ATP. Contudo, em pH fisiológico, a interação do ATP com o motivo Walker A foi maior na NS3 íntegra, demonstrando que a localização do domínio protease sobre o motivo de formação do complexo ATP-Mg2+ não interfere na interação. E finalmente, analisando a fenda de interação do ATP na NS3 íntegra dos 4 sorotipos do DENV, identificamos oito resíduos altamente conservados (R64, E66, G80, K201, R202, R209, K213 e R236) circundando esta região de ligação, que podem ser importantes no desenvolvimento de inibidores específicos desta enzima.

The aim of this work was the cloning, expression, and purification of the full-length NS3 protein of the DENV-2 as well as the its domains and protein complexes, aiming to study i) the thermodynamic stability of the full-length NS3, and of its protease and helicase domains; ii) the effect of the protease domain on ATPase activity; and iii) the effect of the NS2B cofactor on the structure and activity of the protease domain. We used fluorescence spectroscopy to monitor the Trp intrinsic fluorescence, and ATPase, helicase and protease activity assays. Our results thermodynamics showed the importance of the protease domain for the stabilization of the full-length NS3 protein of interdomain peptide (E169-K185) for activity, the interaction of the protease domain with cofactor NS2B, and the stability of the protease domain. We observed that the cofactor NS2B promoted not only a higher stability and cooperativity to the protease domain at basic pH, but also conformational changes that decreased the exposure of hydrophobic residues, disfavoring the interaction of dibasic residues with the catalytic triad. When we evaluated whether the decrease ATPase activity of the full-length NS3, in comparison with NS3 helicase, would be associated or not with the occlusion of the Walker A motif by protease domain, our results showed that the NS3 helicase increased ATPase activity at pH 6.0 because of the hydrophobic residue exposure favoring the ATP binding. On the other hand, the interaction of the ATP with Walker A motif was higher in the full-length NS3 than in the NS3helicase, suggesting that the localization of the protease domain on the helicase domain does not interfere with the interaction of the latter with the ATP-Mg+2. Finally, by analyzing the ATP binding site cleft in the full-length NS3 protein from the four DENV serotypes (DENV-1 to 4), we identified eight highly conserved residues (R64, E66, G80, K201, R202, R209, K213 and R236) surrounding this cleft that can be important in the development of specific inhibitors of that enzyme.