Desenvolvimento de um teste colorimétrico para triagem da atividade leishmanicida de compostos utilizando Leishmania amazonensis expressando a enzima beta-galactosidase

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-graduação em Biotecnologia e Biociências, Florianópolis, 2013No presente trabalho transfectamos uma cepa de Leishmania amazonensis com um plasmídeo contendo o gene da ?-galactosidase, visando estabelecer um ensaio colorimétrico para triagem de compostos ativos contra amastigotas intracelulares de leishmania. A transfecção não alterou o crescimento de promastigotas em cultura, a infectividade de células THP-1 e de camundongos Balb/c. A atividade da enzima foi diretamente proporcional ao número de parasitos. A inibição do crescimento dos parasitos intracelulares pelo fármaco de referência Anfotericina B, determinada tanto pelo método colorimétrico quanto por contagem microscópica dos parasitos, produziu resultados semelhantes, validando o ensaio. O teste colorimétrico também foi aplicado com sucesso para avaliar a atividade de uma classe de moléculas análogas e derivadas do ácido gálico. O estudo da correlação estrutura atividade mostrou que estas moléculas possuem uma atividade leishmanicida pouco seletiva. Algumas melhorias como a transfecção integrativa e estável, bem como padronização deste método com outras espécies de Leishmania ainda são desejáveis. Contudo, o ensaio com L. amazonensis expressando ?-galactosidase foi robusto, sensível, reprodutível e rápido na avaliação da atividade leishmanicida de compostos <br

Branched late-steps of the cytosolic iron-sulphur cluster assembly machinery of Trypanosoma brucei

Support from the Czech Grant Agency (16-18699S to JL) and partial funding by CAPES/Science without Borders (BEX1333/13-5 to MLT) is kindly acknowledged. We are grateful to the University of St. Andrews mass spectrometry facility for collecting and processing MS data and to other members of the TKS and SM groups for their assistance with this project. Work of RL and AJP was supported by the Deutsche Forschungsgemeinschaft (Koselleck grant (to RL) and SPP 1927) and a Coordenação de aperfeiçoamento de pessoal de nivel superior (CAPES - 1333/2013-05) for the financial support to this project. We acknowledge networking support from the COST Action FeSBioNet (Contract CA15133). ERD Funds, The Czech Ministry of Education, project OPVVV 16_019/0000759 to JL.Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.Publisher PDFPeer reviewe

Anti-Infective Pregnane Steroid from the Octocoral Carijoa riisei Collected in South Brazil

SUMMARY. In the present work, fractions of the ethanolic crude extract of Carijoa riisei (Octocorallia) collected at South Brazil (Santa Catarina island) were tested against different bacterial, fungal and protozoal pathogens. The n-hexane fraction (HF) showed a moderate activity against S. aureus in the disk diffusion method, and inhibited 43.4 and 35.9 % the growing of T. cruzi epimastigotes and L. braziliensis promastigotes, respectively. The steroid pregna-1,4,20-trien-3-one was isolated from HF and presented in vitro antiprotozoal activity against the extracellular forms of the parasites at 50 μM, showing 50.4 % growth inhibition of L. braziliensis and 42.4 % growth inhibition of T. cruzi.Fil: Rojo de Almeida, Maria Tereza. Universidade Federal de Santa Catarina; BrasilFil: Tonini, Maiko L.. Universidade Federal de Santa Catarina; BrasilFil: Guimaraes, Tatiana. Universidade Federal de Santa Catarina; BrasilFil: Bianco, Everson. Universidade Federal de Santa Catarina; BrasilFil: Moritz, Maria I.. Universidade Federal de Santa Catarina; BrasilFil: Oliveira, Simone. Universidade Federal de Santa Catarina; BrasilFil: Cabrera, Gabriela Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Palermo, Jorge Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Regginato, Flavio. Universidade Federal de Santa Catarina; BrasilFil: Steindel, Mario. Universidade Federal de Santa Catarina; BrasilFil: Schenkel, Eloir P.. Universidade Federal de Santa Catarina; Brasi

Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis : optimization using a full factorial design

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 22 full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant – soybean lecithin or sorbitan monooleate and type of co-surfactants – polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential -39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 μg⋅g-1) – the main response of interest – confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 μM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment

Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis : optimization using a full factorial design

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 22 full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant – soybean lecithin or sorbitan monooleate and type of co-surfactants – polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential -39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 μg⋅g-1) – the main response of interest – confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 μM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment

The economic burden of households affected by tuberculosis in Brazil: First national survey results, 2019-2021.

BackgroundOne of the three main targets of the World Health Organization (WHO) End TB Strategy (2015-2035) is that no tuberculosis (TB) patients or their households face catastrophic costs (defined as exceeding 20% of the annual household income) because of the disease. Our study seeks to determine, as a baseline, the magnitude and main drivers of the costs associated with TB disease for patients and their households and to monitor the proportion of households experiencing catastrophic costs in Brazil.MethodsA national cross-sectional cluster-based survey was conducted in Brazil in 2019-2021 following WHO methodology. TB patients of all ages and types of TB were eligible for the survey. Adult TB patients and guardians of minors (ResultsWe interviewed 603 patients, including 538 (89%) with drug-sensitive (DS) and 65 (11%) with drug-resistant (DR) TB. Of 603 affected households, 48.1% (95%CI: 43-53.2) experienced costs above 20% of their annual household income during their TB episode. The proportion was 44.4% and 78.5% among patients with DS- and DR-TB, respectively. On average, patients incurred costs of US$1573 (95$317.6, 95%CI: 232.7-402.6) followed by medical costs (US$85.5, 95$79.2, 95%CI: 61.9-96.5). In multivariate analysis, predictors of catastrophic costs included positive HIV status (aOR = 3.0, 95%CI:1.1-8.6) and self-employment (aOR = 2.7, 95%CI:1.1-6.5); high education was a protective factor (aOR = 0.1, 95%CI:0.0-0.9).ConclusionsAlthough the services offered to patients with TB are free of charge in the Brazilian public health sector, the availability of free diagnosis and treatment services does not alleviate patients' financial burden related to accessing TB care. The study allowed us to identify the costs incurred by patients and suggest actions to mitigate their suffering. In addition, this study established a baseline for monitoring catastrophic costs and fostering a national policy to reduce the costs to patients for TB care in Brazil