Antiretroviral activity of protease inhibitors against Toxoplasma gondii

The introduction of highly active antiretroviral therapy (HAART) has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE). These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API) on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

Mind the gap: characterization of periplasmic cytochromes from Shewanella oneidensis involved in extracellular electron transfer

Dissertation presented to obtain the Ph.D degree in Biochemistry

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001) and among the strains from different sites of origin (p=0.014). Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%). Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901). CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

Analysis of metabolic flux distributions in relation to the extracellular environment in Avian cells

Continuous cell lines that proliferate in chemically defined and simple media have been highly regarded as suitable alternatives for vaccine production. One such cell line is the AG1.CR.pIX avian cell line developed by PROBIOGEN. This cell line can be cultivated in a fully scalable suspension culture and adapted to grow in chemically defined, calf serum free, medium [1]–[5]. The medium composition and cultivation strategy are important factors for reaching high virus titers. In this project, a series of computational methods was used to simulate the cell’s response to different environments. The study is based on the metabolic model of the central metabolism proposed in [1]. In a first step, Metabolic Flux Analysis (MFA) was used along with measured uptake and secretion fluxes to estimate intracellular flux values. The network and data were found to be consistent. In a second step, Flux Balance Analysis (FBA) was performed to access the cell’s biological objective. The objective that resulted in the best predicted results fit to the experimental data was the minimization of oxidative phosphorylation. Employing this objective, in the next step Flux Variability Analysis (FVA) was used to characterize the flux solution space. Furthermore, various scenarios, where a reaction deletion (elimination of the compound from the media) was simulated, were performed and the flux solution space for each scenario was calculated. Growth restrictions caused by essential and non-essential amino acids were accurately predicted. Fluxes related to the essential amino acids uptake and catabolism, the lipid synthesis and ATP production via TCA were found to be essential to exponential growth. Finally, the data gathered during the previous steps were analyzed using principal component analysis (PCA), in order to assess potential changes in the physiological state of the cell. Three metabolic states were found, which correspond to zero, partial and maximum biomass growth rate. Elimination of non-essential amino acids or pyruvate from the media showed no impact on the cell’s assumed normal metabolic state.

Modeling and optimization of extracellular polysaccharides production by Enterobacter A47

Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters.

The role of Pep4p, the vacuolar yeast protease ortholog of human cathepsin D, in mitochondria-dependent apoptosis

Doctoral Thesis (PhD Programm on Molecular and Environmental Biology)

Seleção de leveduras da Região Amazônica para produção de protease extracelular

O objetivo deste trabalho foi identificar 50 leveduras isoladas a partir de diferentes substratos da Região Amazônica e selecionar uma espécie de maior atividade proteolítica. Entre as 26 espécies identificadas predominaram Candida aquatica (12%) e Candida tropicalis (10%). A fermentação submersa foi realizada em Extrato de Malte suplementado com gelatina 1%, Candida intermedia foi a que expressou maior atividade proteolítica, halo = 25 mm e 273 U/mL, crescimento máximo a 30 ºC, viabilidade celular 6,2 x 10(6) UFC, em 48 horas, não demonstrou características de patogenicidade e a melhor densidade do inóculo foi 3%, utilizando-se culturas com 24 horas de crescimento em ágar Malte.

Mechanism of extracellular silver nanoparticles synthesis by Stereum hirsutum and Fusarium oxysporum

The increasing interest for greener and biological methods of synthesis has led to the development of non-toxic and comparatively more bioactive nanoparticles. Unlike physical and chemical methods of nanoparticle synthesis, microbial synthesis in general and mycosynthesis in particular is cost-effective and environment-friendly. However, different aspects, such as the rate of synthesis, monodispersity and downstream processing, need to be improved. Many fungal-based mechanisms have been proposed for the formation of silver nanoparticles (AgNPs), mainly those involving the presence of nitrate reductase, which has been detected in filtered fungus cell used for AgNPs production. There is a general acceptance that nitrate reductase is the main responsible for the reduction of Ag ions for the formation of AgNPs. However, this generally accepted mechanism for fungal AgNPs production is not totally understood. In order to elucidate the molecules participating in the mechanistic formation of metal nanoparticles, the current study is focused on the enzymes and other organic compounds involved in the biosynthesis of AgNPs. The use of each free fungal mycelium of both Stereum hirsutum and Fusarium oxysporum will be assessed. In order to identify defective mutants on the nitrate reductase structural gene niaD, fungal cultures of S.hirsutum and F.oxysporum will be selected by chlorate resistance. In addition, in order to verify if each compound identified as key-molecule influenced on the production of nanoparticles, an in vitro assay using different nitrogen sources will be developed. Lately, fungal extracellular enzymes will be measured and an in vitro assay will be done. Finally, The nanoparticle formation and its characterization will be evaluated by UV-visible spectroscopy, electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transforms infrared spectroscopy (FTIR), and LC-MS/MS.

Development of hyaluronic acid, dextrin and extracellular matrix hydrogels for cell expansion

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Produção de protease colagenolítica por Bacillus stearothermophillus de solo amazônico

As proteases colagenolíticas são enzimas capazes de hidrolisar as ligações peptídicas de vários tipos de colágeno e têm grande importância na medicina e em aplicações terapêuticas. O objetivo desta pesquisa foi avaliar a produção de proteases colagenolíticas por Bacillus stearothermophilus. Os tratamentos foram realizados por meio de um planejamento fatorial completo 2³, a fim de avaliar a significância dos efeitos e interações das variáveis - pH inicial, concentração de substrato e temperatura - sobre a produção de protease colagenolítica. O ponto central foi executado em quadruplicata para fornecer uma estimativa dos erros experimentais. Ensaios enzimáticos com colágeno e azocaseína como substratos foram realizados para determinação das atividades colagenolítica e proteolítica respectivamente. A maior atividade enzimática colagenolítica foi 79,38 U mL-1, correspondendo a atividade específica de 136,86 U mg-1, em condições iniciais de fermentação, na concentração de substrato a 1% (p/v), pH 7,2 e 25 °C. A atividade proteolítica da enzima foi mais ativa em pH 9,0 e 50 °C e foi estável nas faixas de pH (6,0 - 9,0) e temperatura (45 °C - 50 °C). Bacillus stearothermophilus apresenta viabilidade para a produção de proteases colagenolíticas e a obtenção dessas enzimas tem grande importância para aplicações biotecnológicas.

Expresión y rol del SLPI (Secretory leukocyte protease inhibitor) en la inmunidad innata del ojo asociado a procesos inflamatorios e infecciosos oculares

En la terapia antimicrobiana, se pretende comprender y desarrollar sinergia con los mecanismos de defensa innatos del huésped, en el área oftalmológica, los relacionados a procesos oculares infecciosos tanto específicos como no específicos. Conservando y estimulando la presencia de los péptidos antimicrobianos, se obtendría una prevención e inhibición de los microorganismos responsables de los principales procesos infecciosos e inflamatorios oculares, que permitirá un tratamiento rápido y eficaz en el control del proceso patológico. De esta forma se podrá evitar secuelas devastadoras de dichas complicaciones que en un gran porcentaje llevan a la ceguera del paciente asociadas al diagnóstico tardío, inespecífico o asociado a resistencia a los antibióticos actuales. Los péptidos antimicrobianos y anti-inflamatorios prometen ser un método terapéutico eficaz, natural, libre de efectos secundarios y adversos en la terapia antimicrobiana e inmuno moduladora futura. Basados en estudios publicados realizados en animales, estospéptidos con capacidad antibacteriana se expresarían en tejidos oculares normales actuando como agentes antimicrobianos de la inmunidad innata del ojo y así también como regulador de la actividad inflamatoria. Estos péptidos antimicrobianos – anti inflamatorios podrían utilizarse para la prevención y tratamiento de procesos infecciosos oculares, en formulaciones simples o combinadas a otras sustancias antibióticas antimicrobianas en forma sinérgica. Secretory leukocyte protease inhibitor (SLPI), terminología anglosajona que se utilizó para definir a una proteína de 12 kDa de peso molecular cuya única función conocida hasta hace unos años era la de ser secretada por leucocitos para inhibir proteasas de la matriz extracelular. El objetivo es estudiar la presencia del péptido antimicrobiano SLPI en relación con el remodelado cicatrizal de la matriz extracelular de la cornea y otras estructuras intra oculares, como así también su expresión en relación a procesos inflamatorios e infecciosos del ojo como agente antimicrobiano de la inmunidad innata del huésped.

Expresión y rol del slpi (secretory leukocyte protease inhibitor) en la inmunidad innata del ojo asociado a procesos inflamatorios e infecciosos oculares.

En la terapia antimicrobiana, se pretende comprender y desarrollar sinergia con los mecanismos de defensa innatos del huésped, en el área oftalmológica, los relacionados a procesos oculares infecciosos tanto específicos como no específicos. Conservando y estimulando la presencia de los péptidos antimicrobianos, se obtendría una prevención e inhibición de los microorganismos responsables de los principales procesos infecciosos e inflamatorios oculares, que permitirá un tratamiento rápido y eficaz en el control del proceso patológico.De esta forma se podrá evitar secuelas devastadoras de dichas complicaciones que en un gran porcentaje llevan a la ceguera del paciente asociadas al diagnóstico tardío, inespecífico o asociado a resistencia a los antibióticos actuales.Los péptidos antimicrobianos y anti-inflamatorios prometen ser un método terapéutico eficaz, natural, libre de efectos secundarios y adversos en la terapia antimicrobiana e inmuno moduladora futura.Basados en estudios publicados realizados en animales, estos péptidos con capacidad antibacteriana se expresarían en tejidos oculares normales actuando como agentes antimicrobianos de la inmunidad innata del ojo y así también como regulador de la actividad inflamatoria. Estos péptidos antimicrobianos – anti inflamatorios podrían utilizarse para la prevención y tratamiento de procesos infecciosos oculares, en formulaciones simples o combinadas a otras sustancias antibióticas antimicrobianas en forma sinérgica.Secretory leukocyte protease inhibitor (SLPI), terminología anglosajona que se utilizo para definir a una proteína de 12 kDa de peso molecular cuya única función conocida hasta hace unos años era la de ser secretada por leucocitos para inhibir proteasas de la matriz extracelular. El objetivo es estudiar la presencia del péptido antimicrobiano SLPI en relación con el remodelado cicatrizal de la matriz extracelular de la cornea y otras estructuras intra oculares, como así también su expresión en relación a procesos inflamatorios e infecciosos del ojo como agente antimicrobiano de la inmunidad innata del huésped

Estudo da reatividade vascular em portadores de HIV com e sem uso de inibidor de protease

FUNDAMENTO: Considerando o alto número de pacientes infectados por HIV em uso de antirretrovirais, evoluindo com alteração da função endotelial e aterotrombose, levando ao alto custo médico social, é importante identificar mecanismos fisiopatológicos envolvidos com a função endotelial em portadores de HIV, para que, precocemente, possamos intervir e evitar a progressão da doença. OBJETIVO: Avaliar a função endotelial pela vasodilatação endotélio dependente e independente em pacientes HIV positivo e em grupo controle. MÉTODOS: O estudo avaliou 27 pacientes HIV positivo e 16 do grupo controle. A avaliação da função endotelial foi realizada por meio da vasodilatação da artéria braquial endotélio dependente (hiperemia reativa) e independente (nitroglicerina SL). RESULTADOS: Pacientes HIV positivo em uso de inibidor de protease (IP) apresentaram vasodilatação endotélio independente significativamente menor que os subgrupos HIV negativo (p = 0,020) e HIV positivo sem uso de IP (p = 0,034). A variação do diâmetro da artéria braquial durante hiperemia reativa não apresentou significância estatística em qualquer subgrupo. A análise de regressão linear múltipla mostrou que apenas o IP estava associado ao delta relativo da reatividade braquial pelo vasodilatador, nos pacientes HIV positivo, aos 60 e 90 segundos. CONCLUSÃO: Os pacientes HIV positivo em uso de IP apresentam disfunção endotélio independente quando comparados a pacientes HIV positivo que não fazem uso de IP e a um grupo controle.