Evaluation of the action of precipitating agents to obtain allergenic proteins from crude mite extracts / Avaliação da ação de agentes precipitantes na obtenção de proteínas alergênicas presentes em extratos brutos de ácaros

Introduction: Allergen proteins found in dust mite extracts, such as Dermatophagoides farinae (DF), Dermatophagoides pteronyssinus (DP) and Tyrophagus putrescentiae (TP), are relevant for scientific studies in the allergy and immunotherapy fields. The precipitation/concentration of protein extracts may favor the aggregation of the allergens in homogenates. Objective and method: This paper investigates the precipitation process by submitting crude mite extracts to compounds such as ammonium sulfate (NH4)2SO4, trichloroacetic acid (TCA) and acetone. Results: The best results were obtained by fractionation with (NH4)2SO4 at 80% (w/v) saturation (~0° C), observing the protein markings on the electrophoresis gel. Major allergens were identified by immunoblot at 25 kDa (cysteine protease) for Der f and Der p; and 25 kDa, 30 kDa (tropomyosin) and Try p 3, near 26 kDa. For this percentage the total protein contents were 12.83 mg mL-1 for Der f, 24.78 mg mL-1 for Der p and 27.35 mg mL-1 for Try. Conclusion: An advantage of precipitation with (NH4)2SO4 over precipitation with acetone was the possibility of gradually obtaining protein fractions, which does not happen when using the latter. The addition of 80% (v/v) acetone to the mite extracts favored total protein precipitation in the concentrations 16.42 mg mL-1, 28.47 mg mL-1 and 13.41 mg mL-1. The use of TCA in concentrations above 20% (w/v) forms peptides that are not retained in the gel under the established experimental conditions, and dilute solutions of this acid are more efficient.

Introdução: As proteínas alergênicas presentes nos extratos dos ácaros de poeira, tais como Dermatofagoides farinae (DF), Dermatofagoides pteronyssinus (DP) e Tyrophagus putrescentiae (TP) são relevantes para estudos científicos na área de alergias e aplicação em imunoterapias. A precipitação/concentração desses extratos proteicos pode favorecer a agregação de alérgenos nos homogenatos. Objetivo e método: O trabalho investiga o processo de precipitação, submetendo os extratos brutos de ácaros de poeira a compostos como sulfato de amônio (NH4)2SO4, ácido tricloroacético (ATC) e acetona. Resultados: Os melhores resultados foram obtidos por fracionamento com (NH4)2SO4 em 80% (m/v) de saturação (~ 0°C), observando as marcações proteicas no gel de eletroforese. Os alérgenos principais foram identificados por immunoblot em 25 kDa (cisteína protease) para Der f 1 e Der p 1; e 25 kDa, 33 kDa (tropomyosin), 11 kDa para Tyr. Para esse percentual, os teores de proteína total foram de 12.83 mg mL-1 para DF; 24,78 mg mL-1 para DP; e 27,35 mg mL-1 para TP. Conclusão: A vantagem da precipitação com (NH4)2SO4 frente à precipitação com acetona foi a possibilidade de gradativamente se obter frações proteicas, o que não acontece quando utilizado esse solvente. A adição de 80% (v/v) de acetona aos extratos de ácaros favoreceu a precipitação total de proteína nas concentrações 16,42 mg mL-1; 28,47 mg mL-1; e 13,41 mg mL-1. O uso de ATC em concentrações acima de 20% (m/v) forma peptídeos que não são retidos no gel nas condições experimentais estabelecidas, sendo eficiente soluções mais diluídas desse ácido.

Use of plant materials for the bioremediation of soil from an industrial site.

Bioremediation is one of the existing techniques applied for treating oil-contaminated soil, which can be improved by the incorporation of low-cost nutritional materials. This study aimed to assess the addition of two low-cost plant residues, sugarcane bagasse (SCB) and leaf litter (LL) of the forest leguminous Mimosa caesalpiniifolia plant (sabiá), either separately or combined, to a contaminated soil from a petroleum refinery area, analyzed after 90 days of treatment. Individually, both amounts of SCB (20 and 40 g kg-1) favored the growth of total heterotrophic bacteria and total fungi, while LL at 20 g kg-1 better stimulated the hydrocarbon-degrading microorganism's activity in the soil. However, no TPH removal was observed under any of these conditions. Higher microbial growth was detected by the application of both plant residues in multicontaminated soil. The maximum TPH removal of 30% was achieved in amended soil with 20 g kg-1 SCB and 20 kg-1 LL. All the experimental conditions revealed changes in the microbial community structure, related to the handling of the soil, with abundance of Alphaproteobacteria. This study demonstrates the effectiveness of the plant residues SCB and LL as low-cost nutritional materials for biodegradation of hydrocarbon in real oil contaminated soil by indigenous populations.

Analysis of methane biodegradation by Methylosinus trichosporium OB3b / Análise da biodegradação de metano por Methylosinus trichosporium OB3b

The microbial oxidation of methane in the atmosphere is performed by methanotrophic bacteria that use methane as a unique source of carbon and energy. The objective of this work consisted of the investigation of the best conditions of methane biodegradation by methanotrophic bacteria Methylosinus trichosporium OB3b that oxidize it to carbon dioxide, and the use of these microorganisms in monitoring methods for methane. The results showed that M. trichosporium OB3b was capable to degrade methane in a more effective way with an initial microorganism concentration of 0.0700 g.L-1, temperature of 30ºC, pH 6.5 and using 1.79 mmol of methane. In these same conditions, there was no bacterial growth when 2.69 mmol of methane was used. The specific rate of microorganism growth, the conversion factor, the efficiency and the volumetric productivity, for the optimized conditions of biodegradation were, respectively, 0.0324 h-1, 0.6830 gcells/gCH4, 73.73% and 2.7732.10-3 gcells/L.h. The final product of methane microbiological degradation, carbon dioxide, was quantified through the use of a commercial electrode, and, through this, the grade of methane conversion in carbon dioxide was calculated.

A oxidação microbiológica de metano na atmosfera é realizada por bactérias metanotróficas, que o utilizam como fonte única de carbono e energia. O objetivo deste trabalho consistiu na investigação das melhores condições de biodegradação do metano por bactérias metanotróficas Methylosinus trichosporium OB3b, que o oxidam a dióxido de carbono, para o emprego destes microrganismos em métodos de monitoração para metano. Os resultados obtidos mostraram que M.trichosporium OB3b foi capaz de degradar o metano de forma mais eficaz partindo-se de uma concentração inicial de microrganismos de 0.0700 g.L-1, a uma temperatura de 30ºC, pH igual a 6.5 e empregando-se 1.79 mmol de metano. Nestas mesmas condições, não houve crescimento bacteriano quando foram empregados 2.69 mmol de metano. A taxa específica de crescimento do microrganismo, o fator de conversão, a eficiência e a produtividade volumétrica para as condições otimizadas de biodegradação foram, respectivamente, 0.0324 h-1, 0.6830 gcélulas/gCH4, 73.73% e 2.7732.10-3 gcélulas/L.h. O produto final da degradação microbiológica do metano, o dióxido de carbono, foi quantificado através do emprego de um eletrodo comercial, e, através desta medida, foi calculado o percentual de conversão de metano em dióxido de carbono.

Amperometric immunosensor for detecting Schistosoma mansoni antibody.

An immunosensor for detecting the antibody anti-apyrase of Schistosoma mansoni based on rigid composite materials, containing graphite powder and epoxy resins, developed in this work, is described. A surface modification strategy for the use of oxidized graphite in the detection of antibody-antigen interaction was developed. This modification strategy is based on silanization of conductive composite. First, the graphite powder-epoxy resin was treated with concentrated hydrogen peroxide to improve surface hydroxyl groups and to form a hydrophilic layer. Second, 3- aminopropyltriethoxysilane was subsequently used to functionalize the treated surface to form amino groups, which were further activated with glutaraldehyde to introduce a layer of aldehyde groups. Contact angle microscopy and scanning electron microscopy were used as a qualitative analysis of the deposition of silane on the surface of the sensor. The effectiveness of the modification strategy was validated by amperometric immunoassays of S. mansoni. Amperometric signals related to concentrations of this immobilized protein were observed, and the effects of pH and incubation times were analyzed. This surface modification strategy provides a platform on which proteins can be directly immobilized for immunosensor and protein array applications.