Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

Cobertura de gotas de pulverização obtida com diferentes pontas e taxas de aplicação na parte aérea da cana-de-açúcar / Spray droplet coverage achieved with different nozzles and application rates in the aerial part of sugar cane

A aplicação de produtos fitosssanitários no setor sucroalcoleiro, até recentemente, estava focada principalmente no controle de plantas daninhas. Com o aparecimento da ferrugem alaranjada, como doença de importância agrícola na cultura da cana-de-açúcar, passou a ser imperioso conhecer como a aplicação chega na parte aérea da planta. Dessa forma, o presente estudo foi desenvolvido com o objetivo de avaliar a qualidade da deposição da calda em pulverização terrestre, conduzido em esquema fatorial 2x2x2 (duas pontas de pulverização, duas taxas de aplicação e duas posições dos cartões de papéis hidrosensíveis no dossel da planta), seis repetições, com dez papéis cada. Foram utilizadas as pontas de pulverizacão XR110.02 (jato plano de uso ampliado) e TTJ60 - 110.02 (jato plano duplo, com ângulo de 60 graus entre si) utilizando-se as taxas de aplicação de 120 e 160L ha-1. Os cartões de papel hidrossensível utilizados para avaliar a deposicão das pulverizações foram distribuidos sobre as folhas de cana-de-açúcar, em duas posições distintas em relação ao dossel da planta (parte superior e inferior, aproximadamente, 0,80 e 0,50 metros do solo, dispostos na horizontal e inclinado em relação à superfície do solo, respectivamente). A cultivar utilizada foi 'RB 96-6928', com altura de 0,80m aos 120 dias do plantio. Um scaner de mesa foi utilizado para digitalizar as imagens dos papéis e avaliou-se as coberturas através do programa Conta-Gotas®. Os dados de deposição/cobertura foram submetidos à análise de variância e as médias comparadas pelo teste Tukey ao nível de 5% de probabilidade de erro. No dossel superior da cana-de-açúcar observou-se maior cobertura, quando comparado ao dossel inferior, sendo que não se observaram diferenças entre as taxas de aplicação de 120 e 160L ha-1,. A ponta TTJ60-110.02 promoveu cobertura superior à ponta XR 110.02 quando se utilizou 160L ha-1 (40,34 e 36,50%, respectivamente).

The terrestrial application of agricultural defensives in the sugar-alchool producer sector until recently was focused on weed control. After the appearance of orange rust, a disease of agricultural importance in sugar cane production, it has become imperative to know how the application arrives at the plant canopy. In such a way, the present study was developed with the objective of evaluate the quality of the deposition in terrestrial spraying, lead in factorial project 2x2x2 (two nozzles of spraying, two rates of application and two positions of the water-sensitive papers in the canopy of plant), six repetitions, with ten papers each. It has been used the nozzles of spraying XR110.02 (plain jet of extended range) and TTJ60 - 110,02 (double plain jet, with angle of 60 degrees between itself) using the rates of application of 120 and 160L ha-1. The water-sensitive papers cards used to evaluate the deposition of the sprayings had been distributed on the leaves of sugar cane in two distinct heights (upper and lower, approximately 0,80 and 0,50 meters above ground level, arranged in horizontal and inclined with respect to the road surface, respectively). A table scanner was used to generate the digital images of the papers and evaluated the coverings through the Conta-Gotas® program. The deposition data/covering had been submitted to the variance analysis and the averages compared with the Tukey test at 5% of significance. It was observed in the superior canopy of the sugarcane a bigger covering when compared with the inferior, there was no difference between the application rates, 120 and 160L ha-1. The TTJ600110.02 nozzle promoted bigger covering the XR110.02 nozzle when using 160L ha-1 (40,34 and 36,50%, respectively).

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

Proteolytic enzymes have a fundamental role in many biological processes and are associated with multiple pathological conditions. Therefore, targeting these enzymes may be important for a better understanding of their function and development of therapeutic inhibitors. Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidases specificity as they allow monitoring of the reaction on a continuous basis, providing a rapid method for the determination of enzymatic activity. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that permits the measurement of the activity of nanomolar concentrations of the enzyme. The assays can be performed directly in a cuvette of the fluorimeter or adapted for determinations in a 96-well fluorescence plate reader. The synthesis of FRET peptides containing ortho-aminobenzoic acid (Abz) as fluorescent group and 2, 4-dinitrophenyl (Dnp) or N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) as quencher was optimized by our group and became an important line of research at the Department of Biophysics of the Federal University of São Paulo. Recently, Abz/Dnp FRET peptide libraries were developed allowing high-throughput screening of peptidases substrate specificity. This review presents the consolidation of our research activities undertaken between 1993 and 2008 on the synthesis of peptides and study of peptidases specificities.

As enzimas proteolíticas têm um papel fundamental em muitos processos biológicos e estão associadas a vários estados patológicos. Por isso, o estudo da especificidade das peptidases pode ser importante para uma melhor compreensão da função destas enzimas e para o desenvolvimento de inibidores. Os substratos com supressão intramolecular de fluorescência constituem uma excelente ferramenta, pois permitem o monitoramento da reação de forma contínua, proporcionando um método prático e rápido para a determinação da atividade enzimática. A hidrólise de qualquer ligação da cadeia peptídica entre o grupo doador e o grupo supressor gera fluorescência que permite detectar concentração nanomolar de enzima. Os ensaios podem ser acompanhados diretamente na cubeta ou adaptados para determinações de fluorescência em leitoras de placa. A síntese dos peptídeos com supressão intramolecular de fluorescência contendo o grupo fluorescente Abz (orto-aminobenzóico) e o grupo supressor EDDnp (N-[2, 4-dinitrofenil]-etilenodiamino ou Dnp (2, 4-dinitrophenyl) foi otimizada pelo nosso grupo e tornou-se uma importante linha de pesquisa no Departamento de Biofísica da Universidade Federal de São Paulo. Recentemente, foram desenvolvidas bibliotecas de peptídeos fluorogênico contendo Abz/Dnp como grupo doador/supressor trazendo um grande avanço no estudo de especificidade das peptidases. Esta revisão apresenta o trabalho desenvolvido pelo nosso grupo entre 1993 e 2008 sobre a síntese de peptídeos e o estudo da especificidade de peptidases.

A glimpse on biological activities of tellurium compounds

Tellurium is a rare element which has been regarded as a toxic, non-essential trace element and its biological role is not clearly established to date. Besides of that, the biological effects of elemental tellurium and some of its inorganic and organic derivatives have been studied, leading to a set of interesting and promising applications. As an example, it can be highlighted the uses of alkali-metal tellurites and tellurates in microbiology, the antioxidant effects of organotellurides and diorganoditellurides and the immunomodulatory effects of the non-toxic inorganic tellurane, named AS-101, and the plethora of its uses. Inasmuch, the nascent applications of organic telluranes (organotelluranes) as protease inhibitors and its applications in disease models are the most recent contribution to the scenario of the biological effects and applications of tellurium and its compounds discussed in this manuscript.

O telúrio é um elemento não-essencial raro que vem sendo considerado tóxico, e o seu papel biológico é ainda pouco esclarecido. Apesar disso, os efeitos biológicos do telúrio elementar e de alguns derivados inorgânicos e orgânicos que têm sido estudados revelam um conjunto de aplicações diversificadas interessantes e promissoras. Como exemplo, pode-se destacar os usos de teluritos e teluratos de metais alcalinos em microbiologia, o efeito antioxidante de teluretos e diteluretos orgânicos, os efeitos imunomodulatórios e a diversidade de usos correlacionados a este efeito de uma telurana inorgânica denominada AS-101. Ademais, as aplicações de teluranas orgânicas (organoteluranas) como inibidoras de proteases e as aplicações em modelos de doenças compõem a mais recente contribuição ao cenário dos efeitos e aplicações biológicas do telúrio e seus compostos discutidas neste manuscrito.

Efeitos da irradiação com o laser HeNe 632.8nm sobre a cicatrização de feridas em ratos / Los efectos de la aplicación del laser HeNe 632.8nm sobre heridas en ratones / Effects of HeNe 632.8nm laser application on wound healing in rats

O objetivo deste estudo foi investigar os efeitos de laser HeNe 632.8nm sobre o conteúdo de fibroblastos e sua influência no processo cicatrizacional de feridas em ratos. Os animais, após incisão intencional, foram submetidos a irradiação com laser HeNe 632.8nm (4J/cm², única e diária) por períodos de 1, 2, 3, 4, 5, 7, 9, 11, 15, 21 e 29 dias. A avaliação histológica e a contagem de fibroblastos foram realizadas através do programa de captura de imagem Image Pro Plus 5. A irradiação com o laser HeNe 632.8nm promoveu uma redução no conteúdo de fibroblastos na área da ferida irradiada, sugerindo um aumento da síntese de colágeno e na diferenciação de fibroblastos. Os dados indicaram que a irradiação pelo laser HeNe 632.8nm interfere no processo de cicatrização de feridas.

The aim of this study was to investigate the effects of HeNe 632.8nm laser on fibroblast content and its influence on the wound healing process in rats. After a deliberate incision, the animals were subjected to irradiation with HeNe laser 632.8nm (4J/cm², once a day) for periods of 1, 2, 3, 4, 5, 7, 9, 11, 15, 21 and 29 days. The histological analysis and fibroblasts count were analyzed using Image Pro Plus 5 imaging software. HeNe laser irradiation promoted a decrease in the fibroblast content in the irradiated wound area, suggesting an increase in collagen synthesis and fibroblasts differentiation. The data indicated that irradiation with HeNe 632.8nm interferes in the wound healing process.

El objetivo del presente estudio fue investigar los efectos de HeNe 632.8nm láser sobre el contenido de fibroblastos y su influencia en el proceso de cicatrización de heridas en ratones. Los animales tras incisión intencional han sido sometidos a irradiación con laser HeNe 632.8nm (4J/cm., una vez por día) por períodos de 1, 2, 3, 4, 5, 7, 9, 11, 15,21 y 29 días. La evaluación histológica y el número de los fibroblastos fueron analizadas por un programa de captura de imagen Image Pro Plus 5. La irradiación con láser HeNe 632.8nm promovió una disminución en el número de los fibroblastos en el área de la herida, lo que sugiere un aumento en el proceso de síntesis de colágeno y de la diferenciación de fibroblastos. Los datos indicaron que la irradiación por el láser HeNe interfirió en el proceso de cicatrización de la herida.

Avaliação das atividades hipoglicemiante e antihiperglicemiante do extrato hidroalcóolico das folhas da Averrhoa carambola L. (Oxalidaceae) em modelos experimentais de hiperglicemia / Evaluation of hypoglycemic and antihyperglycemic activities in hydroethanolic extract of Averrhoa carambola L. (Oxalidaceae) in experimental models of hyperglycemia

As atividades hipoglicemiante e antihiperglicemiante do extrato hidroalcóolico obtido das folhas da Averrhoa carambola L. (Oxalidaceae) foram avaliadas em ratos, utilizandose diferentes modelos experimentais de hiperglicemia: diabetes tipo 1 induzido por aloxana, intolerância à glicose e resistência aguda à insulina induzidas pela dexametasona e resistência crônica à insulina em modelo de obesidade induzida pelo monoglutamato de sódio. O extrato hidroalcóolico da Averrhoa carambola, na dose de 800 mg/Kg de peso corporal, não apresentou atividades hipoglicemiantes e antihiperglicemiantes nos modelos de hiperglicemia testados