Current Microscopy Strategies to Image Fungal Vesicles: From the Intracellular Trafficking and Secretion to the Inner Structure of Isolated Vesicles.

Extracellular vesicles (EVs) are nano-sized structures that play important roles in a variety of biological processes among members of the Eukaryota domain. They have been studied since the 1940s and a broader use of different microscopy techniques to image either isolated vesicles or vesicles within the intracellular milieu (trafficking) has been limited by their nanometric size, usually below the resolution limit of most standard light microscopes. The development of genetically encoded fluorescent proteins and fluorescent probes able to switch between "on" and "off" states, as well the improvement in computer-assisted microscopy, photon detector devices, illumination designs, and imaging strategies in the late Twentieth century, boosted the use of light microscopes to provide structural and functional information at the sub-diffraction resolution, taking advantage of a nondestructive analytical probe such light, and opening new possibilities in the study of life at the nanoscale. As well, traditional and novel electron microscopy techniques have been widely used in the characterization of subcellular compartments, either isolated or in situ, providing a comprehensive understanding of their functional role in many cellular processes. Here, we present basic aspects of some of these techniques that have already been applied and their potential application to the study of fungal vesicles.

Nutrição Esportiva como Componente Curricular nos Cursos de Nutrição e Educação Física do Centro-Oeste do Brasil / Sports Nutrition as Curricular Component in the Nutrition and Physical Education Courses of the Center-West of Brazil

Esse estudo objetivou caracterizar a disciplina de Nutrição Esportiva nos cursos de Graduação em Nutrição e Educação Física das Instituições de Ensino Superior do Centro-Oeste do Brasil e Distrito Federal. Material e Métodos: Adotou-se como critério de inclusão os cursos presenciais ou de educação à distância de Graduação em Nutrição (Bacharelado) e Educação Física (Bacharelado e Licenciatura) das Instituições de Ensino Superior reconhecidas pelo Ministério da Educação. Foram analisados os três estados que compõem a Região Centro-Oeste do Brasil e o Distrito Federal. As Instituições foram selecionadas por meio da Base de Dados do Sistema de Regulação do Ensino Superior ­ Sistema e-MEC e para a avaliação considerou-se a presença da disciplina Nutrição Esportiva e sua caracterização de oferta como sua denominação; natureza (obrigatória ou optativa); carga horária e período ministrado. Resultados: Observou-se que para 100% dos cursos de Nutrição a disciplina é de natureza obrigatória, ao passo para que para a Educação Física esse percentual foi de 95,23%. Ambos os cursos apresentaram ampla variabilidade da carga horária total e maior carga horária teórica, quando comparada à carga horária prática. Também, alguns estados não destinam nenhuma carga horária prática para sua realização. Conclusão: Apesar de os cursos apresentarem similaridade em seu perfil de oferta para a maioria dos aspectos avaliados, faz-se necessário entender se as diferenças encontradas impactam no processo de ensino-aprendizagem da disciplina estudada. (AU)

Objective: This study aimed to characterize the discipline of Sports Nutrition in the Nutrition and Physical Education Undergraduate courses of the Institutions of Higher Education of the Center-West of Brazil and the Federal District. Material and Methods: The adopted inclusion criteria were the presential or distance education courses of Undergraduate Nutrition (Bachelor) and Physical Education (Bachelor and Graduation) of Higher Education Institutions recognized by the Ministry of Education. The three states that compose the Central-West Region of Brazil and the Federal District were analyzed. The Institutions were selected through the Database of the System of Regulation of Higher Education - e-MEC System and for the evaluation, it was considered the presence of the discipline of Sports Nutrition and its offer characterization as its denomination; nature (required or optional); workload and time period. Results: It was observed that for 100% of the Nutrition courses the discipline is required, whereas for Physical Education this percentage was 95.23%. Both courses presented wide variability of the total workload and higher theoretical workload compared to the practical workload. Also, some states do not allocate any practical workload for their realization. Conclusion: Although the courses present similarity in their offer profile for most aspects evaluated, it is necessary to understand if the differences found, impact on the teaching-learning process of the studied discipline. (AU)

Selective and cost-effective protocol to separate bioactive triterpene acids from plant matrices using alkalinized ethanol: Application to leaves of Myrtaceae species.

BACKGROUND: Triterpenes as betulinic (BA), oleanolic (OA) and ursolic acids (UA) have increasingly gained therapeutic relevance due to their wide scope of pharmacological activities. To fit large-scale demands, exploitable sources of these compounds have to be found and simple, cost-effective methods to extract them developed. Leaf material represents the best plant sustainable raw material. To obtain triterpene acid-rich extracts from leaves of Eugenia, Psidium and Syzygium species (Myrtaceae) by directly treating the dry plant material with alkalinized hydrated ethanol. This procedure was adapted from earlier methods to effect depolymerization of the leaf cutin. MATERIALS AND METHODS: Extracts were prepared by shaking the milled dry leaves in freshly prepared 2% NaOH in 95% EtOH solution (1:4 w/v) at room temperature for 6 h. Working up the product in acidic aqueous medium led to clear precipitates in which BA, OA and UA were quantified by gas chromatography. RESULTS: Pigment-free and low-polyphenol content extracts (1.2-2.8%) containing 6-50% of total triterpene acids were obtained for the six species assayed. UA (7-20%) predominated in most extracts, but BA preponderated in Eugenia florida (39%). Carried out in parallel, n-hexane defatted leaves led to up to 9% enhancement of total acids in the extracts. The hydroalcoholate treatment of Myrtaceae species dry leaves proved to be a cost-effective and environmentally friendly method to obtain triterpene acids, providing them be resistant to alkaline medium. These combined techniques might be applicable to other plant species and tissues.

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 µgkg(-1)) to 18.6 (erythromycin at 25 µgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 µgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCß) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 µgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 µgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.

A liquid chromatography-tandem mass spectrometry confirmatory assay for the simultaneous determination of several tetracyclines in milk considering keto-enol tautomerism and epimerization phenomena.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method for the analysis of several tetracyclines residues in bovine milk has been developed. Milk deproteinization/extraction of samples was performed with acidified acetonitrile. After diluting and purification by solid-phase extraction (SPE), the extracts were injected into the instrument operated in Multiple Reaction Monitoring (MRM) acquisition mode. The reversible epimerization at C-4 of oxytetracycline, tetracycline and chlortetracycline and the keto-enol tautomerism of chlortetracycline between C-11a and C-12 were considered for reliable quantification. Degradation was also taken in account and minimized for the same purpose. A central composite (response surface) design with desirability function was employed for the optimization of extraction and clean-up steps. The optimization improved the extraction efficiency of the more polar analytes reaching 93.9% for 4-epioxytetracycline and 95.8% for oxytetracycline at 100 microg L(-1). The validation was performed following the criteria established by Commission Decision 2002/657/EC.