The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

Abstract The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

Produtos da reação de Maillard em alimentos industrializados / Maillard reaction products in processed food

The ingestion of Maillard-reaction products (MRP) has increased over thelast decades in the urban areas and there are evidences that these substancesmay be absorbed and can play an important role in pathologies. PRM determination in processed foods, as well as their ingestion level from the daily diet, have been evaluated by several studies in countries within the Northern hemisphere, but there are no equivalent studies in South America. In this study, we evaluated the contents of furosine (FUR), hydroxymethylfurfural(HMF), carboxymethyllysine (CML) and fluorescent substances in flake cereals, granola-type cereals, medium-roasted coffee and powder milk from different brands. Fluorescent spectrophotometry, high performance liquid chromatography and immunoenzymatic methods were employed. Data were analyzed by descriptive statistics and principal component analysis. A great variation in MRP content for the same food product was observed. Powder milk, despite being the food product with the highest lipid content, has a low MRP content (average 7.6 mg/100g), while coffee has the highest amount (278.6 mg/100g) due to the severe thermal treatment it undergoes.These data are representative of these food products available in the marketand may be used for estimating MRP intake by the population.

El consumo de productos de la reacción de Maillard (PRM) en las áreas urbanas aumentó en las últimas décadas y hay evidencias de que estas substancias son absorbidas y pueden participar en procesos patológicos. La determinación del teor de PRM en alimentos industrializados, así como el consumo de estos compuestos a partir de la dieta ha sido evaluado en estudios conducidos en países del hemisferio Norte, pero no hay estudios equivalente en la America del Sur. En este trabajo analizamos los tenores de substancias fluorescentes, furosina, hidroximetilfurfural y carboximetillisina encereales del tipo escamas, cereales del tipogranola, café en polvo de torrefacción media, gelatina "diet" y leche en polvo integral dediferentes marcas y lotes. Las técnicas utilizadas fueron espectrofotometría de fluorescencia, cromatografía líquida de alto desempeño y teste inmunoenzimático. Los datos fueron analizados mediante estadística descriptiva y análisis decomponentes principales (PCA). Se observó grande variación en el contenido de PRM en los alimentos analizados. La leche en polvo, apesar de ser al alimento con mayor contenido de lípidos, presentó baja concentración de PRM (tenor medio 7,6mg/100g) mientras el café en polvo presentó el mayor contenido de PRM (278,6mg/100g) debido a la severidad del tratamiento térmico a que es sometido. Los datos obtenidos son representativos de los productos disponibles al consumidor y pueden ser utilizados para estimar el consumo de PRM por la población.

O consumo de produtos da reação de Maillard (PRM), nas áreas urbanas, aumentou nas últimas décadas e há evidências de que estas substâncias são absorvidas e podem tomar parte em processos patológicos. A determinação do teor de PRM em alimentos industrializados, assim como o consumo destes compostos a partir da dieta, têm sido avaliados em estudos conduzidos em países do hemisfério Norte. Não há estudos equivalentes na América do Sul. Neste trabalho, analisamos os teores de substâncias fluorescentes, furosina, hidroximetilfurfural e carboximetillisina em cereais do tipo flocos e do tipo granola, café em pó torra média, gelatina diet e leite integral em pó integral de diferentes marcas e lotes. As técnicas utilizadas foram espectrofotometria defluorescência, cromatografia líquida de alta eficiência e teste imunoenzimático. Os dados foram submetidos à análise estatística descritiva e Análise de Componentes Principais (ACP). Observou-se grande variabilidade no teor de PRM, nos alimentos analisados. O leite em pó, apesar de ser o alimento com maior teor delipídios, apresentou baixa concentração de PRM(teor médio 7,6mg/100g) enquanto o café em pó, apresentou o maior teor de PRM (278,6mg/100g) devido à severidade do tratamento térmico ao qual é submetido. Os dados obtidos são representativos dos produtos disponíveis ao consumidor e podem ser utilizados para estimar o consumo de PRM pela população. Palavras-chave: Reação de Maillard.

Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis / 대한진단검사의학회지

BACKGROUND: Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species. METHODS: The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C. haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates. RESULTS: The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3+/-18.5, 126.9+/-6.6, 88.5+/-18.5, 50.8+/-7.0, and 38.1+/-3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF. CONCLUSIONS: This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II

Abstract: One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment.A promising method to do this is by use of pathogen-specific molecular probes. However,this approach has been little used to date.We constructed,and validated in the laboratory,a fluoro-chrome-labeled molecular probe specific to Aurantimonas coralicida ,the bacterial pathogen of the Caribbean coral disease white plague type II (WPII).We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen.Probe design was based on a unique subset (25 nucleotides)of the complete16S rRNA gene sequence derived from a pure culture of the pathogen.The pathogen-specific probe was labeled with the fluorochrome GreenStar*™FITC (fluorescein isothiocyanate,GeneDetect Ltd,New Zealand).As a control, we used the universal eubacterial probe EUB 338,labeled with a different fluorochrome (TRITC,tetra-methyl-rhodamine isothiocyanate).Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II),healthy controls,and corals with an uncharacterized disease ("patchy necrosis ").All samples were analyzed using fluorescence in situ hybridization (FISH).We have determined that the probe is specific to our laboratory culture of the coral pathogen,and does not react with other bacterial species (the eubacterial probe does).The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n=9 samples)exhibiting signs of both WPI and WPII.Diseased (and healthy)tissue samples (n=4)from corals exhibiting signs of "patchy necrosis "were also assayed.In this case the results were negative, indicating that the same pathogen is not involved in the two diseases.Incorporation and use of pathogen-specific probes can...

Aplicaçäo do método fluorescente (soluçäo de diacetato de fluoresceína e brometo de etídio) no estudo da viabilidade de Metarhizium anisopliae / Fluorescent method (fluorescein diacetate and ethidium bromide) for the viability study of Metarhizium anisopliae

Padronizou-se o método de fluorescência (soluçäo de diacetato de fluoresceína e brometo de etídio), para análise de viabilidade de Metarhizium anisopliae var. anisopliae (Metech.) Sorokin, cultivado em ágar Sabouraud-dextrosea 25-C. Após estudar diferentes períodos de contato entre as células e as soluçöes de corantes, verificou-se que uma perfeita diferenciaçäo entre células viáveis (fluorescência verde) e näo viáveis (fluorescência vermelha) foi obtida após 30 minutos, na concentraçäo final de 2 microng/ml para o diacetato de fluoresceína e 50 microng/ml para o brometo de etídio

Método fluorescente para estudo da viabilidade de células fúngicas em materiais clínicos / Fluorescent method to study the viability of fungi cells in clinical materials

Padronizou-se o método de fluorescência (soluçäo de diacetato de fluoresceína e brometo de etídio), para análise da viabilidade de células fúngicas, em 150 amostras de materiais biológicos (urina, escarro, raspado de mucosa oral, pus, raspado de pele e pelo), provenientes de casos comprovados de micoses humanas diagnosticadas mediante técnicas micológicas clássicas. Após o processamento adequado dos diversos tipos de materiais biológicos, foram retiradas alíquotas de 0,1ml das suspensöes obtidas e misturadas a volumes iguais da soluçäo DF-BE (2,0 microng/ml). Para pesquisa de dermaófitos em pelos, colocou-se o material sobre lâmina de vidro contendo 0,1 ml de corantes fluorescentes nas mesmas concentraçöes anteriormente citadas. O tempo de contato ideal entre as células e os corantes foi de 30 minutos, para todos os fungos estudados, resultando perfeita diferenciaçäo entre microrganismos viáveis (fluorescência verde) e näo viáveis (fluorescência vermelha)