Biochemical characterization and analysis of gene expression of an α-mannosidase secreted by Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the Paracoccidioides genus, being endemic in Latin America and with the highest number of cases in Brazil. Paracoccidioides spp. release a wide range of molecules, such as enzymes, which may be important for PCM establishment. Here, we identified the 85- and 90-kDa proteins from the supernatants of P. brasiliensis cultures as being an α-mannosidase. Because the expected mass of this α-mannosidase is 124.2-kDa, we suggest that the proteins were cleavage products. Indeed, we found an α-mannosidase activity in the culture supernatants among the excreted/secreted antigens (ESAg). Moreover, we determined that the enzyme activity was optimal in buffer at pH 5.6, at the temperature of 45ºC, and with a concentration of 3 mM of the substrate p-NP-α-D-Man. Remarkably, we showed that the gene expression of this α-mannosidase was higher in yeasts than hyphae in three P. brasiliensis isolates with different virulence degrees that were grown in Ham's F12 synthetic medium for 15 days. But in complex media YPD and Fava Netto, the significantly higher gene expression in yeasts than in hyphae was seen only for the virulent isolate Pb18, but not for intermediate virulence Pb339 and low virulence Pb265 isolates. These results about the high expression of the α-mannosidase gene in the pathogenic yeast form of P. brasiliensis open perspectives for studying this α-mannosidase concerning the virulence of P. brasiliensis isolates. LAY SUMMARY: Paracoccidioides brasiliensis causes deep mycosis, paracoccidioidomycosis. We determined for the first time the biochemical properties of an α-mannosidase released by this fungus. We suggest that the enzyme gene expression in the fungus is associated with fungal morphology, stress, and virulence.

Assessing driving risk in patients with glaucoma / Avaliação do risco de dirigir em pacientes com glaucoma

ABSTRACT Glaucoma is the leading cause of irreversible blindness worldwide and can affect a broad array of daily activities, including driving. Recently, studies investigating the relationship between driving performance and glaucoma have received a great deal of interest. Assessment of driving behavior is not straightforward because driving is a complex skill involving significant multi-tasking ability. In this review, we summarize recent work from clinical studies investigating how glaucoma can affect driving performance. Patients with glaucoma are more likely to be involved in motor vehicle collisions when compared to healthy subjects. Here we describe how conventional functional tests performed in glaucoma patients, such as visual field measurements via standard automatic perimetry, are associated with driving performance. However, the risk of motor vehicle collisions is not entirely attributable to visual field impairment in glaucoma, suggesting that other factors also account for both driving safety and performance. Finally, we show different studies suggesting that parameters from driving simulators can be helpful because they can identify the impact of visual loss on complex situations.

RESUMO O glaucoma é a principal causa de cegueira irreversível em todo o mundo e pode afetar uma ampla gama de atividades diárias, incluindo a direção veicular. Recentemente, estudos que investigam a relação entre o desempenho na condução veicular e o glaucoma têm recebido grande interesse. A avaliação do comportamento de dirigir não é direta porque dirigir é uma habilidade complexa que envolve habilidade multitarefa significativa. Nesta revisão, resumimos trabalhos recentes de estudos clínicos que investigam como o glaucoma pode afetar o desempenho na direção. Pacientes com glaucoma têm maior probabilidade de se envolverem em colisões de veículos motorizados quando comparados a indivíduos saudáveis. Descrevemos aqui como os testes funcionais convencionais realizados em pacientes com glaucoma, como as medições de campo visual por meio de perimetria automática padrão, estão associados ao desempenho na direção. No entanto, o risco de colisão de veículo motorizado não é totalmente atribuível ao comprometimento do campo visual no glaucoma, sugerindo que outros fatores também são responsáveis pela segurança e pelo desempenho. Finalmente, mostramos diferentes estudos sugerindo que os parâmetros dos simuladores de direção podem ser úteis porque eles podem identificar o impacto da perda visual em situações de complexas.

Assessing driving risk in patients with glaucoma.

Glaucoma is the leading cause of irreversible blindness worldwide and can affect a broad array of daily activities, including driving. Recently, studies investigating the relationship between driving performance and glaucoma have received a great deal of interest. Assessment of driving behavior is not straightforward because driving is a complex skill involving significant multi-tasking ability. In this review, we summarize recent work from clinical studies investigating how glaucoma can affect driving performance. Patients with glaucoma are more likely to be involved in motor vehicle collisions when compared to healthy subjects. Here we describe how conventional functional tests performed in glaucoma patients, such as visual field measurements via standard automatic perimetry, are associated with driving performance. However, the risk of motor vehicle collisions is not entirely attributable to visual field impairment in glaucoma, suggesting that other factors also account for both driving safety and performance. Finally, we show different studies suggesting that parameters from driving simulators can be helpful because they can identify the impact of visual loss on complex situations.

Quantitation of pulmonary fungal burden in Paracoccidioides brasiliensis-infected mice by real-time PCR.

Although colony-forming unit (CFU) counting is widely used to quantify fungal load in tissue from animal experimentally infected with Paracoccidioides brasiliensis, several technical disadvantages have been described. Here we developed highly accurate quantitative PCR (qPCR) assays to determine the relative P brasiliensis load in lungs from infected mice. SYBR Green- and TaqMan-based assays using primers and probe for the 43-kDa glycoprotein (gp43) gene detected as little as 270 gene copies (about 2 fg of DNA) per reaction. Although qPCR assays cannot distinguish between living and dead yeasts, we found a highly positive linear correlation between CFU and qPCR.

Study of the Effectiveness of Multipurpose Solutions on the Bacterial Disinfection of Silicone Hydrogel Contact Lenses In Vitro.

OBJECTIVES: To assess the antimicrobial effectiveness of multipurpose solutions in regard to the disinfection of silicone hydrogel contact lenses (CL) using a study of clinical bacterial isolates from ocular material. METHODS: Three multipurpose solutions (solution A: polyhexamethylene biguanide 0.00025 g/100 mL; solution B: polyquaternary-1 0.001% and myristamidopropyl dimethylamine 0.0006%; and solution C: polyaminopropyl biguanide 0.00013% and polyquaternary 0.0001%) were used as a 3-phase disinfection on silicone hydrogel CL contaminated with bacteria from clinical isolates that were divided into five groups (group 1: Pseudomonas aeruginosa; group 2: Staphylococcus aureus; group 3: Staphylococcus epidermidis; group 4: Streptococcus spp; and group 5: enterobacteria). RESULTS: No differences were observed between the 24- and 48-hr measurements in any of the samples, and the positivity of microorganisms in T0 was 100% for all solutions; it was 0% in T3. Therefore, only steps T1 (rubbing followed by rinsing) and T2 (rubbing followed by rinsing and immersion of CL into solution) were considered for analysis at the 24-hr measurement time. Throughout the phases, a decrease in the number of bacteria was observed, culminating in the elimination (no recovery) of all microorganisms in the three solutions. CONCLUSIONS: At the end of the proposed process, the tested solutions were effective.