Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance - that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound α-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Deciphering the contribution of lipid droplets in leprosy: multifunctional organelles with roles in Mycobacterium leprae pathogenesis

Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves, presenting a singular clinical picture. Across the leprosy spectrum, lepromatous leprosy (LL) exhibits a classical hallmark: the presence of a collection of M. leprae-infected foamy macrophages/Schwann cells characterised by their high lipid content. The significance of this foamy aspect in mycobacterial infections has garnered renewed attention in leprosy due to the recent observation that the foamy aspect represents cells enriched in lipid droplets (LD) (also known as lipid bodies). Here, we discuss the contemporary view of LD as highly regulated organelles with key functions in M. leprae persistence in the LL end of the spectrum. The modern methods of studying this ancient disease have contributed to recent findings that describe M. leprae-triggered LD biogenesis and recruitment as effective mycobacterial intracellular strategies for acquiring lipids, sheltering and/or dampening the immune response and favouring bacterial survival, likely representing a fundamental aspect of M. leprae pathogenesis. The multifaceted functions attributed to the LD in leprosy may contribute to the development of new strategies for adjunctive anti-leprosy therapies.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Early postnatal hospital discharge: the consequences of reducing length of stay for women and newborns

The objective of this study is to examine the literature and identify most salient outcomes of early postnatal discharge for women, newborns and the health system. An electronic search strategy was designed including the following sources: Web of Science, Scopus, ProQuest and PubMed/MEDLINE, using the following terms: (early AND discharge) OR (length AND stay) AND (postpartum OR postnatal) AND (effect* OR result OR outcome). Content analysis was used to identify and summarise the findings and methods of the research papers. The evidence available is not enough to either reject or support the practice of early postnatal discharge; different studies have reported different outcomes for women and newborns. The need of systematic clinical research is discussed.

Response of Epidendrum Ibaguense (orchidaceae) to the application of lime rates to the pot

In the best cultivation methods of orchids, in particular of the genus Phalaenopsis, liming is a common practice. The objective of this study was to evaluate the influence of lime rates (0.0; 1.0; 2.0; 3.0; 4.0; and 5.0 g dm-3 of substrate) applied to the cultivation substrate (xaxim) on the growth of Epidendrum ibaguense seedlings. In a greenhouse, 1-L plastic pots filled with 0.8 dm³ of xaxim were irrigated such that no leachate was lost during the experiment. N, P, K, Ca, Mg, S, Fe, Zn, B, and Mn contents in roots, stems and leaves were measured. Leachate was collected by applying a sufficient water volume to obtain 25 mL from each pot. Fourteen days after lime application of 3 g dm-3, the pH of the collected leachate reached values above 7 and a value of 6.29 with the highest lime rate at the end of the experiment. The lime rate did not influence plant height, probably due to a Zn deficiency at high pH levels and a Ca deficiency in the control. Nevertheless, there was a large increase in leaf production, for number as well as for dry matter mass. There was no statistical difference between treatments in root dry matter production. Maximum dry matter production was obtained at a lime rate of 4.09 g dm-3. Zinc concentrations diminished linearly with increasing lime rates; the concentrations in all treatments were below the levels suggested as adequate in the literature (25-200 mg kg-1). Nutrient concentrations in leaves indicated deficiency of N, S, and B at the highest lime rates (4.0 and 5.0 g dm-3), and of Ca in the treatment without liming.

Morphostructural characterization of soil conventionally tilled with mechanized and animal traction with and without cover crop

The structural stability and restructuring ability of a soil are related to the methods of crop management and soil preparation. A recommended strategy to reduce the effects of soil preparation is to use crop rotation and cover crops that help conserve and restore the soil structure. The aim of this study was to evaluate and quantify the homogeneous morphological units in soil under conventional mechanized tillage and animal traction, as well as to assess the effect on the soil structure of intercropping with jack bean (Canavalia ensiformis L.). Profiles were analyzed in April of 2006, in five counties in the Southern-Central region of Paraná State (Brazil), on family farms producing maize (Zea mays L.), sometimes intercropped with jack bean. The current structures in the crop profile were analyzed using Geographic Information Systems (GIS) and subsequently principal component analysis (PCA) to generate statistics. Morphostructural soil analysis showed a predominance of compact units in areas of high-intensity cultivation under mechanized traction. The cover crop did not improve the structure of the soil with low porosity and compact units that hamper the root system growth. In areas exposed to animal traction, a predominance of cracked units was observed, where roots grew around the clods and along the gaps between them.