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.     

Molecular modeling studies on the interactions of aflatoxin B1 and its metabolites with the peripheral anionic site of human acetylcholinesterase

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them, aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic for mammals. It can contaminate many kinds of food like seeds, oil, olives, milk, dairy products, corn and meat, causing acute and chronic damages to the organism, especially in the liver, being, for this reason, considered highly hepatotoxic. AFB1 is also a mixed inhibitor of the enzyme acetylcholinesterase (AChE). This fact, together with its high toxicity and carcinogenicity, turns AFB1 into a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited molecular modeling studies on the interactions of AFB1 and its metabolites inside the peripheral anionic site of human AChE (HssAChE), to verify their stability, suggest the preferential ways of inhibition, and compare their behavior to each other. Our results suggest that all metabolites can be better inhibitors of HssAChE than AFB1 and that AFBO and AFM1, the most toxic and carcinogenic metabolites of AFB1, are also the most effective HssAChE inhibitors among the AFB1 metabolites.

Communicated by Ramaswamy H. Sarma     

Functional metagenomics of oil-impacted mangrove sediments reveals high abundance of hydrolases of biotechnological interest

Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms. For this, an in-depth survey of the taxonomic and functional microbial diversity recovered in a fosmid library (Library_Oil Mgv) constructed from oil-impacted Brazilian mangrove sediment was carried out. Fosmid DNA of the whole library was extracted and submitted to Illumina HiSeq sequencing. The resulting Library Oil_Mgv dataset was further compared with those obtained by direct sequencing of environmental DNA from Brazilian mangroves (from distinct regions and affected by distinct sources of contamination), focusing on hydrolases with potential use in biotechnological processes. The most abundant hydrolases found were proteases, esterases and amylases, with similar occurrence profile in all datasets. The main microbial groups harboring such hydrolase-encoding genes were distinct in each mangrove, and in the fosmid library these enzymes were mainly assigned to Chloroflexaceae (for amylases), Planctomycetaceae (for esterases) and Bradyrhizobiaceae (for proteases). Assembly and analysis of Library_Oil Mgv reads revealed three potentially novel enzymes, one epoxide hydrolase, one xylanase and one amylase, to be further investigated via heterologous expression assays.     

Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification

The Neotropical armored catfish genus Harttia presents a wide variation of chromosomal rearrangements among its representatives. Studies indicate that translocation and Robertsonian rearrangements have triggered the karyotype evolution in the genus, including differentiation of sex chromosome systems. However, few studies used powerful tools, such as comparative whole chromosome painting, to clarify this highly diversified scenario. Here, we isolated probes from the X₁ (a 5S rDNA carrier) and the X₂ (a 45S rDNA carrier) chromosomes of Harttia punctata, which displays an X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system. Those probes were applied in other Harttia species to evidence homeologous chromosome blocks. The resulting data reinforce that translocation events played a role in the origin of the X₁X₂Y sex chromosome system in H. punctata. The repositioning of homologous chromosomal blocks carrying rDNA sites among ten Harttia species has also been demonstrated. Anchored to phylogenetic data it was possible to evidence some events of the karyotype diversification of the studied species and to prove an independent origin for the two types of multiple sex chromosomes, XX/XY₁Y₂ and X₁X₁X₂X₂/X₁X₂Y, that occur in Harttia species. The results point to evolutionary breakpoint regions in the genomes within or adjacent to rDNA sites that were widely reused in Harttia chromosome remodeling.     

The Search for Cancer Biomarkers: Assessing the Distribution of INDEL Markers in Different Genetic Ancestries

Cancer is a multifactorial group of diseases, being highly incident and one of the leading causes of death worldwide. In Brazil, there is a great variation in cancer incidence and impact among the different geographic regions, partly due to the genetic heterogeneity of the population in this country, composed mainly by European (EUR), Native American (NAM), African (AFR), and Asian (ASN) ancestries. Among different populations, genetic markers commonly present diverse allelic frequencies, but in admixed populations, such as the Brazilian population, data is still limited, which is an issue that might influence cancer incidence. Therefore, we analyzed the allelic and genotypic distribution of 12 INDEL polymorphisms of interest in populations from the five Brazilian geographic regions and in populations representing EUR, NAM, AFR, and ASN, as well as tissue expression in silico. Genotypes were obtained by multiplex PCR and the statistical analyses were done using R, while data of tissue expression for each marker was extracted from GTEx portal. We highlight that all analyzed markers presented statistical differences in at least one of the population comparisons, and that we found 39 tissues to be differentially expressed depending on the genotype. Here, we point out the differences in genotype distribution and gene expression of potential biomarkers for risk of cancer development and we reinforce the importance of this type of study in populations with different genetic backgrounds.     

Impact and Cost-Effectiveness of Culture for Diagnosis of Tuberculosis in HIV-Infected Brazilian Adults

Background

Culture of Mycobacterium tuberculosis currently represents the closest “gold standard” for diagnosis of tuberculosis (TB), but operational data are scant on the impact and cost-effectiveness of TB culture for human immunodeficiency (HIV-) infected individuals in resource-limited settings.

Methodology/Principal Findings

We recorded costs, laboratory results, and dates of initiating TB therapy in a centralized TB culture program for HIV-infected patients in Rio de Janeiro, Brazil, constructing a decision-analysis model to estimate the incremental cost-effectiveness of TB culture from the perspective of a public-sector TB control program. Of 217 TB suspects presenting between January 2006 and March 2008, 33 (15%) had culture-confirmed active tuberculosis; 23 (70%) were smear-negative. Among smear-negative, culture-positive patients, 6 (26%) began TB therapy before culture results were available, 11 (48%) began TB therapy after culture result availability, and 6 (26%) did not begin TB therapy within 180 days of presentation. The cost per negative culture was US$17.52 (solid media)–$23.50 (liquid media). Per 1,000 TB suspects and compared with smear alone, TB culture with solid media would avert an estimated eight TB deaths (95% simulation interval [SI]: 4, 15) and 37 disability-adjusted life years (DALYs) (95% SI: 13, 76), at a cost of $36 (95% SI: $25, $50) per TB suspect or $962 (95% SI: $469, $2642) per DALY averted. Replacing solid media with automated liquid culture would avert one further death (95% SI: −1, 4) and eight DALYs (95% SI: −4, 23) at $2751 per DALY (95% SI: $680, dominated). The cost-effectiveness of TB culture was more sensitive to characteristics of the existing TB diagnostic system than to the accuracy or cost of TB culture.

Conclusions/Significance

TB culture is potentially effective and cost-effective for HIV-positive patients in resource-constrained settings. Reliable transmission of culture results to patients and integration with existing systems are essential.     

Hysteresivity of the lung and tissue strip in the normal rat: effects of heterogeneities.

We measured lung impedance in rats in closed chest (CC), open chest (OC), and isolated lungs (IL) at four transpulmonary pressures with a optimal ventilator waveform. Data were analyzed with an homogeneous linear or an inhomogeneous linear model. Both models include tissue damping and elastance and airway inertance. The homogeneous linear model includes airway resistance (Raw), whereas the inhomogeneous linear model has a continuous distribution of Raw characterized by the mean Raw and the standard deviation of Raw (SDR). Lung mechanics were compared with tissue strip mechanics at frequencies and operating stresses comparable to those during lung impedance measurements. The hysteresivity (eta) was calculated as tissue damping/elastance. We found that 1) airway and tissue parameters were different in the IL than in the CC and OC conditions; 2) SDR was lowest in the IL; and 3) eta in IL at low transpulmonary pressure was similar to eta in the tissue strip. We conclude that eta is primarily determined by lung connective tissue, and its elevated estimates from impedance data in the CC and OC conditions are a consequence of compartment-like heterogeneity being greater in CC and OC conditions than in the IL.     

Removal of some polycyclic aromatic hydrocarbons from petrochemical wastewater using low-cost adsorbents of natural origin

Removal of polycyclic aromatic hydrocarbons (PAHs) from petrochemical wastewater was investigated using various low-cost adsorbents of natural origin including sugar cane bagasse, green coconut shells, chitin, and chitosan. Adsorption experiments of mixtures of PAHs (5.0-15.0 mg/L) have been carried out at ambient temperature (28+/-2 degrees C) and pH 7.5. The adsorption isotherms of PAHs were in agreement with a Freundlich model, while the uptake capacity of PAHs followed the order: green coconut shells>sugar cane bagasse>chitin>chitosan. The adsorption properties of green coconut shells were comparable to those of some conventional adsorbents such as Amberlite T. The partition coefficients in acetone:water, the adsorption constants at equilibrium, and the molecular masses of the PAHs could be linearly correlated with octanol-water partition coefficients.     

Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis

The diagnosis of chronic pulmonary aspergillosis (CPA) depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA) and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID) test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA) and four chronic fibrosing pulmonary aspergillosis (CFPA); G2: 28 patients with pulmonary tuberculosis (TB); G3: 23 patients with histoplasmosis (HST); G4: 50 patients with paracoccidioidomycosis (PCM); G5: 20 patients with cryptococcosis (CRC); and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations—Aspergillus fumigatus (DID₁, ELISA₁) and a pool of A. fumigatus, A. flavus and A. niger antigens (DID₂, ELISA₂). The Platélia Aspergillus Enzyme Immunoassay (EIA) kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio–especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA₁ and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA₁ and ELISA₂. The detection of serum galactomannan showed high sensitivity, comparable to ELISA₂. The immunodiffusion test showed an excellent relationship with the progression after treatment, which made it the reaction of choice for patient follow-up.