Heterologous expression of an alternative oxidase from Moniliophthora perniciosa in Saccharomyces cerevisiae: Antioxidant function and in vivo platform for the study of new drugs against witches' broom disease.

The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition.

Chronic cutaneous lupus erythematosus presenting as atypical acneiform and comedonal plaque: case report and literature review.

Introduction Chronic cutaneous lupus erythematosus (CCLE) usually presents as characteristic erythematous patches and infiltrated coin-shaped plaques. However, there are some atypical clinical variants that may mimic other dermatological conditions. Haroon et al. reported in 1972 an unusual presentation of CCLE with hypertrophic follicular scars seen in acne vulgaris. Acneiform presentation is one of the most rarely reported and one of the most confusing, as it resembles a very common inflammatory skin disease. A brief review of the literature using PubMed found only nine other reports. Case report A 32-year-old woman presented with two-year pruritic infiltrated acneiform and comedonal eruption on the right chin treated as acne with isotretinoin without improvement. On examination the patient presented with erythematous-infiltrated plaque, papules, open comedones, pitting scars and hypopigmented atrophic scars on the right chin area and scalp hair loss. An incisional skin biopsy on the chin and scalp lesions was performed and the anatomopathological and immunofluorescence exam showed findings that are consistent with CCLE. Additional tests ruled out systemic involvement. The patient was treated with prednisone and chloroquine diphosphate with great improvement. After four years the lesion is stable, with some scarring. Discussion In a literature review we found nine other cases of acneiform presentation of lupus erythematosus: Three cases were systemic lupus erythematosus (SLE) and seven others were diagnosed as CCLE (including our patient). All three patients who had SLE tested positive for antinuclear antibodies (ANA), and only one patient with CCLE, had a low titer of positive ANA (1:80). Ages varied from 24 to 60 years old, with a median of 32 years old, the same as our patient's age and consistent with the literature. Seven were females and three were males, with a ratio of 2.3:1. Most cases, such as our patient, showed acneiform lesions mainly on the face, a common site of typical CCLE. The present case and literature review illustrates the need to expand the differential diagnosis of atypical acneiform and comedonal lesions. CCLE should be considered especially in a localized lesion, which can be itchy and does not improve with conventional treatment for acne vulgaris.

Socio-epidemiological characterisation of blood donors with asymptomatic Leishmania infantum infection from three Brazilian endemic regions and analysis of the transfusional transmission risk of visceral leishmaniasis.

OBJECTIVES: This study assessed the prevalence of visceral leishmaniasis in blood donors from three endemic regions in Brazil and evaluated the risk of transmission by transfusion. BACKGROUND: Despite strong evidence of the transmission of visceral leishmaniasis through blood transfusion, the real risk, an essential condition for taking effective measures to control this serious disease, has not been determined. METHODS: A multicentre study was performed in highly endemic areas. Candidates eligible for their first blood donation underwent a socio-epidemiological interview, and blood samples were collected for enzyme-linked immunosorbent assay (ELISA) analysis, Western blot and polymerase chain reaction (PCR). Patients transfused with red blood cells or random platelet concentrates collected from these donors were also studied. The results were analysed using descriptive statistics and prevalence estimates, with significance defined as p-values <0·05. RESULTS: Of the 608 eligible donors, 37 (6·1%) were positive for visceral leishmaniasis as per ELISA. The socio-epidemiological analysis showed a significantly higher prevalence in non-Caucasians (p = 0·008). Among 296 patients who received blood components from these donors, the pre-transfusion seropositivity was 7·7%, and 13 patients received blood positive for Leishmania infantum. Six patients were followed up for 90 days, of which two (33·3%) had serological conversion at 60 days. CONCLUSIONS: The results confirm the high prevalence of L. infantum seropositivity among donors in the three regions; the seroconversion in a short period of time in two of six patients suggests the possibility of transmission of the infection by transfusion.

Identification and characterization of genes related to cellulolytic activity in basidiomycetes.

Enzymes produced by basidiomycetes that are involved in the cellulose degradation process, and their respective codifying genes, must be identified to facilitate the development of novel biotechnological strategies and applications in the agro-industry. The objective of this study was to identify prospective cellulase-producing genes and characterize their cellulolytic activity, in order to elucidate the potential biotechnological applications (with respect to vegetal residues) of basidiomycetes. The basidiomycete strains Lentinula edodes U8-1, Lentinus crinitus U9-1, and Schizophyllum commune U6-7 were analyzed in this study. The cellulolytic activities of these fungi were evaluated based on the halo formation in carboxymethyl cellulose culture medium after dyeing with Congo red. The presence of cellulase-codifying genes (cel7A, cel6B, cel3A, and egl) in these fungal strains was also evaluated. L. edodes and S. commune presented the highest cellulolytic halo to mycelial growth radius ratio, followed by L. crinitus. Four genes were amplified in the L. edodes strain, whereas three and one genes were isolated from L. crinitus and S. commune, respectively. The cel6B gene (L. edodes) presented the conserved domain glyco_hydro_6 and characterized as cellobiohydrolase gene. The results of this study contribute to the existing knowledge on cellulases in basidiomycetes, and serve as a basis for future studies on the expression of these genes and the characterization of the catalytic activity of these enzymes. This allows for better utilization of these fungi in degrading vegetal fibers from agro-industrial residues and in other biotechnological applications.

Diphtheria outbreak in Maranhão, Brazil: microbiological, clinical and epidemiological aspects.

We describe microbiological, clinical and epidemiological aspects of a diphtheria outbreak that occurred in Maranhão, Brazil. The majority of the 27 confirmed cases occurred in partially (n = 16) or completely (n = 10) immunized children (n = 26). Clinical signs and characteristic symptoms of diphtheria such as cervical lymphadenopathy and pseudomembrane formation were absent in 48% and 7% of the cases, respectively. Complications such as paralysis of lower limbs were observed. Three cases resulted in death, two of them in completely immunized children. Microbiological analysis identified the isolates as Corynebacterium diphtheriae biovar intermedius with a predominant PFGE type. Most of them were toxigenic and some showed a decrease in penicillin G susceptibility. In conclusion, diphtheria remains endemic in Brazil. Health professionals need to be aware of the possibility of atypical cases of C. diphtheriae infection, including pharyngitis without pseudomembrane formation.

Effective encapsulation of reuterin-producing Limosilactobacillus reuteri in alginate beads prepared with different mucilages/gums.

The mainly aim of this study was to use mucilaginous solutions obtained from tamarind, mutamba, cassia tora, psyllium and konjac powdered to encapsulate reuterin-producing Limosilactobacillus reuteri in alginate beads by extrusion technique. In the particles were determined the bacterial encapsulation efficiency, cell viability during storage and survival under simulated gastric and intestinal conditions. Moreover, the reuterin production, its entrapment into the beads and the influence on viability of encapsulated microorganism were evaluated. Scanning electron microscopy and Fourier Transform Infrared spectroscopy were employed to characterize the produced particles. The beads showed a relatively spherical shape with homogenous distribution of L. reuteri. The use of gums and mucilages combined with alginate improved the encapsulation efficiency (from 93.2 to 97.4%), the viability of encapsulated bacteria during refrigerated storage (especially in prolonged storage of 20, 30 and 60 days) and the survival after exposure to gastric and enteric environments (from 67.7 to 76.6%). The L. reuteri was able to produce reuterin via bioconversion of glycerol in the film-forming solutions, and the entrapment of the metabolite was improved using konjac, mutamba and tamarind mucilaginous solutions in the encapsulation process (45, 44.57 and 41.25%, respectively). Thus, our findings confirm the great potential of these hydrocolloids to different further purposes, enabling its application as support material for delivery of chemical or biological compounds.

Analysis of the workers head transcriptome of the Asian subterranean termite, Coptotermes gestroi.

The lower termite, Coptotermes gestroi (Isoptera: Rhinotermitidae), is originally from Southeast Asia and has become a pest in Brazil. The main goal of this study was to survey C. gestroi transcriptome composition. To accomplish this, we sequenced and analyzed 3003 expressed sequence tags (ESTs) isolated from libraries of worker heads. After assembly, 695 uniESTs were obtained from which 349 have similarity with known sequences. Comparison with insect genomes demonstrated similarity, primarily with genes from Apis mellifera (28%), Tribolium castaneum (28%) and Aedes aegypti (10%). Notably, we identified two endogenous cellulases in the sequences, which may be of interest for biotechnological applications. The results presented in this work represent the first genomic study of the Asian subterranean termite, Coptotermes gestroi.

Validation of a food frequency questionnaire for women with osteoporosis.

The importance of nutrient intakes in osteoporosis prevention in treatment is widely recognized. The objective of the present study was to develop and validate a FFQ for women with osteoporosis. The questionnaire was composed of 60 items, separated into 10 groups. The relative validation was accomplished through comparison of the 3-Day Food Record (3DR) with the FFQ. The 3DR was applied to 30 elderly women with confirmed osteoporosis, and after 45 days the FFQ was administrated. Statistical analysis comprised the Kolmogorov-Smirnov, Student T test and Pearson correlation coefficient. The agreement between two methods was evaluated by the frequency of similar classification into quartiles, and by the Bland-Altman method. No significant differences between methods were observed for the mean evaluated nutrients, except for carbohydrate and magnesium. Pearson correlation coefficients were positive and statistically significant for all nutrients. The overall proportion of subjects classified in the same quartile by the two methods was on average 50.01% and in the opposite quartile 0.47%. For calcium intake, only 3% of subjects were classified in opposite extreme quartiles by the two methods. The Bland-Altman analysis demonstrated that the differences obtained by the two methods in each subject were well distributed around the mean of the difference, and the disagreement increases as the mean intake increases. These results indicates that the FFQ for elderly women with osteoporosis presented here is highly acceptable and is an accurate method that can be used in large-scale or clinical studies for evaluation of nutrient intakes in a similar population.

Corynebacterium pseudodiphtheriticum isolated from relevant clinical sites of infection: a human pathogen overlooked in emerging countries.

AIMS: To examine the occurrence of and to determine the antimicrobial susceptibility of Corynebacterium pseudodiphtheriticum among patients with bacterial infections at a teaching hospital. METHODS AND RESULTS: A total of 113 Coryne. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups: 65.48% adults (18 to < or =59 years old), 9.73% aged (> or =60 years old); 14.15% infants (<18 years old); 4.42% newborns (0-7 days). Micro-organisms were mostly related to infections in the urinary (29.2%) and respiratory tracts (27.45%) and intravenous sites (18.6%). Clinical samples were obtained only from 32.7% patients (26 adults, four aged, four infants and three newborns) presenting at least one of the predisposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains (>50%) were resistant to oxacillin, erythromycin and clindamycin. CONCLUSIONS: Despite significant differences in age and functional status of patients Coryne. pseudodiphtheriticum may be implicated as a cause of respiratory and nonrespiratory human infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Data are valuable for practitioners indicating the occurrence of multiresistant phenotypes and the possibility of severe infections due to Coryne. pseudodiphtheriticum, a pathogen usually overlooked in emerging countries.

DNase test as a novel approach for the routine screening of Corynebacterium diphtheriae.

AIMS: To examine the value of the DNase test as an alternative procedure for differentiating Corynebacterium diphtheriae from Corynebacterium-like colonies. METHODS AND RESULTS: DNase test medium was inoculated by spotting a loopful of bacterial growth and incubated aerobically at 37 degrees C. The DNase production was detectable following both 24 and 48 h incubation periods. The DNase activity was detected in all 91 C. diphtheriae (37 toxigenic and 54 nontoxigenic) strains examined, previously identified by both conventional biochemical methods and API Coryne System. Conversely, DNase test results were negative in 93.9% of the 564 nondiphtherial Gram-positive rod clinical strains. CONCLUSIONS: The DNase test emerged as an easily interpretable and cost-effective alternative screening procedure for C. diphtheriae laboratory identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The method should facilitate routine laboratory diagnosis of toxigenic and nontoxigenic C. diphtheriae.

First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil.

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

Assessment of basal and gonadotropin-releasing hormone-stimulated gonadotropins by immunochemiluminometric and immunofluorometric assays in normal children.

CONTEXT: Recently, new methodologies have been applied to commercial immunofluorometric (IFMA) and immunochemiluminometric (ICMA) LH and FSH assays. OBJECTIVE: The objective of the study was to use ICMA to establish basal and GnRH-stimulated LH and FSH reference values in normal subjects of different ages and sexual development, compared with IFMA. DESIGN AND METHODS: We established basal and GnRH-stimulated LH and FSH levels of 315 prepubertal and pubertal children (170 males and 145 females) divided into five groups according to Tanner stage. Of these, 106 subjects (59 males and 47 females) were submitted to GnRH test. The prepubertal upper limit of normal for basal LH, determined by the 95th percentiles of the prepubertal population, were 0.2 IU/liter (ICMA) and 0.6 IU/liter (IFMA) in both genders. RESULTS: No overlap of basal LH levels determined by ICMA was observed between prepubertal and pubertal males, but basal LH determined by IFMA overlapped in 11.8% of subjects. In girls, both methods yielded overlapping values (10.4%, ICMA; and 84.6%, IFMA). The LH peak after GnRH stimulation that defined puberty was 4.1 IU/liter (ICMA) and 3.3 IU/liter (IFMA) in boys and 3.3 IU/liter (ICMA) and 4.2 IU/liter (IFMA) in girls. After GnRH stimulation, values determined by the two methods overlapped in both genders. CONCLUSIONS: We conclude that ICMA is more sensitive and precise than IFMA, permitting differentiation of pubertal and prepubertal stage in boys under basal conditions. However, in girls the overlap of basal values was marked, indicating the need for the GnRH test to establish maturity of the hypothalamus-pituitary-gonadal axis.

Seasonality, biology and threats to Sicalis luteola (Sparrman, 1789) (Aves, Thraupidae) in northeastern Brazil.

Sicalis luteola occurs from Mexico to South America. In the Northeastern Brazilian region its biology, population dynamics and threats are poorly known. The subspecies S. luteola luteiventris apparently migrates northward during austral winter. The specific aims of this work were: 1) to verify the seasonality of the species; 2) to analyze information on molting and breeding, and 3) to check the main threats to this bird in northeastern Brazil. The highest number of records were in March, April, and May (51.12%) and the lowest in November and December (3%).We found a greater population peak in April in the states of Rio Grande do Norte and Ceará, and in April and May in Pernambuco. In Paraíba there was not an apparent seasonal variation in frequency. We captured 66 individuals, all adults, being 38 males and 28 females. 30% of the captured birds showed contour feathers molt, mainly on the head. A total of 23 individuals had brood patch, all being females. Six nests were found and four contained two to three eggs. We found that the main threat is the illegal trade. Our findings may support conservation plans for this bird in the region.

Distribution and conservation of three important bird groups of the Atlantic Forest in north-east Brazil.

The Pernambuco Endemism Center in north-east Brazil has the most fragmented forest cover and the largest number of threatened birds of the whole Atlantic Forest. We analyzed the distribution of three groups of bird species: forest-dependent, endemic and/or threatened using the interpolation method of Inverse Distance Weighting. We also checked the concentration of these birds in protected and unprotected areas, suggesting new sites that need to be protected. The richness concentration of forest-dependent, endemic and/or threatened birds in 123 sites were analysed. There was a greater concentration of the three groups in north Alagoas, south and north Pernambuco, and north and west Paraíba. The distribution of the three groups was almost regular in different vegetation types, although a lower concentration was found in the pioneer formation. There was a greater concentration of birds from all three groups between Pernambuco and Alagoas, and this must be due to the presence of more forest fragments with better structure and vegetation heterogeneity. The protected and unprotected areas hosted important records of endemic and/or threatened birds. We suggested some important places for implementation of new protected areas due to the larger concentrations of the target birds and because they are located within the boundaries of the Important Bird Areas.

Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria.

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H(2)O(2) in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H(2)O(2). In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.

Cell surface components and adhesion in Corynebacterium diphtheriae.

Main primary approaches and new developments in the study of the molecular basis of the adhesive process of Corynebacterium diphtheriae are reviewed along with a discussion of the potential importance of hemagglutinins, exposed sugar residues, hydrophobins and trans-sialidase enzymes as adhesins of strains of the sucrose fermenting and non-fermenting biotypes.

Portopulmonary hypertension syndrome in schistosomiasis mansoni.

Portopulmonary hypertension syndrome (PPHS) is a complication of portal hypertension where the substrate is micro-vessel lesions which are indicative of plexogenic arteriopathy. PPHS has not been linked to pulmonary schistosomiasis. We report, to the best of our knowledge for the first time, a case of PPHS associated with schistosomiasis mansoni.

Influence of early postnatal gonadal hormones on anxiety in adult male rats.

Behavioral sex differences have been linked to the presence of testosterone secretion during a critical perinatal period. The present experiment tested whether or not castration at different ages (early postnatal period and adulthood) would alter performance in the plus maze, a behavioral test of anxiety. Intact adult male rats (n = 17) were compared to intact adult females (n = 17); adult castrated males (n = 7) to sham-operated adult male rats (n = 9); and newborn castrated males (n = 7) to sham-operated male offspring (n = 8). When adult, the subjects were left on an elevated plus maze for 5 min. Females made a higher percentage of entries onto the open arms and showed a greater number of scans over the edge of an open arm than males. There were no differences in the percentage of arm entries or time spent on the open arms when adult castrated males were compared to sham-operated rats. On the other hand, newborn castrated males showed a significantly higher number of open arm entries and spent a greater percentage of time on the open arms than sham-operated offspring. The results demonstrate that the absence of male gonadal hormones during the perinatal period decreases anxiety, as assessed in the elevated plus maze, leading to a behavioral pattern that resembles that of females. These data provide evidence for the organizational role of gonadal hormones in the development of behavioral inhibitory systems.

Corynebacterium diphtheriae threats in cancer patients.

The aim of this study was to determine the bacteriological properties of Corynebacterium diphtheriae strains isolated from bronchiole washing and cancer lesions. Bacteriological characterization included fluorescence/double sugar urease (King/DSU) screening tests, pyrazinamidase (PYZ), CAMP-reactions and radial immunodiffusion toxigenicity assay. Microorganisms produced fluorescence under ultraviolet light and were catalase positive; urea and aesculin hydrolysis negative; fermentation of glucose, maltose and sucrose and no fermentation of mannitol and xylose; PYZ and CAMP reaction negative. The API-Coryne system was used for bacterial preliminary identification at local hospital laboratory and produced numerical profiles 1010325 and 0010325 for sucrose positive C. diphtheriae var. mitis (nitrate positive) and C. diphtheriae var. belfanti (nitrate negative), respectively. The hemagglutination, adherence to glass and polystyrene assays evaluated adhesive characteristics. Strains were toxigenic and able to adhere to glass, polystyrene and human erythrocyte surfaces (titer 4). C. diphtheriae strains isolated from cancer patients expressed adhesive characteristics similar to strains isolated from immunocompetent hosts. Circulation of toxigenic C. diphtheriae continues to present a threat for children and adults including patients with cancer in hospital environment. Laboratories should remain alert to the possibility of isolation of diphtheria bacilli from adults with neoplastic disease.