Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Clonal relationships determined by multilocus sequence typing among enteropathogenic Escherichia coli isolated in Brazil

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Multilocus sequence typing (MLST) characterizes bacterial strains based on the sequences of internal fragments in housekeeping genes. Little is known about strains of EPEC analyzed by MLST from Brazil. In this study, a diverse collection of 29 EPEC strains isolated from patients with diarrhea, admitted to the University Hospital of Ribeirao Preto, was characterized by MLST. Strain analysis demonstrated 22 different sequence types (STs), of which almost half (48%) were new, indicating a high genotype diversity. The 22 STs were divided by eBURST into 12 clonal complexes. It was not possible to correlate typical and atypical EPEC with other strains in the MLST database. This is the first study that analyzed EPEC strains from South America that are included in the E. coli MLST database. Nine (31%) out of 29 strains are part of the CC10 clonal complex, the major clonal complex in the database, which comprises 174 strains and 86 different STs, suggesting that these strains might be the most important intestinal pathogenic E. coli worldwide. Genetic relationships between typical and atypical EPEC, enterohemorrhagic E. coli, and enteroaggregative E. coli strains were not established by MLST.

Colonization sequence of non-pollinating fig wasps associated with Ficus citrifolia in Brazil

Mutualisms, such as the fig-agaonid wasp association, are susceptible to colonization by parasitic species, which exploit the resources involved therein. In most cases, they oviposit into the figs from outside without providing any pollination service. In this study, we used several different methods (adhesive traps and direct standardized field observations) to assess the colonization sequence of a diverse fig wasp fauna associated with Ficus citrifolia, section Americana, in Brazil. They consistently showed a temporal partitioning in colonization among non-pollinating fig wasp species. Idarnes species belonging to the flavicollis and incerta groups colonized figs just before or during the fig receptive phase. In contrast, Idarnes females belonging to the carme group oviposited one to three weeks later, mainly in the middle of the inter-floral phase. Eurytoma, Heterandrium, Physothorax and Torymus were later colonizers, and laid eggs either in the middle or during the late inter-floral phase. The results suggest that these Neotropical fig wasps have different strategies of resource exploitation, even among species belonging to the same genus.

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. (C) 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 573-584.

Mitochondrial DNA Variability Among Eight Tikuna Villages: Evidence for an Intratribal Genetic Heterogeneity Pattern

To study the genetic structure of the Tikuna tribe, four major Native American mitochondrial DNA (mtDNA) founder haplogroups were analyzed in 187 Amerindians from eight Tikuna villages located in the Brazilian Amazon. The central position of these villages in the continent makes them relevant for attempts to reconstruct population movements in South America. In this geographic region, there is particular concern regarding the genetic structure of the Tikuna tribe, formerly designated ""enigmatic"" due to its remarkable degree of intratribal homogeneity and the scarcity of private protein variants. In spite of its large population size and geographic distribution, the Tikuna tribe presents marked genetic and linguistic isolation. All individuals presented indigenous mtDNA haplogroups. An intratribal genetic heterogeneity pattern characterized by two highly homogeneous Tikuna groups that differ considerably from each other was observed. Such a finding was unexpected, since the Tikuna tribe is characterized by a social system that favors intratribal exogamy and patrilocality that would lead to a higher female migration rate and homogenization of the mtDNA gene pool. Demographic explosions and religious events, which significantly changed the sizes and compositions of many Tikuna villages, may be reflected in the genetic results presented here. Am J Phys Anthropol 140:526-531,2009. (C) 2009 Wiley-Liss, Inc

Age and Method-dependent Variability of Predicted Oxygen Consumption in Congenital Heart Disease

Background and Purpose. There has been a lot of debate about the use of predicted oxygen consumption to calculate pulmonary vascular resistance using the Fick principle. We therefore comparatively analyzed predicted oxygen consumption in infants and children in specific age groups, using different methods (formulas), as an attempt to better understand the usefulness and limitations of predictions. Methods and Results. Four models (LaFarge & Miettinen, Bergstra et al., Lindahl, and Lundell et al.) were used to predict oxygen consumption in 200 acyanotic patients with congenital cardiac defects aged 0-2.0, > 2.0-4.0, > 4.0-6.0, and > 6.0-8.75 years (median 2.04 years). Significant differences were observed between the age groups (P < .001) and between the methods (P < .001), not related to diagnoses. Differences between methods were more impressive in the first age group (P < .01). In patients aged 0-2.0 years, the lowest values of oxygen consumption (corresponding to the highest estimates of pulmonary vascular resistance) were obtained with the method of Lindahl; above this age, any method except that of Lundell et al. Conclusions. Although measuring oxygen consumption is always preferable, a rational use of predictions, using different methods, may be of help in situations where measurements are definitely not possible.

Molecular epidemiology and genetic diversity of hepatitis B virus genotype E in an isolated Afro-Colombian community

Hepatitis B virus (HBV) infection is a significant public health concern with 350 million chronic carriers worldwide. Eight HBV genotypes (A-H) have been described so far. Genotype E (HBV/E) is widely distributed in West Africa and has rarely been found in other continents, except for a few cases in individuals with an African background. In this study, we characterized HBV genotypes in Quibdo, Colombia, by partial S/P gene sequencing, and found, for the first time, HBV/E circulating in nine Afro-Colombian patients who had no recent contact with Africa. The presence of HBV/E in this community as a monophyletic group suggests that it was a result of a recent introduction by some Afro-descendent contact or, alternatively, that the virus came with slaves brought to Colombia. By using sequences with sampling dates, we estimated the substitution rate to be about 3.2x10(-4) substitutions per site per year, which resulted in a time to the most recent common ancestor (TMRCA) of 29 years. In parallel, we also estimated the TMRCA for HBV/E by using two previously estimated substitution rates (7.7x10(-4) and 1.5x10(-5) substitutions per site per year). The TMRCA was around 35 years under the higher rate and 1500 years under the slower rate. In sum, this work reports for the first time the presence of an exclusively African HBV genotype circulating in South America. We also discuss the time of the entry of this virus into America based on different substitution rates estimated for HBV.

Relative Bioavailability of Two Oral Formulations of Risperidone 2 mg: A Single-Dose, Randomized-Sequence, Open-Label, Two-Period Crossover Comparison in Healthy Brazilian Volunteers

Background: Risperidone (RSP) is a benzisoxazole antipsychotic agent used to treat schizophrenia and other psychiatric illnesses in adults and children (including those with autism). After oral administration, RSP is completely absorbed from the gastrointestinal tract and undergoes hydroxylation to yield 9-hydroxyrisperidone (9-OH-RSP), an active metabolite that has a pharmacologic profile and potency similar to RSP. Objectives: The aims of this study were to compare the relative bioavailability of a pharmaceutical-equivalent (test) formulation with a reference formulation of oral RSP 2 mg, both available commercially on the Brazilian pharmaceutical market, and to generate data regarding the oral bioavailability of the tested drug in healthy Brazilian volunteers. Methods: This single-dose, randomized-sequence, open-label, 2-period crossover study was conducted in healthy Brazilian volunteers from August to December 2008. Subjects were randomly assigned to receive the test formulation followed by the reference formulation or vice versa, with a 30-day washout period between doses. Study drugs were administered after a 12-hour overnight fast. For pharmacokinetic analysis, blood samples were drawn at 0 (baseline), 0.25, 0.5, 1, 1.5, 3, 5, 8, 12, 24, 48, 72, 96, and 120 hours after administration. Plasma concentrations of RSP and 9-OH-RSP were determined using LC-MS/MS. The test and reference formulations were to be considered bioequivalent if the 90% CIs for the geometric mean test/reference ratios were within a predetermined range of 80% to 125%, in accordance with the policies of the Brazilian Sanitary Surveillance Agency and the US Food and Drug Administration. Tolerability was determined using clinical assessments, monitoring of vital signs, analysis of laboratory test results, and subject interviews regarding adverse events. Results: A total of 22 subjects were enrolled (11 men, 11 women; mean [SD] age, 32 [12] years [range, 18-58 years]; weight, 70.4 [11.9] kg [range, 50-103 kg]; height, 1.67 [0.08] m [range, 1.56-1.80 m]; and body mass index, 25 [4] kg/m(2) [range, 18-29 kg/m(2)]). For RSP, mean (SD) C(max) values were 12.6 (2.7) and 16.0 (2.3) ng/mL for the test and reference formulations, respectively. For 9-OH-RSP, mean C(max) values were 17.8 (1.3) and 21.0 (1.7) ng/mL for the test and reference formulations. The 90% CIs for the mean test/reference ratios for RSP C(max), AUC(0-120), and AUC(0-infinity) were 74% to 82%, 75% to 85%, and 76% to 85%, respectively, and 83% to 87%, 75% to 79%, and 75% to 78% for 9-OH-RSP. The related adverse events (headache, low back pain, drowsiness, standing hypotension, local postvenipuncture ecchymoses, insomnia, nausea, and vomiting) were transient and mild. Conclusions: This single-dose study found that the test and reference formulations of oral RSP 2 mg did not meet the Brazilian and US regulatory criteria for bioequivalence in these fasting, healthy volunteers. The study formulations appeared to be well tolerated. (Clin Ther 2010;32:2106-2115) (C) 2010 Elsevier HS Journals, Inc.

Changes in plasma free fatty acid levels in septic patients are associated with cardiac damage and reduction in heart rate variability

Free fatty acids (FFAs) have been shown to produce alteration of heart rate variability (HRV) in healthy and diabetic individuals. Changes in HRV have been described in septic patients and in those with hyperglycemia and elevated plasma FFA levels. We studied if sepsis-induced heart damage and HRV alteration are associated with plasma FFA levels in patients. Thirty-one patients with sepsis were included. The patients were divided into two groups: survivors(n = 12) and nonsurvivors (n = 19). The following associations were investigated: (a) troponin I elevation and HRV reduction and (b) clinical evolution and HRV index, plasma troponin, and plasma FFA levels. Initial measurements of C-reactive protein and gravity Acute Physiology and Chronic Health Evaluation scores were similar in both groups. Overall, an increase in plasma troponin level was related to increased mortality risk. From the first day of study, the nonsurvivor group presented a reduced left ventricular stroke work systolic index and a reduced low frequency (LF) that is one of HRV indexes. The correlation coefficient for LF values and troponin was r(2) = 0.75 (P < 0.05). All patients presented elevated plasma FFA levels on the first day of the study (5.11 +/- 0.53 mg/mL), and this elevation was even greater in the nonsurvivor group compared with the survivors (6.88 +/- 0.13 vs. 3.85 +/- 0.48 mg/mL, respectively; P < 0.05). Cardiac damage was confirmed by measurement of plasma troponin I and histological analysis. Heart dysfunction was determined by left ventricular stroke work systolic index and HRV index in nonsurvivor patients. A relationship was found between plasma FFA levels, LFnu index, troponin levels, and histological changes. Plasma FFA levels emerged as possible cause of heart damage in sepsis.

Hepatitis Delta virus genotype 8 infection in Northeast Brazil: Inheritance from African slaves?

Hepatitis Delta virus (HDV) is endemic worldwide, but its prevalence varies in different geographical areas. While in the Brazilian Amazon, HDV is known to be endemic and to represent a significant public health problem, few studies have assessed its prevalence in other regions in the country. This study evaluated the seroprevalence of HDV among HBsAg chronic carriers from Maranhao state, a region located in the Northeast of Brazil. Among 133 patients, 5 had anti-HD, of whom 3 had HDV RNA. HDV genotypes were characterized by Bayesian phylogenetic analysis of nucleotide sequences from the HDAg coding region. HDV-3 was identified in one patient who lives in Maranhao, but was born in Amazonas state (Western Amazon basin). Phylogenetic analysis shows that this HDV-3 sequence grouped with other HDV-3 sequences isolated in this state, which suggests that the patient probably contracted HDV infection there. Surprisingly, the other two patients were infected with HDV-8, an African genotype. These patients were born and have always lived in Urbano Santos, a rural county of Maranhao state, moreover they had never been to Africa and denied any contact with people from that continent. This is the first description of the HDV-8 in non-native African populations. This genotype may have been introduced to Brazil through the slaves brought to the country from the West Africa regions during the 16-18th centuries. Our results indicate that the need of clinical and epidemiological studies to investigate the presence of this infection in other areas in Brazil. (C) 2011 Elsevier B.V. All rights reserved.

The ceja-1 sequence as a potential new molecular marker for Paracoccidioides brasiliensis infection

P>We have developed a two-step PCR assay that amplifies a region of the ceja-1 sequence that is specific for virulent strains of Paracoccidioides brasiliensis. An internal region of the ceja-1 sequence was chosen for designing primers that were utilised in a single tube heminested PCR protocol to amplify DNA from six virulent strains. PCR specificity was determined by the absence of amplified products with genomic DNA from four non-virulent strains of P. brasiliensis and from eight fungal pathogens, one bacterium, two protozoa, one worm and mouse and human genomic DNA (leucocytes). The fact that the PCR product was only obtained with the genetic material from virulent isolates of P. brasiliensis suggested that this partial amplified sequence might be a marker of virulence for this fungus. The diagnostic potential of this PCR was confirmed by the successful amplification of this fragment with genomic DNA obtained in lymph node aspirate from a patient with paracoccidioidomycosis.

Brain Structural Variability due to Aging and Gender in Cognitively Healthy Elders: Results from the Sao Paulo Ageing and Health Study

BACKGROUND AND PURPOSE: Several morphometric MR imaging studies have investigated age- and sex-related cerebral volume changes in healthy human brains, most often by using samples spanning several decades of life and linear correlation methods. This study aimed to map the normal pattern of regional age-related volumetric reductions specifically in the elderly population. MATERIALS AND METHODS: One hundred thirty-two eligible individuals (67-75 years of age) were selected from a community-based sample recruited for the Sao Paulo Ageing and Health (SPAH) study, and a cross-sectional MR imaging investigation was performed concurrently with the second SPAH wave. We used voxel-based morphometry (VBM) to conduct a voxelwise search for significant linear correlations between gray matter (GM) volumes and age. In addition, region-of-interest masks were used to investigate whether the relationship between regional GM (rGM) volumes and age would be best predicted by a nonlinear model. RESULTS: VBM and region-of-interest analyses revealed selective foci of accelerated rGM loss exclusively in men, involving the temporal neocortex, prefrontal cortex, and medial temporal region. The only structure in which GM volumetric changes were best predicted by a nonlinear model was the left parahippocampal gyrus. CONCLUSIONS: The variable patterns of age-related GM loss across separate neocortical and temporolimbic regions highlight the complexity of degenerative processes that affect the healthy human brain across the life span. The detection of age-related Ill GM decrease in men supports the view that atrophy in such regions should be seen as compatible with normal aging.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

Evaluation of RET polymorphisms in a six-generation family with G533C RET mutation: specific RET variants may modulate age at onset and clinical presentation

P>Context We previously described a six-generation family with G533C RET mutation and medullary thyroid carcinoma, in the largest family reported do date. Of particular interest, phenotype variability regarding the age of onset and clinical presentation of the disease, was observed. Objective We evaluate whether single SNPs within RET oncogene or haplotype comprising the RET variants (defined by Haploview) could predispose to early development of MTC in this family and influence the clinical manifestation. Design Eight SNPs were selected based on their previous association with the clinical course of hereditary or sporadic MTC, in particular promoting an early onset of disease. The variants were initially tested in 77 G533C-carriers and 100 controls using either PCR-direct sequencing or PCR-RFLP. Association between a SNP or haplotype and age at diagnosis or presence of lymph node metastasis was tested in 34 G533C-carries with MTC. Different bioinformatic tools were used to evaluate the potential effects on RNA splicing. Results An association was found between IVS1-126G > T and age at diagnosis. The variant [IVS8 +82A > G; 85-86 insC] was associated with the presence of lymph node metastases at diagnosis. In silico analysis suggested that this variant may induce abnormal splicing. This in silico analysis predicted that the [IVS8 +82A > G; 85-86 insC] could alter the splicing by disrupting and/or creating exonic splicing enhancer motifs. Conclusions We here identified two RET variants that were associated with phenotype variability in G533C-carriers, which highlights the fact that the modifier effect of a variant might depend on the type of mutation.

Karyotype variability in KP1(+) and KP1(-) strains of Trypanosoma rangeli isolated in Brazil and Colombia

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(-) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T rangeli strains into two groups coinciding with the KP1(+) and KP1(-) genotypes. Southern blot hybridization showed that, except for the hexose transporter probe, all other probes produced distinct patterns able to differentiate the KP1(+) and KP1(-) genotypes. The UH, STK and An-1A04 probes exclusively hybridized to the chromosomes of KP1(+) strains and can be used as markers of this group. In addition, the UBE2, rRNAmtr and PSMD6 markers, which are present in a conserved region in all trypanosomatid species sequenced so far, co-hybridized to the same T. rangeli chromosomal bands, suggesting the occurrence of gene synteny in these species. The finding of distinct molecular karyotypes in KP1(+) and KP1 (-) strains of T rangeli is noteworthy and might be used as a new approach to the study of genetic variability in this parasite. Together with the Southern blot hybridization results, these findings demonstrate that differences at the kDNA level might be associated with variations in nuclear DNA. (c) 2009 Elsevier BY. All rights reserved.