Early expression of the aflatoxin gene cluster in Aspergillus nomiae isolated from Brazil nut.

Aspergillus nomiae is the most important contaminant in Brazil nut due to its high incidence in these nuts and its strong production of carcinogenic metabolites: aflatoxins (AF). Aflatoxin biosynthesis pathway in A. nomiae is poorly studied. Thus, in present investigation, aflatoxin production and gene cluster (aflC, aflQ, aflU, and aflX) expression profile were evaluated on two strains of A. nomiae isolated from Brazil nut samples collected in the Amazon region, and cultivated on Brazil nut-based medium. The results showed that the expression of the aflatoxin gene cluster in A. nomiae, started at day 2 and occurred before the production of aflatoxins. Aflatoxin production (AFB1 and AFG1) was detected on day 3 on both strains. From day 4 onwards, all four toxins were detected and their production kept at similar proportions (AFG1>AFB1>AFG2>AFB2). Thus, the anticipated information obtained through early expression profile results of aflC, aflQ, aflU, and aflX gene cluster in A. nomiae may foresee aflatoxin production before its detection in Brazil nuts.

Brazil Nut Supplementation Does Not Regulate PPARß/δ Signaling Pathway in Peripheral Blood Mononuclear Cells from Coronary Artery Disease Patients.

Background: Peroxisome proliferator-activated receptor (PPAR)ß/δ activation is a potential target for modulation of inflammation in cardiovascular disease. PPARß/δ activation depends on the presence of a ligand, which may be pharmacological or natural, such as bioactive compounds and nutrients. Due to its composition, rich in selenium and unsaturated fatty acids, Brazil nuts have been related to reduced oxidative stress and inflammation in chronic non-communicable diseases and could regulate PPARß/δ. This study aimed to evaluate the effects of Brazil nut supplementation on PPARß/δ mRNA expression in patients with Coronary Artery Disease (CAD).Methods: A secondary analysis of a randomized controlled clinical trial was performed with 36 CAD patients. Patients were randomly assigned to either the Supplementation group or the control group and followed up for three months. The Supplementation group consumed 1 Brazil nut/day; the control group did not receive any intervention. At the baseline and after three months, analysis of gene expression and biochemical parameters linked to inflammatory biomarkers and oxidative stress was carried out.Results: In the supplementation group, no significant change was observed in PPARß/δ (0.9 ± 0.5 vs 1.2 ± 0.6; p = 0.178) and NF-κB (1.6 ± 1.5 vs 0.8 ± 0.30, p = 0.554) mRNA expression. There were no significant changes in both groups concerning all the other biochemical parameters.Conclusion: One Brazil nut per day for three months was not able to increase the PPARß/δ expression in CAD patients.

Target sequence capture in the Brazil nut family (Lecythidaceae): Marker selection and in silico capture from genome skimming data.

Reconstructing species trees from multi-loci datasets is becoming a standard practice in phylogenetics. Nevertheless, access to high-throughput sequencing may be costly, especially with studies of many samples. The potential high cost makes a priori assessments desirable in order to make informed decisions about sequencing. We generated twelve transcriptomes for ten species of the Brazil nut family (Lecythidaceae), identified a set of putatively orthologous nuclear loci and evaluated, in silico, their phylogenetic utility using genome skimming data of 24 species. We designed the markers using MarkerMiner, and developed a script, GoldFinder, to efficiently sub-select the best makers for sequencing. We captured, in silico, all designed 354 nuclear loci and performed a maximum likelihood phylogenetic analysis on the concatenated sequence matrix. We also calculated individual gene trees with maximum likelihood and used them for a coalescent-based species tree inference. Both analyses resulted in almost identical topologies. However, our nuclear-loci phylogenies were strongly incongruent with a published plastome phylogeny, suggesting that plastome data alone is not sufficient for species tree estimation. Our results suggest that using hundreds of nuclear markers (i.e. 354) will significantly improve the Lecythidaceae species tree. The framework described here will be useful, generally, for developing markers for species tree inference.

Pollen and seed dispersal of Brazil nut trees in the southwestern Brazilian Amazon / Dispersão de pólen e sementes da castanheira no sudoeste da Amazônia brasileira

Pollen and seed dispersal patterns greatly influence the spatial distribution of plant genetic diversity. Microsatellite-based parentage analysis provides accurate estimates of contemporary gene dispersal. Although most tropical trees have been shown to exhibit widespread pollen dispersal, few studies have estimated contemporary gene dispersal after seedling establishment. Bertholletia excelsa (Lecythidaceae) is pollinated by large-bodied bees, while previous seed-tracking experiments suggest their seeds are mainly dispersed across very short distances by scatter-hoarding rodents, who primarily act as seed predators. Here we used parentage analysis to provide contemporary estimates of pollen and seed dispersal in B. excelsa recruits. We examined six 25-ha plots located in two natural stands in the Acre River valley, in the southwestern Brazilian Amazon. We used 11 microsatellite markers to estimate genetic diversity and fixation index parameters in adults, seedlings and saplings. Genetic diversity was moderate and did not differ across size classes or sampling locations. We assigned pollen and seed parents for < 20% of the recruits, indicating that most events of realized gene flow occurred beyond our 25-ha plots. Only 10 parentage assignments were confirmed with 80% confidence. Pollen distance ranged from 33 to 372 m and seed dispersal from 58 to 655 m. Actual seed-dispersal distances were far greater than the estimates obtained in previous seed-tracking experiments. Thus, studies encompassing larger sampling areas are necessary to determine a more representative spatial scale of B. excelsas pollen and seed dispersal capacity in natural stands.

Os padrões de dispersão de pólen e sementes influenciam a distribuição espacial da diversidade genética. Muitas espécies arbóreas tropicais apresentam ampla dispersão de pólen, mas poucos estudos avaliaram fluxo gênico a partir de plântulas. Bertholletia excelsa (Lecythidaceae) é polinizada por abelhas e as sementes são dispersas por roedores do tipo scatter-hoarders (que estocam recursos em diferentes pontos de sua área de vida), que atuam primariamente como predadores de sementes. Experimentos de remoção de sementes tem mostrado que a dispersão de sementes por esses roedores é espacialmente limitada. Nosso objetivo foi obter estimativas de dispersão de pólen e sementes em B. excelsa a partir da análise de parentesco de regenerantes. Nós estudamos seis parcelas de 25 ha, em duas áreas de floresta nativa no vale do Rio Acre, no sudoeste da Amazônia brasileira. Parâmetros de diversidade genética e índice de fixação foram estimados em adultos, varetas e plântulas com 11 marcadores microssatélites. A diversidade genética foi moderada e não diferiu entre classes de tamanho ou entre localidades. A paternidade foi determinada em menos de 20% dos regenerantes, indicando que a maioria dos eventos de fluxo gênico ocorreu em distâncias maiores que as encontradas nas parcelas de 25 ha. As distâncias de pólen variaram de 33 a 372 m e as de dispersão de sementes variaram de 58 a 655 m. As distâncias de dispersão obtidas neste estudo excedem em muito as estimativas obtidas em experimentos de remoção de sementes. Estudos envolvendo áreas maiores são necessários para que possamos aprofundar nosso conhecimento sobre capacidade de dispersão de pólen e sementes em populações naturais de B. excelsa.

Contemporary pollen and seed dispersal in natural populations of Bertholletia excelsa (Bonpl.).

Due to the nutritional content and commercial value of its seeds, Bertholletia excelsa is one of the most important species exploited in the Amazon region. The species is hermaphroditic, insect pollinated, and its seeds are dispersed by barochory and animals. Because the fruit set is dependent on natural pollinator activity, gene flow plays a key role in fruit production. However, to date, there have been no studies on pollen and seed flow in natural populations of B. excelsa. Herein, we used microsatellite loci and parentage analysis to investigate the spatial genetic structure (SGS), realized pollen and seed dispersal, and effective pollen dispersal for two B. excelsa populations in the Brazilian Amazon forest. Two plots were established in natural forests from which adults, juveniles, and seeds were sampled. Realized and effective pollen flow was greater than realized seed flow. The distance of realized pollen dispersal ranged from 36 to 2060 m, and the distance of realized seed dispersal ranged from 30 to 1742 m. Both pollen and seeds showed a dispersal pattern of isolation by distance, indicating a high frequency of mating among near-neighbor trees and seed dispersal near to mother trees. Both populations present SGS up to 175 m, which can be explained by isolation by distance pollen and seed dispersal patterns. Our results suggested that fragmentation of these forest populations may result in a significant decrease in gene flow, due to the isolation by distance pollen and seed dispersal patterns.

Minimum number of measurements for evaluating Bertholletia excelsa.

Repeatability studies on fruit species are of great importance to identify the minimum number of measurements necessary to accurately select superior genotypes. This study aimed to identify the most efficient method to estimate the repeatability coefficient (r) and predict the minimum number of measurements needed for a more accurate evaluation of Brazil nut tree (Bertholletia excelsa) genotypes based on fruit yield. For this, we assessed the number of fruits and dry mass of seeds of 75 Brazil nut genotypes, from native forest, located in the municipality of Itaúba, MT, for 5 years. To better estimate r, four procedures were used: analysis of variance (ANOVA), principal component analysis based on the correlation matrix (CPCOR), principal component analysis based on the phenotypic variance and covariance matrix (CPCOV), and structural analysis based on the correlation matrix (mean r - AECOR). There was a significant effect of genotypes and measurements, which reveals the need to study the minimum number of measurements for selecting superior Brazil nut genotypes for a production increase. Estimates of r by ANOVA were lower than those observed with the principal component methodology and close to AECOR. The CPCOV methodology provided the highest estimate of r, which resulted in a lower number of measurements needed to identify superior Brazil nut genotypes for the number of fruits and dry mass of seeds. Based on this methodology, three measurements are necessary to predict the true value of the Brazil nut genotypes with a minimum accuracy of 85%.

Diversity and genetic structure of the native Brazil nut tree (Bertholletia excelsa Bonpl.) population.

The present study was carried out in a native Brazil nut tree population (Bertholletia excelsa Bonpl., Lecythidaceae) to assess its genetic diversity and structure. Ten microsatellite markers were used to genotype 198 adult trees (B. excelsa). The population presented high genetic diversity and inbreeding absence rates. The empirical Bayesian method showed three distinct groups in the structure of this population. Molecular analysis of variance showed 98% variability within groups, and 2% between groups. The genetic divergence (FST) indicated little difference between groups; thus, suggesting efficient gene flow between the analyzed B. excelsa adult trees.

Isolation and characterization of microsatellite markers for Bertholletia excelsa (Lecythidaceae) population genetic analysis.

Seven polymorphic microsatellite markers were developed and validated for Bertholletia excelsa (Brazil nut tree) population genetic studies. This species is a widespread monotypic Amazonian tree with high non-timber economic value. Unfortunately, Brazil nut production is currently less than 25% of historical production levels, because of extensive deforestation. All pairs of primers produced clearly interpretable and polymorphic bands. No linkage disequilibrium was observed in an analysis of 46 individuals from one population, three to seven alleles per locus were observed; the expected heterozygosity ranged from 0.378 to 0.978, with significant heterozygote excess for four loci. An analysis of individuals from two populations showed private alleles at all loci. These primer pairs will be useful for population studies, especially for comparing samples from different parts of the Amazon forest.

Selection of recombinant antibodies by phage display technology and application for detection of allergenic Brazil nut (Bertholletia excelsa) in processed foods.

Current immunological methods for detection of Brazil nut allergens in foods are based on polyclonal antibodies raised in animals. Phage display technology allows the procurement of high-affinity antibodies avoiding animal immunization steps and therefore attaining the principle of replacement supported by animal welfare guidelines. In this study, we screened Tomlinson I and J libraries for specific binders against Brazil nut by employing a Brazil nut protein extract and a purified Brazil nut 2S globulin, and we successfully isolated a phage single chain variable fragment (named BE95) that specifically recognizes Brazil nut proteins. The selected phage scFv was further used as affinity probe to develop an indirect phage-ELISA for detection of Brazil nut in experimental binary mixtures and in commercial food products, with a limit of detection of 5 mg g(-1). This study describes for the first time the isolation of recombinant antibody fragments specific for an allergenic tree nut protein from a naïve library and paves the way to develop new immunoassays for food analysis based on probes that can be produced in vitro when required and do not rely on animal immunization.

Morphological and molecular characteristics do not confirm popular classification of the Brazil nut tree in Acre, Brazil.

In the State of Acre, the Brazil nut tree, Bertholletia excelsa (Lecythidaceae), is classified by the local population into two types according to morphological characteristics, including color and quality of wood, shape of the trunk and crown, and fruit production. We examined the reliability of this classification by comparing morphological and molecular data of four populations of Brazil nut trees from Vale do Rio Acre in the Brazilian Amazon. For the morphological analysis, we evaluated qualitative and quantitative information of the trees, fruits, and seeds. The molecular analysis was performed using RAPD and ISSR markers, with cluster analysis. Significant differences were found between the two types of Brazil nut trees for the characters diameter at breast height, fruit yield, fruit size, and number of seeds per fruit. Despite the significant correlation between the morphological characteristics and the popular classification, we observed all possible combinations of morphological characteristics in both types of Brazil nut trees. In some individuals, the classification did not correspond to any of the characteristics. The results obtained with molecular markers showed that the two locally classified types of Brazil nut trees did not differ genetically, indicating that there is no consistent separation between them.

Aspergillus bertholletius sp. nov. from Brazil nuts.

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial ß-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial ß-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).

Variabilidade genética para caracteres morfométricos de matrizes de castanha-do-brasil da Amazônia Mato-grossense / Genetic variability for morphometric characteristics in Brazilian nut parent trees from northern Mato Grosso, Amazon rain forest

O presente estudo objetivou estudar a variabilidade genética de matrizes de Bertholletia excelsa através da estimação de parâmetros e ganhos genéticos para os caracteres peso/ouriço (g), peso de sementes/ouriço (g) e número de sementes/ouriço no pré-melhoramento da espécie. Foram utilizadas 90 matrizes de polinização aberta, sendo 30 matrizes de cada tipo, denominadas localmente de rajada, mirim e rosa, no município de Cotriguaçu, noroeste de Mato Grosso, região amazônica. O experimento foi estabelecido sob delineamento inteiramente ao acaso, com 90 tratamentos (matrizes) e seis ouriços por matriz, com suas respectivas sementes. As variáveis foram analisadas usando-se a metodologia de modelo linear misto do software SELEGEN-REML/BLUP. Os coeficientes de herdabilidades individuais no sentido amplo dos efeitos genotípicos totais (0,21, 0,14 e 0,34) para os caracteres peso/ouriço (g), peso de sementes/ouriço (g) e número de sementes/ouriço, respectivamente, são considerados moderados para os dois primeiros caracteres e alto para o caráter número de sementes/ouriço, sugerindo expressivo controle genético. A seleção das 10 melhores matrizes revelou predominância da procedência do tipo rosa, proporcionando ganhos genéticos expressivos de pelo menos 24,16% para peso/ouriço (g), 27,44% para peso de sementes/ouriço e 16,92% para o caráter número de sementes por ouriço. Os valores expressivos das matrizes do tipo rosa estimulam a utilização desses germoplasmas em programas de melhoramento genético da espécie, na seqüência das avaliações, bem como apontando para a possibilidade de obtenção de híbridos intraespecíficos para caracteres desejáveis.

The goal of the study was to detect genetic variability in Brazilian nuts parent trees through parameters estimation and genetic gains for the following indexes: fruit weight (g) seed weight per fruit (g) and number of seeds per fruit at species pre-improvement. Ninety (90) open pollination parent trees were used, 30 of which being of each type, locally called "rajada", "mirim" and "rosa" at the Cotriguacu municipality, Northeastern of Mato Grosso State. A completely randomized design was used, with 90 treatments (parent trees) and six fruits per parcel with their respective seeds. Variables were analyzed using the method of mixed univariate additive linear model from the software SELEGEN-REML/BLUP. Individual heritability coefficients in the broad sense for total genotypic effects (0,21, 0,14 e 0,34) for fruit weight (g) seed weight per fruit (g) and number of seeds per fruit (g) respectively, are considered moderate for the two first indexes and high for number of seeds per fruit, suggesting substantial genetic control. Selection of the 10 best provenances and parent trees showed predominance of the "rosa" type, allowing substantial genetic gains of at least 24,16% for fruit weight (g), 27,44% for seed weight per fruit and 16,92% for the number of seeds per fruit. The remarkable values for the "rosa" type suggest utilization of such germoplasms in genetic improvement programs of this species in the following evaluations. The results also point out the possibility of obtaining intraspecific hybrids for desirable characteristics.

A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods.

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.

A novel real-time polymerase chain reaction method for the detection of Brazil nuts in food.

A qualitative real-time PCR-based method for the detection of the Brazil nut (Bertholletia excelsa) component in food is described. The method consists of DNA isolation by chaotropic SPE and the subsequent PCR with Brazil nut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the gene encoding for the 11s globulin of the Brazil nut. The method was positive for eight Brazil nut samples from the market and negative for all other tested plant and meat materials used in the food industry (36 samples). The intrinsic LOD of the method was 10 pg Brazil nut DNA. Using a series of model nut paste mixtures with defined Brazil nut contents, a practical detection limit of 0.1% (w/w) Brazil nut was estimated. Practical applicability of the entire method was tested by qualitative analysis of nine food samples; no discrepancies between the declared and detected Brazil nut contents were found. The presented PCR-based method is useful for sensitive and selective detection of Brazil nut in food samples.

Parallel rate heterogeneity in chloroplast and mitochondrial genomes of Brazil nut trees (Lecythidaceae) is consistent with lineage effects.

We investigated whether relative rates of divergence were correlated between the mitochondrial and chloroplast genomes as expected under lineage effects or were genome specific as expected with locus-specific effects. Five mitochondrial noncoding regions (nad1B_C, nad4exon1_2, nad7exon2_3, nad7exon3_4, and rps14-cob) for 21 samples from Lecythidaceae were sequenced. Three chloroplast regions (rpl20-5'rps12, trnS-trnG, and psbA-trnH) were sequenced to expand the taxa in an existing data set. Absolute rates of nucleotide and insertion and deletion (indel) changes were 13 times faster in the chloroplast genome than in the mitochondrial genome. Similar indel length frequency distributions for both organelles suggested that common mechanisms were responsible for generating indels. Molecular clock tests applied to phylogenetic trees estimated from mitochondrial and chloroplast sequences revealed global rate heterogeneity of nucleotide substitution. Maximum likelihood and Tajima's 1D relative rate tests show that Lecythis zabucajo exhibited a rate acceleration for both the mitochondrial and chloroplast sequences. Whereas Eschweilera romeu-cardosoi showed a significant rate slowdown for chloroplast sequences, the mitochondrial sequences for 3 Eschweilera taxa showed evidence for a rate slowdown only when compared with L. zabucajo. Significant rate heterogeneity was also observed for indel changes in the mitochondrial genome but not for the chloroplast. The lack of mitochondrial nucleotide changes for some taxa as well as chloroplast indel homoplasy may have limited the power of relative rate tests to detect rate variation. Relative ratio tests consistently indicated rate proportionality among branch lengths between the mitochondrial and chloroplast phylogenetic trees. The relative ratio tests showed that taxa possessing rate heterogeneity had parallel relative divergence rates in both mitochondrial and chloroplast sequences as expected under lineage effects. A neutral replication-dependent model of rate heterogeneity for both nucleotide and indel changes provides a simple explanation for common patterns of rate heterogeneity across the 2 organelle genomes in Lecythidaceae. The lineage effects observed here were uncoupled from annual/perennial habit because all the species from this study are perennial.

Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa).

Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms ( approximately 10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one >or=7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly alpha-helical in nature, but that the Mr 13400 species was structurally distinct, with a higher proportion of alpha-helical structure.