DGGE with genomic DNA: Suitable for detection of numerically important organisms but not for identification of the most abundant organisms

Identification of all important community members as well as of the numerically dominant members of a community are key aspects of microbial community analysis of bioreactor samples. A systematic study was conducted with artificial consortia to test whether denaturing gradient gel electrophoresis (DGCE) is a reliable technique to obtain such community data under conditions where results would not be affected by differences in DNA extraction efficiency from cells. A total of 27 consortia were established by mixing DNA extracted from Escherichia coli K12, Burkholderia cepacia and Stenotrophomonas maltophilia in different proportions. Concentrations of DNA of single organisms in the consortia were either 0.04, 0.4 or 4 ng/mu l. DGGE-PCR of genomic DNA with primer sets targeted at the V3 and V6-V8 regions of the 16S rDNA failed to detect the three community members in only 7% of consortia, but provided incorrect information about dominance or co-dominance for 85% and 89% of consortia with the primer sets for the V6-V8 and V3 regions, respectively. The high failure rate in detection of dominant B. cepacia with the primers for the V6-V8 region was attributable to a single nucleoticle primer mismatch in the target sequences of both, the forward and reverse primer. Amplification bias in PCR of E. coli and S. maltophilia for the V6-V8 region and for all three organisms for the V3 region occurred due to interference of genomic DNA in PCR-DGGE, since a nested PCR approach, where PCR-DGGE was started from mixtures of 16S rRNA genes of the organisms, provided correct information about the relative abundance of original DNA in the sample. Multiple bands were not observed in pure culture amplicons produced with the V6-V8 primer pair, but pure culture V3 DGGE profiles of E. coli, S. maltophilia and B. cepacia contained 5, 3 and 3 bands, respectively. These results demonstrate DGGE was suitable for identification of all important community members in the three-membered artificial consortium, but not for identification of the dominant organisms in this small community. Multiple DGGE bands obtained for single organisms with the V3 primer pair could greatly confound interpretation of DGGE profiles. (C) 2008 Elsevier Ltd. All rights reserved.

Analysis of the genome of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV-19) and of the high genomic heterogeneity in group II nucleopolyhedroviruses

The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-1 9), was completely sequenced and shown to comprise 132 565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus; promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-1 9 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-1 9 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-1 9 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes and rearrangements. Synthenic maps for the genomes of group 11 NPVs revealed that gene collinearity was observed only within certain clusters. Analysis of the dynamics of gene gain and loss along the phylogenetic tree of the NPVs showed that group 11 had only five defining genes and supported the hypothesis that these viruses form ten highly divergent ancient lineages. Crucially, more than 60% of the gene gain events followed a power-law relation to genetic distance among baculoviruses, indicative of temporal organization in the gene accretion process.

Abnormal Corpus Callosum Integrity in Bipolar Disorder: A Diffusion Tensor Imaging Study

Objective: Abnormalities in the anterior interhemispheric connections provided by the corpus callosum (CC) have long been implicated in bipolar disorder (BID). In this study, we used complementary diffusion tensor imaging methods to study the structural integrity of the CC and localization of potential abnormalities in BD. Methods: Subjects included 33 participants with BID and 40 healthy comparison participants. Fractional anisotropy (FA) measures were compared between groups with region of interest (ROD methods to investigate the anterior, middle, and posterior CC and voxel-based methods to further localize abnormalities. Results: In ROI-based analyses, FA was significantly decreased in the anterior and middle CC in the BID group (p <.05). Voxel-based analyses similarly localized group differences to the genu, rostral body, and anterior midbody of CC (p <.05, corrected). Conclusion: The findings demonstrate abnormalities in the structural integrity of the anterior CC in BID that might contribute to altered interhemispheric connectivity in this disorder.

Abnormal anterior cingulum integrity in bipolar disorder determined through diffusion tensor imaging

Background Convergent evidence implicates white matter abnormalities in bipolar disorder. The cingulum is an important candidate structure for study in bipolar disorder as it provides substantial white matter connections within the corticolimbic neural system that subserves emotional regulation involved in the disorder. Aims To test the hypothesis that bipolar disorder is associated with abnormal white matter integrity in the cingulum. Method Fractional anisotropy in the anterior and posterior cingulum was compared between 42 participants with bipolar disorder and 42 healthy participants using diffusion tensor imaging. Results Fractional anisotropy was significantly decreased in the anterior cingulum in the bipolar disorder group compared with the healthy group (P=0.003); however, fractional anisotropy in the posterior cingulum did not differ significantly between groups. Conclusions Our findings demonstrate abnormalities in the structural integrity of the anterior cingulum in bipolar disorder. They extend evidence that supports involvement of the neural system comprising the anterior cingulate cortex and its corticolimbic gray matter connection sites in bipolar disorder to implicate abnormalities in the white matter connections within the system provided by the cingulum.

Genomic organization and transcription analysis of the 195-bp satellite DNA in Trypanosoma cruzi

The 195-bp satellite DNA is the most abundant Trypanosoma cruzi repetitive sequence. Here we show by RNA blotting and RT-PCR that 195 SAT is intensely transcribed. We observed a positive correlation between the level of satellite RNA and the abundance of the satellite copies in the genome of T cruzi strains and that the satellite expression is not developmentally regulated. By analyzing CL Brener individual reads, we estimated that 195 SAT corresponds to approximately 5% of the CL Brener genome. 195 SAT elements were found in only 37 annotated contigs, indicating that a large number of satellite copies were not incorporated into the assembled data. The assembled satellite units are distributed in non-syntenic regions with Trypanosoma brucei and Leishmania major genomes, enriched with surface proteins, retroelements, RHS and hypothetical proteins. Satellite repeats were not observed in annotated subtelomeric regions. We report that 12 satellite sequences are truncated by the retroelement VIPER. (C) 2008 Elsevier B.V. All rights reserved.

Novel Statistical Methods in Quantitative Genetics : Modeling Genetic Variance for Quantitative Trait Loci Mapping and Genomic Evaluation

This thesis develops and evaluates statistical methods for different types of genetic analyses, including quantitative trait loci (QTL) analysis, genome-wide association study (GWAS), and genomic evaluation. The main contribution of the thesis is to provide novel insights in modeling genetic variance, especially via random effects models. In variance component QTL analysis, a full likelihood model accounting for uncertainty in the identity-by-descent (IBD) matrix was developed. It was found to be able to correctly adjust the bias in genetic variance component estimation and gain power in QTL mapping in terms of precision.  Double hierarchical generalized linear models, and a non-iterative simplified version, were implemented and applied to fit data of an entire genome. These whole genome models were shown to have good performance in both QTL mapping and genomic prediction. A re-analysis of a publicly available GWAS data set identified significant loci in Arabidopsis that control phenotypic variance instead of mean, which validated the idea of variance-controlling genes.  The works in the thesis are accompanied by R packages available online, including a general statistical tool for fitting random effects models (hglm), an efficient generalized ridge regression for high-dimensional data (bigRR), a double-layer mixed model for genomic data analysis (iQTL), a stochastic IBD matrix calculator (MCIBD), a computational interface for QTL mapping (qtl.outbred), and a GWAS analysis tool for mapping variance-controlling loci (vGWAS).

Defining the role of common variation in the genomic and biological architecture of adult human height

Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated ∼2,000, ∼3,700 and ∼9,500 SNPs explained ∼21%, ∼24% and ∼29% of phenotypic variance. Furthermore, all common variants together captured 60% of heritability. The 697 variants clustered in 423 loci were enriched for genes, pathways and tissue types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/β-catenin and chondroitin sulfate-related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants.2014

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Brazilian coffee genome project: an EST-based genomic resource

O café é um dos principais produtos agrícolas, sendo considerado o segundo item em importância do comércio internacional de commodities. O gênero Coffea pertence à família Rubiaceae que também inclui outras plantas importantes. Este gênero contém aproximadamente 100 espécies, mas a produção comercial é baseada somente em duas espécies, Coffea arabica e Coffea canephora, que representam aproximadamente 70 % e 30 % do mercado total de café, respectivamente. O Projeto Genoma Café Brasileiro foi desenvolvido com o objetivo de disponibilizar os modernos recursos da genômica à comunidade científica e aos diferentes segmentos da cadeia produtiva do café. Para isso, foram seqüenciados 214.964 clones escolhidos aleatoriamente de 37 bibliotecas de cDNA de C. arabica, C. canephora e C. racemosa representando estádios específicos do desenvolvimento de células e de tecidos do cafeeiro, resultando em 130.792, 12.381 e 10.566 seqüências de cada espécie, respectivamente, após processo de trimagem. Os ESTs foram agrupados em 17.982 contigs e em 32.155 singletons. A comparação destas seqüências pelo programa BLAST revelou que 22 % não tiveram nenhuma similaridade significativa às seqüências no banco de dados do National Center for Biotechnology Information (de função conhecida ou desconhecida). A base de dados de ESTs do cafeeiro resultou na identificação de cerca de 33.000 unigenes diferentes. Os resultados de anotação das seqüências foram armazenados em base de dados online em http://www.lge.ibi.unicamp.br/cafe. Os recursos desenvolvidos por este projeto disponibilizam ferramentas genéticas e genômicas que podem ser decisivas para a sustentabilidade, a competitividade e a futura viabilidade da agroindústria cafeeira nos mercados interno e externo.

Evaluation of the Integrity of Horse Hoof Dermal and Epidermal Tissues Collected by Dorsal Transmural Access

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Role of the oblique ligament in the integrity of the medial collateral ligament of the canine elbow joint

Foram estudados o arranjo e o diâmetro médio das fibrilas colágenas do ligamento colateral medial da articulação do cotovelo do cão, isolado ou associado ao ligamento oblíquo e tracionado até a ruptura. Dezoito articulações foram divididas em três grupos. O primeiro grupo teve o ligamento colateral medial coletado, mas não tracionado; o segundo grupo teve o ligamento colateral medial tracionado isoladamente; o terceiro grupo teve os ligamentos colateral medial e oblíquo tracionados associadamente. O ligamento colateral medial não submetido ao ensaio de tração apresentou um padrão ondulado das fibras colágenas, o qual não foi totalmente destruído quando foi tracionado, associado ao ligamento oblíquo, e perdeu totalmente o padrão reticular das fibras colágenas quando testado isoladamente. Quando o ligamento colateral medial foi submetido à tensão isoladamente, o diâmetro médio das fibrilas colágenas aumentou em relação ao grupo não submetido à tensão. Associado ao ligamento oblíquo, o diâmetro médio das fibrilas colágenas foi o maior na região de inserção e o menor na região média, em relação aos outros grupos. Concluiu-se que o ligamento oblíquo pode favorecer a integridade da região de inserção do ligamento colateral medial, aumentando a eficácia de sua reconstrução após a lesão.

Saccharomyces Cerevisiae cell wall dietary supplementation on the performance and intestinal mucosa development and integrity of broiler chickens vaccinated against coccidiosis

This study was carried out to verify if Saccharomyces cerevisiae cell wall (SCCW) dietary supplementation (0.2%) was capable of protecting the intestinal mucosa of broiler chickens vaccinated against coccidiosis. Body weight gain, feed intake, feed conversion and intestinal mucosa morphometric parameters and epithelial loss were evaluated. In the experiment,400 day-old male chicks were distributed according to a completely randomized design in a 2x2 factorial arrangement. The following treatments were applied: T1 - no vaccination/ no SCCW supplementation; T2 - no vaccination/SCCW supplementation; T3 - vaccination/no SCCW supplementation; and T4 - vaccination/SCCW supplementation to four replicates of 25 birds each. Birds were vaccinated on the first day of age using a spray vaccine (Coccivac B®, Coopers), containing E. acervulina, E. maxima, E. mivati and E. tenella. S. cerevisiae cell wall was supplied from the first day of age. Live performance, intestinal morphometric parameters and epithelial loss were evaluated at 14, 21 and 28 days of age. Performance was affected by vaccination only at 21-days of age, when body weight gain was reduced in the vaccinated birds, but no body weight difference was observed on day 28. Vaccine also increased the crypt depth (p<0.05) in the duodenum and jejunum, suggesting a high cell activity in the crypt:villus transition area to maintain the epithelial cell turnover. Villi number/area (103,269 µm²) was not affected (p>0.05) by vaccine or cell wall supplementation, and epithelial loss was more pronounced in the duodenum and jejunum. In conclusion, the findings of this study suggest that S. cerevisiae cell wall supplementation may be an useful management tool to maintain the intestinal integrity of broilers vaccinated against coccidiosis.