RESUMO
Background: For more than 25 years, the golden mussel, Limnoperna fortunei, has aggressively invaded South American freshwaters, having travelled more than 5000 km upstream across 5 countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it. Findings: We assembled the 1.6-Gb genome into 20 548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60 717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina paired-end, mate-paired, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei's genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.
Assuntos
Mapeamento de Sequências Contíguas/métodos , Genoma , Espécies Introduzidas , Mytilidae/genética , Proteínas/genética , Transcriptoma , Animais , Brasil , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Mytilidae/classificação , Fases de Leitura Aberta , Controle de Pragas , Filogenia , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismoRESUMO
Currently, twelve validated genetic variants have been identified that are associated with urinary bladder cancer (UBC) risk. However, those validated variants explain only 5-10% of the overall inherited risk. In addition, there are more than 100 published polymorphisms still awaiting validation or disproval. A particularly promising of the latter unconfirmed polymorphisms is rs2854744 that recently has been published to be associated with UBC risk. The [A] allele of rs2854744 has been reported to be associated with a higher promoter activity of the insulin-like growth factor-binding protein-3 (IGFBP3) gene, which may lead to increased IGFBP-3 plasma levels and cancer risk. Therefore, we investigated the association of rs2854744 with UBC in the IfADo case-control series consisting of 1,450 cases and 1,725 controls from Germany, Hungary, Venezuela and Pakistan. No significant association of rs2854744 with UBC risk was obtained (all study groups combined: unadjusted P = 0.4446; adjusted for age, gender and smoking habits P = 0.6510), besides a small effect of the [A] allele in the Pakistani study group opposed to the original findings (unadjusted P = 0.0508, odds ratio (OR) = 1.43 for the multiplicative model) that diminished after adjustment for age, gender and smoking habits (P = 0.7871; OR = 0.93). Associations of rs2854744 with occupational exposure to urinary bladder carcinogens and smoking habits were also not present. A meta-analysis of all available case-control series including the original discovery study resulted in an OR of 1.00 (P = 0.9562). In conclusion, we could not confirm the recently published hypothesis that rs2854744 in the IGFBP3 gene is associated with UBC risk.
Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença/etnologia , Alemanha , Humanos , Hungria , Masculino , Pessoa de Meia-Idade , Paquistão , Polimorfismo de Nucleotídeo Único , Neoplasias da Bexiga Urinária/etnologia , VenezuelaRESUMO
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s.
Assuntos
Vacina BCG/genética , Genoma Bacteriano/genética , Mycobacterium bovis/genética , Dados de Sequência Molecular , Tuberculose/imunologiaRESUMO
BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens. RESULTS: We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite. CONCLUSIONS: In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and triacylglycerol lipase (EC 3.1.1.3), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets.
Assuntos
Antiparasitários/uso terapêutico , Doença de Chagas/tratamento farmacológico , Modelos Moleculares , Proteínas de Protozoários/química , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Trypanosoma cruzi/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Sequência de Aminoácidos , Antiparasitários/farmacologia , Doença de Chagas/parasitologia , Bases de Dados de Proteínas , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/classificação , Proteínas de Protozoários/metabolismo , Especificidade da Espécie , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
Single nucleotide polymorphism (SNP) rs710521[A], located near TP63 on chromosome 3q28, was identified to be significantly associated with increased bladder cancer risk. To investigate the association of rs710521[A] and bladder cancer by new data and by meta-analysis including all published data, rs710521 was studied in 1,425 bladder cancer cases and 1,740 controls that had not been included in previous studies. Blood samples were collected from 1995 to 2010 in Germany (n = 948/1,258), Hungary (n = 262/65), Venezuela (n = 112/190) and Pakistan (n = 103/227) supplemented by a meta-analysis of 5,695 cases and 40,187 controls. Detection of a A/G substitution (rs710521) on chromosome 3q28, position 191128627 was done via fast real-time polymerase chain reaction (rt-PCR). Rs710521[A] is associated with increased risk in the unadjusted analysis (OR = 1.21; 95% Cl = 1.04-1.40; P = 0.011) and in the recessive model adjusted for age, gender, smoking habits and ethnicity (OR = 1.23; 95% Cl = 1.05-1.44; P = 0.010). No difference between individuals occupationally exposed versus not occupationally exposed to urinary bladder carcinogens was observed concerning the relevance of rs710521[A]. Similarly, rs710521[A] did not confer different susceptibility in smokers and non-smokers. Performing a meta-analysis of 5,695 cases and 40,187 controls including all published studies on rs710521, a convincing association with bladder cancer risk was obtained (OR = 1.18; 95% Cl = 1.12-1.25; P < 0.0001). However, the odds ratio is relatively small.
Assuntos
Cromossomos Humanos Par 3 , Genes , Polimorfismo de Nucleotídeo Único , Transativadores/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Estudos de Casos e Controles , Feminino , Alemanha , Humanos , Hungria , Masculino , Razão de Chances , Paquistão , Reação em Cadeia da Polimerase , Risco , Fumar/efeitos adversos , Fumar/genética , Fatores de Transcrição , VenezuelaRESUMO
Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes.
Assuntos
Cisteína Proteases/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Leishmania braziliensis/enzimologia , Fatores de Virulência/biossíntese , Animais , Cisteína Proteases/genética , Immunoblotting , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/patogenicidade , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inoculações Seriadas , Virulência , Fatores de Virulência/genéticaRESUMO
MOTIVATION: Many analyses in modern biological research are based on comparisons between biological sequences, resulting in functional, evolutionary and structural inferences. When large numbers of sequences are compared, heuristics are often used resulting in a certain lack of accuracy. In order to improve and validate results of such comparisons, we have performed radical all-against-all comparisons of 4 million protein sequences belonging to the RefSeq database, using an implementation of the Smith-Waterman algorithm. This extremely intensive computational approach was made possible with the help of World Community Grid, through the Genome Comparison Project. The resulting database, ProteinWorldDB, which contains coordinates of pairwise protein alignments and their respective scores, is now made available. Users can download, compare and analyze the results, filtered by genomes, protein functions or clusters. ProteinWorldDB is integrated with annotations derived from Swiss-Prot, Pfam, KEGG, NCBI Taxonomy database and gene ontology. The database is a unique and valuable asset, representing a major effort to create a reliable and consistent dataset of cross-comparisons of the whole protein content encoded in hundreds of completely sequenced genomes using a rigorous dynamic programming approach. AVAILABILITY: The database can be accessed through http://proteinworlddb.org
Assuntos
Bases de Dados de Proteínas , Genômica/métodos , Proteínas/química , Alinhamento de Sequência/métodos , Software , Algoritmos , Genoma , Filogenia , Proteínas/genéticaRESUMO
BACKGROUND: Enzymes are responsible for the catalysis of the biochemical reactions in metabolic pathways. Analogous enzymes are able to catalyze the same reactions, but they present no significant sequence similarity at the primary level, and possibly different tertiary structures as well. They are thought to have arisen as the result of independent evolutionary events. A detailed study of analogous enzymes may reveal new catalytic mechanisms, add information about the origin and evolution of biochemical pathways and disclose potential targets for drug development. RESULTS: In this work, we have constructed and implemented a new approach, AnEnPi (the Analogous Enzyme Pipeline), using a combination of bioinformatics tools like BLAST, HMMer, and in-house scripts, to assist in the identification, annotation, comparison and study of analogous and homologous enzymes. The algorithm for the detection of analogy is based i) on the construction of groups of homologous enzymes and ii) on the identification of cases where a given enzymatic activity is performed by two or more proteins without significant similarity between their primary structures. We applied this approach to a dataset obtained from KEGG Comprising all annotated enzymes, which resulted in the identification of 986 EC classes where putative analogy was detected (40.5% of all EC classes). AnEnPi is of considerable value in the construction of initial datasets that can be further curated, particularly in gene and genome annotation, in studies involving molecular evolution and metabolism and in the identification of new potential drug targets. CONCLUSION: AnEnPi is an efficient tool for detection and annotation of analogous enzymes and other enzymes in whole genomes. It is available for academic use at: http://bioinfo.pdtis.fiocruz.br/AnEnPi/
Assuntos
Biologia Computacional/métodos , Enzimas/química , Algoritmos , Animais , Catálise , Análise por Conglomerados , Interpretação Estatística de Dados , Bases de Dados de Proteínas , Desenho de Fármacos , Genoma , Humanos , Leishmania major , Modelos Biológicos , Conformação Proteica , SoftwareRESUMO
BACKGROUND: Genome survey sequences (GSS) offer a preliminary global view of a genome since, unlike ESTs, they cover coding as well as non-coding DNA and include repetitive regions of the genome. A more precise estimation of the nature, quantity and variability of repetitive sequences very early in a genome sequencing project is of considerable importance, as such data strongly influence the estimation of genome coverage, library quality and progress in scaffold construction. Also, the elimination of repetitive sequences from the initial assembly process is important to avoid errors and unnecessary complexity. Repetitive sequences are also of interest in a variety of other studies, for instance as molecular markers. RESULTS: We designed and implemented a straightforward pipeline called ReRep, which combines bioinformatics tools for identifying repetitive structures in a GSS dataset. In a case study, we first applied the pipeline to a set of 970 GSSs, sequenced in our laboratory from the human pathogen Leishmania braziliensis, the causative agent of leishmaniosis, an important public health problem in Brazil. We also verified the applicability of ReRep to new sequencing technologies using a set of 454-reads of an Escheria coli. The behaviour of several parameters in the algorithm is evaluated and suggestions are made for tuning of the analysis. CONCLUSION: The ReRep approach for identification of repetitive elements in GSS datasets proved to be straightforward and efficient. Several potential repetitive sequences were found in a L. braziliensis GSS dataset generated in our laboratory, and further validated by the analysis of a more complete genomic dataset from the EMBL and Sanger Centre databases. ReRep also identified most of the E. coli K12 repeats prior to assembly in an example dataset obtained by automated sequencing using 454 technology. The parameters controlling the algorithm behaved consistently and may be tuned to the properties of the dataset, in particular to the length of sequencing reads and the genome coverage. ReRep is freely available for academic use at http://bioinfo.pdtis.fiocruz.br/ReRep/.
Assuntos
Algoritmos , Mapeamento Cromossômico/métodos , Genoma/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA/métodos , Software , Sequência de Bases , Dados de Sequência MolecularRESUMO
Esta dissertação trata do desenvolvimento de novas abordagens e ferramentas computacionais focadas nas seguintes aplicações: aperfeiçoamento do fluxo de informação de Plataformas Tecnológicas de Seqüenciamento e de Bioinformática; montagem de seqüências em projetos genomas; detecção de seqüências repetitivas; análise comparativa e anotação de genomas. A maioria das ferramentas foram desenvolvidas e/ou aplicadas no projeto genoma do Mycobacterium bovis cepa BCG Moreau-RJ, que foi seqüenciado, montado e anotado em nosso laboratório. Uma parte dos resultados deste projeto genoma foi utilizada no desenvolvimento de ferramentas de identificação e controle de qualidade que possam ser utilizadas na manutenção e produção da vacina BCG Moreau-RJ, e foram objetos de um depósito de patente, no âmbito nacional e internacional. Além disto, vários sistemas independentes, porém complementares, foram desenvolvidos para a análise de genomas em geral, os quais permitem uma série de aplicações na interface in silico- in vitro. (1) ReRep (Read Repeat Finder) é um sistema que detecta seqüências repetitivas em um conjunto de dados oriundos de seqüenciamento aleatório (GSS), visando a caracterização de repetições e permitindo um auxílio na montagem de seqüências genômicas, tendo sido aplicado em um conjunto de GSS com cobertura de 1,4por cento do genoma de Leishmania braziliensis. (2) AnEnPi (Analogous Enzyme Pipeline), composto por um banco de dados de enzimas para a anotação de vias metabólicas e detecção de possíveis alvos terapêuticos, com várias possibilidades de comparação e geração de mapas metabólicas, aplicado em L. major e Trypanosoma cruzi. (3) Conjunto de ferramentas para assistir a montagem e a anotação do genoma BCG Moreau-RJ: páginas web do projeto, incluindo monitoramento de estatísticas de montagem e visualização do mapeamento das leituras contra o genoma do Mycobacterium bovis; fluxo...ao Programa de Desenvolvimento Tecnológico em Insumos para Saúde (PDTIS).