Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes

A simple and efficient method for direct in vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts were translated in vivo in Xenopus laevis oocytes and in vitro in reticulocyte and in wheat germ cell-free protein synthesizing systems

RATT: Rapid Annotation Transfer Tool

Second-generation sequencing technologies have made large-scale sequencing projects commonplace. However, making use of these datasets often requires gene function to be ascribed genome wide. Although tool development has kept pace with the changes in sequence production, for tasks such as mapping, de novo assembly or visualization, genome annotation remains a challenge. We have developed a method to rapidly provide accurate annotation for new genomes using previously annotated genomes as a reference. The method, implemented in a tool called RATT (Rapid Annotation Transfer Tool), transfers annotations from a high-quality reference to a new genome on the basis of conserved synteny. We demonstrate that a Mycobacterium tuberculosis genome or a single 2.5 Mb chromosome from a malaria parasite can be annotated in less than five minutes with only modest computational resources. RATT is available at http://ratt.sourceforge.net

Qualificação e Percepção de Riscos de Trabalhadores

Este estudo analisa alguns elementos relativos à formação e capacitação de trabalhadores que atuam na área biotecnológica. Buscou-se perceber a relação entre o conhecimento acerca de biossegurança e a percepção sobre os riscos envolvidos nos diferentes processos de trabalho. Para tanto, foram realizadas entrevistas, baseadas em um roteiro previamente definido, em uma empresa privada e uma instituição pública. Das conclusões emerge uma série de similaridades e de diferenças entre os setores público e privado. Entretanto, constata-se que existem deficiências de ordens diversas na informação dos trabalhadores, em ambos os setores. Esta situação pode interferir na desejada eficiência da aplicação de programas de biossegurança. PALAVRAS-CHAVE: Biossegurança, biotecnologia, percepção de risco, informação sobre risco, gerenciamento de risco. QUALIFICATION AND RISK PERCEPTION AMONG WORKERS OF THE BIOTECHNOLOGICAL AREA: PUBLIC AND PRIVATE SECTORES Carmem Marinho Carlos Minayo-Gomez Wim Degrave This study analyzes some elements related to formation and training of workers of biotechnological areas. Its objective is to identify the relationship between workers' knowledge about Biosafety and their perception of the risks involved in different work processes. For that, interviews, based on a predetermined guideline, were carried on among staff of both the private and the public sectors, which – conclusions point out - hold a series of similarities and differences. However, it has been verified that deficiencies regarding workers' information concerning Biosafety in both sectors are as varied as significant, and can interfere in the desired efficiency of Biosafety programs to be applied. KEY WORDS: Biosafety; Biotechnology; Risk Perception; Risk Information; Risk Management. Publicação Online do Caderno CRH: http://www.cadernocrh.ufba.b

AnEnPi: Identification and annotation of analogous enzymes

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

Squid – a simple bioinformatics grid

BACKGROUND: BLAST is a widely used genetic research tool for analysis of similarity between nucleotide and protein sequences. This paper presents a software application entitled "Squid" that makes use of grid technology. The current version, as an example, is configured for BLAST applications, but adaptation for other computing intensive repetitive tasks can be easily accomplished in the open source version. This enables the allocation of remote resources to perform distributed computing, making large BLAST queries viable without the need of high-end computers. RESULTS: Most distributed computing / grid solutions have complex installation procedures requiring a computer specialist, or have limitations regarding operating systems. Squid is a multi-platform, open-source program designed to "keep things simple" while offering high-end computing power for large scale applications. Squid also has an efficient fault tolerance and crash recovery system against data loss, being able to re-route jobs upon node failure and recover even if the master machine fails. Our results show that a Squid application, working with N nodes and proper network resources, can process BLAST queries almost N times faster than if working with only one computer. CONCLUSION: Squid offers high-end computing, even for the non-specialist, and is freely available at the project web site. Its open-source and binary Windows distributions contain detailed instructions and a "plug-n-play" instalation containing a pre-configured example

Structural modelling and comparative analysis of homologous, analogous and specific proteins from Trypanosoma cruzi versus Homo sapiens: putative drug targets for chagas' disease treatment

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma cruzi </it>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 <it>Trypanosoma cruzi </it>versus <it>Homo sapiens</it>.</p> <p>Results</p> <p>We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of <it>T</it>. <it>cruzi</it>. In combination with comparative genome analysis to <it>Homo sapiens</it>, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite.</p> <p>Conclusions</p> <p>In this work, we present a set of 397 enzyme models of <it>T</it>. <it>cruzi </it>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 <it>H. sapiens </it>enzymes, were identified as new potential molecular targets.</p

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

<p>Abstract</p> <p>Background</p> <p>Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from <it>M. bovis </it>BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.</p> <p>Results</p> <p>The 2DE proteomic map of <it>M. bovis </it>BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.</p> <p>Conclusions</p> <p>Here we report the detailed 2DE profile of CFPs from <it>M. bovis </it>BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.</p

Biodegradação de matéria orgânica em lama sedimentada - definição de parâmetros na lagoa de piratininga, RJ: Biodegradation of organic matter in the sedimented mud - parameters definition at the piratininga lagoon, RJ

Entre março e julho/22, duas tecnologias complementares, EM® e Pulmão™, foram testadas para estudar a biorremediação na camada de lama da Lagoa de Piratininga, Niterói, RJ. Dados ortométricos, matéria orgânica, carbono orgânico, nitrogênio Kjeldahl total, fósforo e biopolímeros foram quantificados na superfície do sedimento. As concentrações crescentes de nitrogênio Kjeldahl indicaram aportes de esgoto doméstico ao sistema. O fósforo orgânico predominou sobre o total, pois foi consumido no processo de biorremediação. Ocorreram mudanças quali- quantitativas na composição da matéria orgânica lábil, com consumo de lipídio, molécula recalcitrante, e produção de proteínas e carboidratos. Os dados altimétricos indicaram maior tendência de consumo da lama na área que reuniu as tecnologias EM1®+Pulmão™, onde o aporte sedimentar foi praticamente anulado. Houve uma diminuição da espessura da lama, refletindo no aumento sutil de profundidade superficial da camada sedimentar. Estas modificações nas áreas experimentais indicam que o processo de restauração ambiental está ocorrendo

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24Sα ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24Sα rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage I was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite