UTILIDAD DE LA PCR RECURSIVA PARA INSERTAR UN APTAMER DE ALTA AFINIDAD EN EL ESPACIADOR MENOR DE CALMODULINA de Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness in Panama. In this parasite, regulation of gene expression is mainly a post transcriptional process. Has been suggested that RNA-binding proteins have a key role in this gene regulation. Calmodulin is a highly conserved calcium sensor protein, encodes by three genes separated by two intergenic spacers (major and minor spacer). The study of molecular basis that led RNA-proteins interactions in T. cruzi could contribute to understand the biology of the parasite to develop novel strategies for disease control. We developed a methodology based on a recursive PCR to insert a streptavidin high affinity aptamer into the sequence of the minor spacer, as the basis for the development of a protein capture system. The complete calmodulin locus sequence was download from GenBank (AAHK01001263.1). Previous reported aptameric sequence (83bp) was manipulated for primer design. Sequences were edited with UGENE bioinformatic software. Calmodulin minor spacer (CMS) sequence was fragmented into two regions. Primers were designed to flank the two regions, the inner forward/reverse oligos attached to half aptamer sequences each. Final recursive PCR was able to amplify the Calmodulin minor spacer incorporating the aptamer. Sequence was confirmed by Sanger sequencing. Recursive PCR represent a useful tool to insert high affinity sequences as aptamers to study specific protein-RNA interactions.Trypanosoma cruzi es el parásito protozoario causante de la enfermedad de Chagas, una enfermedad endémica en Panamá. En este parásito, la regulación de la expresión génica es principalmente un proceso postranscripcional. Se ha sugerido que las proteínas de unión al ARN tienen un papel clave en esta regulación génica. La calmodulina es una proteína sensora de calcio altamente conservada, codificada por tres genes separados por dos espaciadores intergénicos (espaciador mayor y menor). El estudio de las bases moleculares que llevaron a las interacciones ARN-proteínas en T. cruzi podría contribuir a comprender la biología del parásito para desarrollar estrategias novedosas para el control de enfermedades. Desarrollamos una metodología basada en una PCR recursiva para insertar un aptámero de alta afinidad de estreptavidina en la secuencia del espaciador menor, como base para el desarrollo de un sistema de captura de proteínas. La secuencia completa del locus de calmodulina se descargó de GenBank (AAHK01001263.1). La secuencia del aptámero reportadas en publicaciones anteriores (83 pb) se manipuló para el diseño del cebador. Las secuencias se editaron con el software bioinformático UGENE. La secuencia del espaciador menor de calmodulina (CMS) se fragmentó en dos regiones. Los cebadores se diseñaron para flanquear las dos regiones, cada uno con los oligonucleótidos internos directo / inverso unidos a secuencias de medio aptámero. La PCR recursiva final pudo amplificar el espaciador menor de calmodulina incorporando el aptámero. La secuencia fue confirmada por secuenciación de Sanger. La PCR recursiva representa una herramienta útil para insertar secuencias de alta afinidad como aptámeros para estudiar interacciones proteína-ARN específicas

The calmodulin intergenic spacer as molecular target for characterization of Leishmania species

Background: Human leishmaniasis is a neglected disease caused by parasites of the genus Leishmania. Clinical aspects of this disease can vary significantly, reflecting the wide range of parasites in the genus Leishmania. Knowing accurately the Leishmania species infecting humans is important for clinical case management and evaluation of epidemiological risk. Calmodulin is an essential gene in trypanosomatids that modulates the calcium metabolism in various cellular activities. Despite its strong conservation in trypanosomatids, it has been recently observed that its untranslated regions (UTR) diverge among species. Methods: In this study we analyzed the sequences and the absolute dinucleotide frequency of the intergenic spacer of the calmodulin gene (containing both, 3′ and 5′UTR) in nine reference Leishmania species and ten clinical isolates obtained from patients with cutaneous leishmaniasis. Results: We show that the short calmodulin intergenic spacers exhibit features that make them interesting for applications in molecular characterization and phylogenetic studies of Leishmania. Dendrograms based on sequence alignments and on the dinucleotide frequency indicate that this particular region of calmodulin gene might be useful for species typing between the Leishmania and Viannia subgenera. Conclusions: Mutations and composition of the calmodulin intergenic spacer from Leishmania species might have taxonomic value as parameters to define if an isolate is identical to a certain species or belongs to one of the two current subgenera

BENZILAÇÃO DO GLICEROL COM AQUECIMENTO POR MICROONDAS

A infinidade de aplicações da glicerina incentivou inúmeras pesquisas voltadas ao reaproveitamento do principal coproduto do biodiesel. Destaca-se a produção de aditivos oxigenados para combustíveis, como os éteres, os acetais e os ésteres de glicerina, processos estes que demandam um tempo de reação muito prolongado. Considerando que a aplicação da irradiação de microondas pode influenciar na cinética das reações químicas, o presente trabalho investigou a reação de benzilação do glicerol via catálise homogênea ácida, sob a irradiação de microondas. Foi constatado que o fator variação da potência das micro-ondas apresentou a maior conversão do glicerol dentre todas as variáveis estudadas. Em contrapartida, esta variável também influenciou uma reação indesejável, a autoeterificação do álcool benzílico.Palavras-chave: Benzilação. Glicerol. Álcool benzílico. Microondas.BENZYLATION OF GLYCEROL WITH HEATING BY MICROWAVEAbstract: The multitude of applications of glycerin has encouraged countless researches directed to the reutilization of the main byproduct of biodiesel. Highlighting the production of additives for oxygenated fuels, such as the ethers, the acetal and the esters of glycerin, those processes require a very long reaction time. Whereas the application of irradiation of microwave can influence on the kinetics of chemical reactions, the present work investigated the benzylation reaction of glycerol via catalysis homogeneous acid, in the irradiation of microwave. It was found that the factor power variation of microwave presented the greatest conversion of glycerol among all the variables studied. In contrast, that variable also influenced an undesirable reaction, the self-etherification of benzyl alcohol.Keywords: Benzylation. Glycerol. Benzyl alcohol. Microwave.BENZILACIÓN GLICERINA CON EL CALOR EN MICROONDASResumen: La grande cantidad de aplicaciones de la glicerina ha alentado numerosos estudios dirigidos a la reutilización del principal subproducto de biodiesel. Destacando la producción de aditivos oxigenados de combustibles para motores, tales como éteres, acetales y ésteres de glicerina, estos procedimientos requieren un tiempo de reacción muy largo. Considerando que la aplicación de irradiación de microondas puede influir en la cinética de las reacciones químicas, este estudio investigó la reacción de bencilación de glicerol a través de catálisis homogénea ácida bajo irradiación con microondas. Se encontró que la variación del factor potencia de microondas mostró la mayor conversión de glicerol entre todas las variables evaluadas. En contraste, esta variable también influenció una reacción indeseable, la auto-eterificación de alcohol bencílico.Palabras clave: Bencilación. Glicerol. Alcohol bencílico. Microondas

The compositional landscape of minicircle sequences isolated from active lesions and scars of American cutaneous leishmaniasis

Submitted by sandra infurna ([email protected]) on 2016-07-14T15:41:26Z No. of bitstreams: 1 octavio_fernandes_etal_IOC_2013.pdf: 998508 bytes, checksum: e842184f76aed4b07fa8930601e0bbe3 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-14T15:51:04Z (GMT) No. of bitstreams: 1 octavio_fernandes_etal_IOC_2013.pdf: 998508 bytes, checksum: e842184f76aed4b07fa8930601e0bbe3 (MD5)Made available in DSpace on 2016-07-14T15:51:04Z (GMT). No. of bitstreams: 1 octavio_fernandes_etal_IOC_2013.pdf: 998508 bytes, checksum: e842184f76aed4b07fa8930601e0bbe3 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Departamento de Imunologia. Laboratório de Imunopatalogia e Biologia Molecular. Recife, PE, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Departamento de Imunologia. Laboratório de Imunopatalogia e Biologia Molecular. Recife, PE, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Departamento de Imunologia. Laboratório de Imunopatalogia e Biologia Molecular. Recife, PE, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Departamento de Imunologia. Laboratório de Imunopatalogia e Biologia Molecular. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Departamento de Imunologia. Laboratório de Imunopatalogia e Biologia Molecular. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil.American cutaneous leishmaniasis (ACL) is characterized by cutaneous lesions that heal spontaneously or after specific treatment. This paper reports on the analysis of kDNA minicircle sequences from clinical samples (typical lesions and scars) that were PCR-amplified with specific primers for Leishmania species of the subgenus Viannia

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed