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Reignault L.C.,Federal University of Rio de Janeiro | Barrias E.S.,Brazilian National Institute of Technology | Soares Medeiros L.C.,Instituto Carlos Chagas ICC | De Souza W.,Federal University of Rio de Janeiro | And 2 more authors.
Parasitology Research | Year: 2014

Trypanosoma cruzi has a complex life cycle where the infective forms for the vertebrate host are trypomastigotes and amastigotes. Both forms invade and lyse their parasitophorous vacuole (PV) membrane, entering into the cytoplasm of its host cells. Galectin-3 (Gal-3) is a protein abundantly distributed in macrophages and epithelial cells. Previous studies demonstrated that Gal-3 binds to a 45KDa mucin of trypomastigotes surface, enhancing its adhesion to the extracellular matrix and even its entry into cells. Gal-3 has another novel cytoplasmic function recently described: a vacuole lyses marker in intracellular bacteria. Considering (1) the importance of Gal-3 during T. cruzi early infection and (2) the importance of T. cruzi PV lyses for parasite differentiation and replication, this study intended to explore a possible recruitment of structures containing Gal-3 (G3CSs) to T. cruzi PVs. Microscopy analyses showed these G3CSs around PVs after 30 and 90 min of amastigotes and trypomastigotes infection, respectively. This recruitment was specific for T. cruzi PVs since we did not observe the same distribution at macrophages vacuoles containing fluorescent microspheres (FM). Concomitantly, this study intended to analyze the participation of actin cytoskeleton in T. cruzi PV maturation. We observed that actin filaments form a "belt-like" structure around trypomastigotes and amastigotes PVs, also labeled for Gal-3. At the time proposed for PV lysis, we observed an actin disassembling while LAMP-1 was recruited to PVs membrane. However, this pattern was maintained in macrophages derived from Gal-3 knockout mice, revealing that the actin belt structure forms independently from Gal-3. Taken together, these data suggest that G3CSs are recruited to vicinity of T. cruzi PV and that actin filaments localize and remain around T. cruzi PVs until the time of its lysis. © 2014 Springer-Verlag.

Rampazzo R.R.C.P.,Institute Biologia Molecular do Parana IBMP | Rampazzo R.R.C.P.,Instituto Carlos Chagas ICC | Cereda M.,STMicroelectronics | Cocci A.,STMicroelectronics | And 10 more authors.
IFMBE Proceedings | Year: 2014

Discovered in 1909, Chagas disease, caused by the parasite Trypanosoma cruzi, continues to be an important tropical disease in Latin America and Caribbean, affecting more than 7 million people. Recently, Chagas disease has also been reported in US and Europe mainly because of unscreened blood donations by immigrants from endemic areas. Current Chagas disease diagnostic relies on search for parasites on blood smears using optical microscopes, screening for serological response, xenodiagnosis, hemoculture and, more recently, polymerase chain reaction (PCR). Each of these tests has its own problems, mainly because of the changes in the immunological profile of the patients and number of circulating parasites in the blood throughout the evolution of the disease. In endemic areas, logistic issues are an additional difficulty for delivering the results to the patients, so a rapid and sensitive point of care test is desirable. In this work, we present results of an on-chip test able to detect equivalent amounts of parasites' DNA such as those typically present in both acute and chronic phase of the disease. We developed a silicon chip-based PCR using a portable thermocycler with fluorescent detectors that has many advantages over the conventional PCR. When compared to the conventional PCR, our on-chip reaction shows similar sensitivity (between 1 and 0.1 genome equivalents), but a shorter reaction time (35 min versus 90 min). The results presented herein are the first step towards the development of a portable diagnostic test using a fast, sensitive and specific reaction for the detection of Trypanosoma cruzi. © Springer International Publishing Switzerland 2014.

Serpeloni M.,Federal University of Paraná | Serpeloni M.,Instituto Carlos Chagas ICC | Moraes C.B.,Institute Pasteur Korea | MunizJoa J.R.C.,University of Oxford | And 12 more authors.
PLoS ONE | Year: 2011

In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes. © 2011 Serpeloni et al.

Wagner G.,Instituto Oswaldo Cruz IOC | Wagner G.,Campus Universitario | Wagner G.,University of West Santa Catarina | Jardim R.,Instituto Oswaldo Cruz IOC | And 13 more authors.
BMC Research Notes | Year: 2014

Background: The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. Findings. STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. Conclusion: STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at and the open source code at. © 2014 Wagner et al.; licensee BioMed Central Ltd.

Rossato D.O.,Federal University of Rio Grande do Sul | Ludwig A.,Instituto Carlos Chagas ICC | Depra M.,Federal University of Rio Grande do Sul | Loreto E.L.S.,Federal University of Rio Grande do Sul | And 3 more authors.
Genome Biology and Evolution | Year: 2014

The hAT superfamily comprises a large and diverse array of DNA transposons found in all supergroups of eukaryotes. Here we characterized the Drosophila buzzatii BuT2 element and found that it harbors a five-exon gene encoding a 643-aa putatively functional transposase. Aphylogeny built with 85hAT transposases yielded, in addition to the two major groups already described, Ac and Buster, a thirdone comprising 20 sequences that includes BuT2, Tip100, hAT-4-BM, and RP-hAT1. This third group is here named Tip. In addition, we studied the phylogenetic distribution and evolution of BuT2 by in silico searches and molecular approaches. Our data revealed BuT2 was, most often, vertically transmitted during the evolution of genus Drosophila being lost independently in several species. Nevertheless, we propose the occurrence of three horizontal transfer events to explain its distribution and conservation amongspecies. Another aspect of BuT2 evolution and life cycle is the presence of short related sequences, which contain similar 50 and 30 regions, including the terminal inverted repeats. These sequences that can be considered asminiature inverted repeat transposable elements probably originated by internal deletion of complete copies and show evidences of recent mobilization. © The Author(s) 2014.

Depra M.,Federal University of Rio Grande do Sul | Ludwig A.,Federal University of Rio Grande do Sul | Ludwig A.,Instituto Carlos Chagas ICC | Ludwig A.,Pontifical Catholic University of Parana | And 3 more authors.
Mobile DNA | Year: 2012

Background: Miniature inverted-repeat transposable elements (MITEs) are short, nonautonomous DNA elements flanked by subterminal or terminal inverted repeats (TIRs) with no coding capacity. MITEs were originally recognized as important components of plant genomes, where they can attain extremely high copy numbers, and are also found in several animal genomes, including mosquitoes, fish and humans. So far, few MITEs have been described in Drosophila. Results: Herein we describe the distribution and evolution of Mar, a MITE family of hAT transposons, in Drosophilidae species. In silico searches and PCR screening showed that Mar distribution is restricted to the willistoni subgroup of the Drosophila species, and a phylogenetic analysis of Mar indicates that this element may have originated prior to the diversification of these species. Most of the Mar copies in D. willistoni present conserved target site duplications and TIRs, indicating recent mobilization of these sequences. We also identified relic copies of potentially full-length Mar transposon in D. tropicalis and D. willistoni. The phylogenetic relationship among transposases from the putative full-length Mar and other hAT superfamily elements revealed that Mar is placed into the recently determined Buster group of hAT transposons. Conclusion: On the basis of the obtained data, we can suggest that the origin of these Mar MITEs occurred before the subgroup willistoni speciation, which started about 5.7 Mya. The Mar relic transposase existence indicates that these MITEs originated by internal deletions and suggests that the full-length transposon was recently functional in D. willistoni, promoting Mar MITEs mobilization. © 2012 Deprá et al.; licensee BioMed Central Ltd.

Serpeloni M.,University Federal do Paran | Serpeloni M.,Instituto Carlos Chagas ICC | Vidal N.M.,University Federal do Paran | Vidal N.M.,Instituto Carlos Chagas ICC | And 7 more authors.
BMC Evolutionary Biology | Year: 2011

Background: The establishment of the nuclear membrane resulted in the physical separation of transcription and translation, and presented early eukaryotes with a formidable challenge: how to shuttle RNA from the nucleus to the locus of protein synthesis. In prokaryotes, mRNA is translated as it is being synthesized, whereas in eukaryotes mRNA is synthesized and processed in the nucleus, and it is then exported to the cytoplasm. In metazoa and fungi, the different RNA species are exported from the nucleus by specialized pathways. For example, tRNA is exported by exportin-t in a RanGTP-dependent fashion. By contrast, mRNAs are associated to ribonucleoproteins (RNPs) and exported by an essential shuttling complex (TAP-p15 in human, Mex67-mtr2 in yeast) that transports them through the nuclear pore. The different RNA export pathways appear to be well conserved among members of Opisthokonta, the eukaryotic supergroup that includes Fungi and Metazoa. However, it is not known whether RNA export in the other eukaryotic supergroups follows the same export routes as in opisthokonts. Methods. Our objective was to reconstruct the evolutionary history of the different RNA export pathways across eukaryotes. To do so, we screened an array of eukaryotic genomes for the presence of homologs of the proteins involved in RNA export in Metazoa and Fungi, using human and yeast proteins as queries. Results: Our genomic comparisons indicate that the basic components of the RanGTP-dependent RNA pathways are conserved across eukaryotes, and thus we infer that these are traceable to the last eukaryotic common ancestor (LECA). On the other hand, several of the proteins involved in RanGTP-independent mRNA export pathways are less conserved, which would suggest that they represent innovations that appeared later in the evolution of eukaryotes. Conclusions: Our analyses suggest that the LECA possessed the basic components of the different RNA export mechanisms found today in opisthokonts, and that these mechanisms became more specialized throughout eukaryotic evolution. © 2011 Serpeloni et al.

Avila A.R.,Instituto Carlos Chagas ICC | Goldenberg S.,Instituto Carlos Chagas ICC
Open Parasitology Journal | Year: 2010

The regulation of gene expression in trypanosomatids is predominantly post-transcriptional. Polycistronic transcripts are processed by the addition of a common 5'-spliced leader and polyadenylation. However, the processed mRNAs are not necessarily functionally related, suggesting the existence of mechanisms for the degradation or storage of untranslatable mRNAs. Determination of the TriTryps (Leishmania major, Trypanosoma brucei and Trypanosoma cruzi) genome sequences has allowed the identification of genes encoding potential regulatory proteins. This review discusses some of the mechanisms and regulatory elements involved in cytoplasmic gene expression regulation in Trypanosoma cruzi. We also discuss how functional genomic tools have contributed toward determining the role played by RNA binding protein complexes, supporting the concept of "post-transcriptional RNA operons" or "RNA regulons". This suggests the existence of interconnected regulatory networks in the parasite, in which RNA granules act as protagonists in cytoplasmic mRNA metabolism. © Ávila and Goldenberg.

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