Evolutionary analyses of myosin genes in trypanosomatids show a history of expansion, secondary losses and neofunctionalization

Submitted by Manoel Barata ([email protected]) on 2019-02-14T13:49:21Z No. of bitstreams: 1 s41598-017-188.pdf: 5208060 bytes, checksum: 284ab473d765391a581c2ad147ddbd1a (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-02-15T18:18:59Z (GMT) No. of bitstreams: 1 s41598-017-188.pdf: 5208060 bytes, checksum: 284ab473d765391a581c2ad147ddbd1a (MD5)Made available in DSpace on 2019-02-15T18:18:59Z (GMT). No. of bitstreams: 1 s41598-017-188.pdf: 5208060 bytes, checksum: 284ab473d765391a581c2ad147ddbd1a (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Programa de Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Programa de Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Programa de Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Myosins are motor proteins that comprise a large and diversified family important for a broad range of functions. Two myosin classes, I and XIII, were previously assigned in Trypanosomatids, based mainly on the studies of Trypanosoma cruzi, T. brucei and Leishmania major, and important human pathogenic species; seven orphan myosins were identified in T. cruzi. Our results show that the great variety of T. cruzi myosins is also present in some closely related species and in Bodo saltans, a member of an early divergent branch of Kinetoplastida. Therefore, these myosins should no longer be considered "orphans". We proposed the classification of a kinetoplastid-specific myosin group into a new class, XXXVI. Moreover, our phylogenetic data suggest that a great repertoire of myosin genes was present in the last common ancestor of trypanosomatids and B. saltans, mainly resulting from several gene duplications. These genes have since been predominantly maintained in synteny in some species, and secondary losses explain the current distribution. We also found two interesting genes that were clearly derived from myosin genes, demonstrating that possible redundant or useless genes, instead of simply being lost, can serve as raw material for the evolution of new genes and functions

Fluoropolymer Studies for Radiation Dosymetry

The polymers Tetrafluoroethylene-hexa-fluoropropylene (FEP) and Tetrafluoroethylene-per-fluoromethoxyethelene (PFA) are normally used as anti-adherent coatings and can also be applied for several applications in research. For example, they can be used as radiation dosimeters for X-ray and gamma photons, electrons, protons and other ionazing particles. In order to determine radiation induced damage, that can compromise applications in dosimetry, FEP and PFA films were bombarded with protons of 1 MeV at fluences from 1 × 10 11 protons/cm 2 to 1 × 10 16 protons/cm 2 . During the bombardment, the chemical species emission was monitored with a Residual Gas Analyzer (RGA), and results show that the CF3 radical is the specie preferentially emitted. The bombarded films were also analyzed with Optical Absorption Photospectrometry (OAP) which shows quantitative chemically specific evidence of the damage caused by the proton bombardment. Our results show that damage to polymers is detectable for all fluences used in this work, but damage that can compromise applications in dosimetry occurs only for fluences greater than 1 × 10 14 protons/cm 2

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses

Profiling the Trypanosoma cruzi phosphoproteome

Submitted by Manoel Barata ([email protected]) on 2019-12-03T17:47:47Z No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2020-02-19T14:39:34Z (GMT) No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5)Made available in DSpace on 2020-02-19T14:39:34Z (GMT). No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis--differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes--were enriched for phosphopeptides using TiO(2) chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve

Submitted by Manoel Barata ([email protected]) on 2019-02-14T12:34:54Z No. of bitstreams: 1 0074-0276-mioc-113-5.pdf: 3046522 bytes, checksum: ca70ac4a197b74c287651a9f0359a489 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-02-14T17:36:01Z (GMT) No. of bitstreams: 1 0074-0276-mioc-113-5.pdf: 3046522 bytes, checksum: ca70ac4a197b74c287651a9f0359a489 (MD5)Made available in DSpace on 2019-02-14T17:36:01Z (GMT). No. of bitstreams: 1 0074-0276-mioc-113-5.pdf: 3046522 bytes, checksum: ca70ac4a197b74c287651a9f0359a489 (MD5) Previous issue date: 2018Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve

<div><p> BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.</p></div