Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite's genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote

Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi

A novel large multigene family was recently identified in the human pathogen Trypanosoma cruzi, causative agent of Chagas disease, and corresponds to ∼6% of the parasite diploid genome. The predicted gene products, mucin-associated surface proteins (MASPs), are characterized by highly conserved N- and C-terminal domains and a strikingly variable and repetitive central region. We report here an analysis of the genomic organization and expression profile of masp genes. Masps are not randomly distributed throughout the genome but instead are clustered with genes encoding mucin and other surface protein families. Masp transcripts vary in size, are preferentially expressed during the trypomastigote stage and contain highly conserved 5′ and 3′ untranslated regions. A sequence analysis of a trypomastigote cDNA library reveals the expression of multiple masp variants with a bias towards a particular masp subgroup. Immunofluorescence assays using antibodies generated against a MASP peptide reveals that the expression of particular MASPs at the cell membrane is limited to subsets of the parasite population. Western blots of phosphatidylinositol-specific phospholipase C (PI-PLC)-treated parasites suggest that MASP may be GPI-anchored and shed into the medium culture, thus contributing to the large repertoire of parasite polypeptides that are exposed to the host immune system

A GINÁSTICA ARTÍSTICA NA PROPOSTA CURRICULAR PARA A EDUCAÇÃO FÍSICA EM SÃO PAULO

O objetivo do presente trabalho foi analisar os Cadernos de Educação Física do Estado de São Paulo, especificamente, com enfoque para o conteúdo de Ginástica Artística. O procedimento metodológico utilizado foi a análise temática (SEVERINO, 1996) dos documentos. Os temas destacados, analisados e discutidos foram as características e os fundamentos da Ginástica Artística. Nos resultados são apresentadas sugestões para a aplicação das tarefas exemplificadas nas aulas, com maiores detalhes e relacionadas aos fundamentos da Ginástica Artística. Isto poderá facilitar o trabalho do professor em dois aspectos: oferecer maior nível de motivação e elevar a autoestima dos praticantes por meio de subsídios para o desenvolvimento coerente, gradual e prazeroso da Ginástica Artística

Desempenho ortográfico de escolares do 2º ao 5º ano do ensino público

OBJETIVOS: Caracterizar, comparar e classificar o desempenho de escolares do 2º ao 5º ano do ensino público segundo a semiologia dos erros. MÉTODOS: Participaram deste estudo 120 escolares do 2º ao 5º ano de escola pública municipal de Marília-SP, sendo 30 de cada série, divididos em quatro grupos: GI (2º ano); GII (3º ano); GIII (4º ano); e GIV (5º ano). Como procedimento foram aplicadas as provas do Pro-Ortografia: versão coletiva (escrita de letras do alfabeto, ditado randomizado das letras do alfabeto, ditado de palavras, ditado de pseudopalavras, ditado com figuras, escrita temática induzida por figura) e versão individual (ditado de frases, erro proposital, ditado soletrado, memória lexical ortográfica). RESULTADOS: Houve diferença na comparação intergrupos, indicando melhor desempenho dos escolares a cada série subsequente, na maior parte das provas da versão coletiva e individual. Com o avanço da seriação escolar, os grupos apresentaram menor média de erros na escrita. CONCLUSÃO: O perfil de aquisição da ortografia do sistema de escrita do Português observado em escolares do ensino público é indicativo do funcionamento normal de desenvolvimento da escrita infantil.PURPOSE: To characterize, compare and classify the performance of 2nd to 5th grade students from public schools according to the semiology of spelling errors. METHODS: Participants were 120 students from 2nd to 5th grades of a public school in Marília (SP), Brazil, 30 students from each grade, who were divided into four groups: GI (2nd grade), GII (3rd grade), GIII (4th grade), and GIV (5th grade). The tasks of the Pro-Ortografia test were applied: collective version (writing of alphabet letters, randomized dictation of letters, words dictation, nonwords dictation, dictation with pictures, thematic writing induced by picture) and individual version (dictation of sentences, purposeful error, spelled dictation, orthographic lexical memory). RESULTS: Significant difference was found in the between-group comparison indicating better performance of students in every subsequent grade in most of the individual and collective version tasks. With the increase of grade level, the groups decreased the average of writing errors. CONCLUSION: The profile of spelling acquisition of the Portuguese writing system found in these public school students indicates normal writing development in this population.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Estadual Paulista Júlio de Mesquita FilhoUniversidad Catolica de ValênciaUniversitat de ValènciaUniversidade Estadual Paulista Júlio de Mesquita Filh

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9- RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability, and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryote

The Nucleosome (Histone-DNA Complex) Is the TLR9-Specific Immunostimulatory Component of Plasmodium falciparum That Activates DCs

The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2−/−, TLR9−/− and MyD88−/− mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development

Trypanosoma cruzi Gene Expression in Response to Gamma Radiation

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress