Proteomics uncovers novel components of an interactive protein network supporting RNA export in trypanosomes

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation

Impactos ambientais do manejo agroecológico da caatinga no Rio Grande do Norte.

O objetivo deste trabalho foi avaliar os impactos ambientais do manejo agroecológico da caatinga, em unidades de produção familiar no Rio Grande do Norte, pelo método Ambitec de produção animal - dimensão ambiental, desenvolvido pela Embrapa Meio Ambiente. Foram avaliadas sete unidades de produção familiar, em quatro projetos de assentamentos de reforma agrária do Município de Apodi, RN. Os dados para o levantamento foram obtidos por meio de questionários aplicados aos representantes das unidades produtivas familiares, que atribuíram, a cada variável estudada, um valor que representou a alteração proporcionada pela implementação da tecnologia. Após a inserção dos coeficientes de alteração de cada variável dos indicadores por unidade de produção, o coeficiente de impacto foi automaticamente calculado por meio da planilha Ambitec. O manejo agroecológico da caatinga resultou num impacto ambiental positivo, e suas maiores contribuições foram relacionadas aos efeitos positivos dos seguintes indicadores: capacidade produtiva do solo, uso de insumos materiais, qualidade do produto e diminuição da emissão de poluentes à atmosfera. Dois indicadores geraram efeitos negativos: o uso de energia e o uso de recursos naturais. Pela superioridade dos benefícios gerados, o manejo agroecológico da caatinga é uma inovação tecnológica geradora de impactos ambientais positivos

An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

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

mRNA export in the apicomplexan parasite Toxoplasma gondii: emerging divergent components of a crucial pathway

Abstract Control of gene expression is crucial for parasite survival and is the result of a series of processes that are regulated to permit fine-tuning of gene expression in response to biological changes during the life-cycle of apicomplexan parasites. Control of mRNA nuclear export is a key process in eukaryotic cells but is poorly understood in apicomplexan parasites. Here, we review recent knowledge regarding this process with an emphasis on T. gondii. We describe the presence of divergent orthologs and discuss structural and functional differences in export factors between apicomplexans and other eukaryotic lineages. Undoubtedly, the use of the CRISPR/Cas9 system in high throughput screenings associated with the discovery of mRNA nuclear export complexes by proteomic analysis will contribute to identify these divergent factors. Ligand-based or structure-based strategies may be applied to investigate the potential use of these proteins as targets for new antiprotozoal agents

Modulation of Virulence Factors during <i>Trypanosoma cruzi</i> Differentiation

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. This protozoan developed several mechanisms to infect, propagate, and survive in different hosts. The specific expression of proteins is responsible for morphological and metabolic changes in different parasite stages along the parasite life cycle. The virulence strategies at the cellular and molecular levels consist of molecules responsible for mediating resistance mechanisms to oxidative damage, cellular invasion, and immune evasion, performed mainly by surface proteins. Since parasite surface coat remodeling is crucial to invasion and infectivity, surface proteins are essential virulence elements. Understanding the factors involved in these processes improves the knowledge of parasite pathogenesis. Genome sequencing has opened the door to high-throughput technologies, allowing us to obtain a deeper understanding of gene reprogramming along the parasite life cycle and identify critical molecules for survival. This review therefore focuses on proteins regulated during differentiation into infective forms considered virulence factors and addresses the current known mechanisms acting in the modulation of gene expression, emphasizing mRNA signals, regulatory factors, and protein complexes

Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism

Dipyridamole Stress myocardial Perfusion by Computed Tomography in Patients with Left Bundle Branch Block

AbstractBackground:Functional tests have limited accuracy for identifying myocardial ischemia in patients with left bundle branch block (LBBB).Objective:To assess the diagnostic accuracy of dipyridamole-stress myocardial computed tomography perfusion (CTP) by 320-detector CT in patients with LBBB using invasive quantitative coronary angiography (QCA) (stenosis ≥ 70%) as reference; to investigate the advantage of adding CTP to coronary computed tomography angiography (CTA) and compare the results with those of single photon emission computed tomography (SPECT) myocardial perfusion scintigraphy.Methods:Thirty patients with LBBB who had undergone SPECT for the investigation of coronary artery disease were referred for stress tomography. Independent examiners performed per-patient and per-coronary territory assessments. All patients gave written informed consent to participate in the study that was approved by the institution’s ethics committee.Results:The patients’ mean age was 62 ± 10 years. The mean dose of radiation for the tomography protocol was 9.3 ± 4.6 mSv. With regard to CTP, the per-patient values for sensitivity, specificity, positive and negative predictive values, and accuracy were 86%, 81%, 80%, 87%, and 83%, respectively (p = 0.001). The per-territory values were 63%, 86%, 65%, 84%, and 79%, respectively (p < 0.001). In both analyses, the addition of CTP to CTA achieved higher diagnostic accuracy for detecting myocardial ischemia than SPECT (p < 0.001).Conclusion:The use of the stress tomography protocol is feasible and has good diagnostic accuracy for assessing myocardial ischemia in patients with LBBB

A Long Way to Go: A Scenario for Clinical Trials of PI3K Inhibitors in Treating Cancer

Background Alterations in PI3K function are directly related to cancer, making PI3K inhibitors suitable options for anticancer therapies. Information on therapy using different types of PI3K inhibitors is available in literature, providing indications of trends in developing new therapies. Although some studies on PI3K inhibitors for cancer treatment provide clinical evidence, they do not allow a careful search for potential PI3K inhibitors conducted by development indicators. Here, we performed a foresight study of clinical trials involving PI3K inhibitors from the past 11 years using indicators of clinical evolution to identify technological trends and provide data for supporting recommendations for new study designs. Methods A comprehensive foresight study was designed based on documents from clinical trials on PI3K inhibitors to perform a systematic and comparative analysis, in order to identify technological trends on new cancer therapies. Results Our results demonstrate that total number of clinical trials has decreased over the years and, currently, there is a clear prevalence of studies using isoform-specific inhibitors in combined interventions. Clinical trials in Phases I and II were the most frequently found in the database, whereas Phase III trials correspond to 7% of studies. The measurement of clinical trials progression using indicators (drugs in Phase III profile, top-10 drugs, and top-10 combined drugs) demonstrated that the 3 new medicines BKM120, IBI-376, and PF-05212384 have a high potential to provide more efficient cancer treatment in combined interventions. These data also include the groups of targets for each drug, providing a useful and reliable source for design new combinations to overcome the resistance and the poor tolerability observed in some PI3K therapies. Conclusions The establishment of development indicators based on clinical trials for cancer treatment was useful to highlight the clinical investment in 3 new PI3K drugs and the advantages of combine therapy using FDA-approved drugs

Stage-regulated GFP expression in Trypanosoma cruzi: applications from host-parasite interactions to drug screening

Submitted by Luciane Willcox ([email protected]) on 2016-09-30T17:52:19Z No. of bitstreams: 1 Stage-Regulated GFP Expression in Trypanosoma cruzi.PDF: 2194571 bytes, checksum: 7ad80c423fdb2e2b11b74f6ec9b0b6b5 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-09-30T17:59:59Z (GMT) No. of bitstreams: 1 Stage-Regulated GFP Expression in Trypanosoma cruzi.PDF: 2194571 bytes, checksum: 7ad80c423fdb2e2b11b74f6ec9b0b6b5 (MD5)Made available in DSpace on 2016-09-30T17:59:59Z (GMT). No. of bitstreams: 1 Stage-Regulated GFP Expression in Trypanosoma cruzi.PDF: 2194571 bytes, checksum: 7ad80c423fdb2e2b11b74f6ec9b0b6b5 (MD5) Previous issue date: 2013-06-20CNPq, Fundação Araucaria, Fiocruz, CapesFundação 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.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.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.Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development