Spatial organization of transcription as a potential gene expression mechanism in Plasmodium falciparum

Crescentes evidências mostram que a organização espacial da transcrição é um fator epigenético importante na regulação gênica em eucariotos. Em células de mamíferos, os genes são transcritos em estruturas nucleares discretas conhecidas como fábricas de transcrição, e genes com funções relacionadas e co-regulados compartilham as mesmas fábricas de transcrição. Plasmodium falciparum apresenta um padrão de expressão gênica bastante complexo durante o ciclo eritrocítico, contrastando paradoxalmente com o baixo número de putativos fatores de transcrição codificados pelo seu genoma. Por outro lado, mecanismos epigenéticos são importantes na regulação gênica neste parasita. Nesta tese, investigamos a organização da transcrição em P. falciparum visando determinar se a organização nuclear está relacionada com a expressão/regulação gênica ao longo do desenvolvimento do parasita. Com este objetivo, marcamos transcritos nascentes com BrUTP em formas eritrocíticas de P. falciparum. Assim como em mamíferos, a transcrição no parasita também está organizada em focos nucleoplásmicos discretos, chamados fábricas de transcrição. Análises automatizadas de imagens em 3D mostram que o número e a intensidade das fábricas de transcrição variam durante o ciclo eritrocítico, estando correlacionados com o número de genes expressos em cada estágio, mas não com o volume nuclear. O baixo número de fábricas indica que os genes ativos compartilham as fábricas enquanto estão sendo transcritos. Surpreendentemente, as fábricas são espacilamente redistribuídas durante o desenvolvimento de anéis para trofozoítas, com a periferia nuclear sendo a zona de transcrição favorita nos anéis, enquanto nos trofozoítas as fábricas estão igualmente distribuídas por todo o nucleoplasma. Também observamos que a transcrição por RNA polimerase I ocorre principalmente nas áreas centrais dos núcleos em trofozoítas, sugerindo que os componentes nucleolares podem ser dispersos devido à entrada na fase S. Assim como nos eucariotos superiores, as fábricas de transcrição em P. falciparum também se localizam em áreas nucleares com baixa densidade de cromatina. Análises de imunofluorescência combinando incorporação de BrUTP com marcadores nucleares mostram que as fábricas de transcrição formam um compartimento exclusivo, diferente do compartimento de silenciamento definido por PfSir2A ou do compartimento de cromatina ativa definido por H4ac ou H3K79me3. Para estudar a organização espacial da cromatina e entender como os genes co-regulados interagem com as fábricas de transcrição, decidimos realizar ensaios de hibridização fluorescente in situ (fluorescence in situ hybridization, FISH). Durante a realização de ensaios de FISH seguindo protocolos publicados, observamos uma grande variação na morfologia nuclear dos parasitas, o que nos moveu a otimizar esta técnica. Utilizando análises automatizadas de imagens, mostramos que os parasitas desidratados por secagem ao ar e fixados por curtos períodos de tempo à temperatura ambiente apresentam uma variação intra-populacional maior em relação à forma e ao volume nucleares após o ensaio de FISH, assim como valores de volume quase duas vezes maiores, do que núcleos de parasitas fixados em suspensão por longos períodos de tempo e em baixas temperaturas. Também observamos que a fixação em suspensão leva a uma melhor conservação da estrutura nuclear, e índices de colocalização mais altos para duas sondas de repetições adjacentes das extremidades cromossômicas, Rep20 e telômeros. Em resumo, nossos resultados mostram que o tipo de protocolo de fixação utilizado antes da realização do desenvolvimento de FISH é um fator crucial para a conservação apropriada da arquitetura nuclear.Growing evidence points that transcription spatial organization is an important epigenetic factor for eukaryotes gene regulation. In mammalian cells, genes are transcribed in discrete nuclear structures known as transcription factories, and developmentally co-regulated, functionally related genes have been shown to share factories. Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the low number of putative transcription factors encoded by its genome. However, epigenetic mechanisms are important for gene regulation in this parasite. In this thesis, we investigated transcription organization of P. falciparum in order to determine if nuclear architecture is related with developmentally regulated gene expression. To this aim, we have labeled nascent transcrips with BrUTP in P. falciparum erythrocytic forms. Transcription in organized in discrete nuceloplasmic foci, the transcription factories. Automated 3D analysis of confocal images shows the number and intensity of transcription factories change during the erythrocytic cycle, correlating with the number of genes expressed at each stage but not with the nuclear volume. The low number of factories indicates that active genes share factories while being transcribed. Unexpectedly, factories spatially redistribute from ring to trophozoites, being the nuclear periphery the preferential transcription zone in rings while in trophozoites factories are equally distributed throughout the nucleoplasm. We also observed that RNApolymerase I transcription occurs mostly at central nuclear areas in trophozoites, suggesting that nucleolar components may be dispersed upon S phase transition. Like in higher eukaryotes, in P. falciparum transcription factories occur on nuclear areas with low chromatin density. Immunofluorescence analysis of P. falciparum nuclear markers combined with BrUTP incorporation show that transcription factories are a novel and exclusive nuclear compartment, different from the silent compartment defined by PfSir2A or the active chromatin compartment defined by H4ac and H3K79me3. In order to study the spatial organization of chromatin, and how co-regulated, functionally related genes interact with transcription factories, we decided to perform fluorescence in situ hybridization (FISH). While performing FISH with published protocols, we observed great variation in the parasite nuclear morphology, prompting us to optimize this technique. Using automated image analysis, we show that parasites dehydrated by air-drying and fixed for short periods at room temperature present after FISH higher intra-population variation of nuclear shape and volume, as well as almost two-times higher relative volume values, than parasite nuclei fixed in suspension for long periods at low temperatures. We also observe that longer fixation in suspension leads to improved conservation of the nuclear structure, with chromosome end clusters preferentially locating at the nuclear periphery, and higher colocalization indexes for two adjacent chromosome end probes, Rep20 and telomere. Overall, our results show that the type of fixation protocol applied prior to FISH is crucial for the conservation of nuclear architecture.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ministério da Educação, Ciência e Tecnologia da Coreia do Sul (MEST)Governo da província de Gyeonggi da Coreia do SulInstituto Coreano de Informação Científica e Tecnológica (KISTI)BV UNIFESP: Teses e dissertaçõe

The translational challenge in chagas disease drug development

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. There is an urgent need for safe, effective, and accessible new treatments since the currently approved drugs have serious limitations. Drug development for Chagas disease has historically been hampered by the complexity of the disease, critical knowledge gaps, and lack of coordinated R&D efforts. This review covers some of the translational challenges associated with the progression of new chemical entities from preclinical to clinical phases of development, and discusses how recent technological advances might allow the research community to answer key questions relevant to the disease and to overcome hurdles in R&D for Chagas disease.Fil: Kratz, Jadel M.. No especifíca;Fil: Gonçalves, Karolina R.. Universidade de Sao Paulo; BrasilFil: Romera, Lavínia M. D.. Universidade de Sao Paulo; BrasilFil: Borsoi Moraes, Carolina. Universidade Federal de Sao Paulo; BrasilFil: Bittencourt Cunha, Paula. Universidade de Sao Paulo; Brasil. Universidade Federal de Sao Paulo; BrasilFil: Schenkman, Sergio. Universidade Federal de Sao Paulo; BrasilFil: Chatelain, Eric. No especifíca;Fil: Sosa-Estani, Sergio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentin

Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation

Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2 alpha), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 mu M and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2 alpha with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2 alpha phosphorylation, as replacement of WT-eIF2 alpha with a non-phosphorylatable eIF2 alpha, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2 alpha phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)CNPqFundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)NIHUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Parasitol Mol, Rio De Janeiro, RJ, BrazilInst Butantan, Sao Paulo, SP, BrazilUniv Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, Sao Paulo, SP, BrazilBrigham & Womens Hosp, Dept Med, Hematol Lab Translat Res, 75 Francis St, Boston, MA 02115 USAHarvard Med Sch, 75 Francis St, Boston, MA 02115 USAUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04039032 Sao Paulo, SP, BrazilFAPESP: 2015/20031-0FAPESP: 2014/01577-2CNPq: 445655/2014-3NIH: R01 CA152312Web of Scienc

Identification of Inhibitors to trypanosoma cruzi sirtuins based on compounds developed to human enzymes

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment

Amazonian plant natural products:perspectives for discovery of new antimalarial drug leads

Plasmodium falciparum and P. vivax malaria parasites are now resistant, or showing signs of resistance, to most drugs used in therapy. Novel chemical entities that exhibit new mechanisms of antiplasmodial action are needed. New antimalarials that block transmission of Plasmodium spp. from humans to Anopheles mosquito vectors are key to malaria eradication efforts. Although P. vivax causes a considerable number of malaria cases, its importance has for long been neglected. Vivax malaria can cause severe manifestations and death; hence there is a need for P. vivax-directed research. Plants used in traditional medicine, namely Artemisia annua and Cinchona spp. are the sources of the antimalarial natural products artemisinin and quinine, respectively. Based on these compounds, semi-synthetic artemisinin-derivatives and synthetic quinoline antimalarials have been developed and are the most important drugs in the current therapeutic arsenal for combating malaria. In the Amazon region, where P. vivax predominates, there is a local tradition of using plant-derived preparations to treat malaria. Here, we review the current P. falciparum and P. vivax drug-sensitivity assays, focusing on challenges and perspectives of drug discovery for P. vivax, including tests against hypnozoites. We also present the latest findings of our group and others on the antiplasmodial and antimalarial chemical components from Amazonian plants that may be potential drug leads against malaria

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

The evaluation of in vitro antichagasic and anti-SARS-CoV-2 potential of inclusion complexes of β- and methyl-β-cyclodextrin with naphthoquinone

Funding Information: The authors thank the Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq) for their financial support. This study was supported by the CAPES — number 88887.505029/2020–00 . Cecilia Gomes Barbosa receives a scholarship funded by CAPES — number 88887.643352/2021–00 . Publisher Copyright: © 2023 Elsevier B.V.The compound 3a,10b-dihydro-1H-cyclopenta[b]naphtho[2,3-d]furan-5,10-dione (IVS320) is a naphthoquinone with antifungal and antichagasic potential, which however has low aqueous solubility. To increase bioavailability, inclusion complexes with β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) were prepared by physical mixture (PM), kneading (KN) and rotary evaporation (RE), and their in vitro anti-SARS-CoV-2 and antichagasic potential was assessed. The formation of inclusion complexes led to a change in the physicochemical characteristics compared to IVS320 alone as well as a decrease in crystallinity degree that reached 74.44% for the IVS320-MβCD one prepared by RE. The IVS320 and IVS320-MβCD/RE system exhibited anti-SARS-CoV-2 activity, showing half maximal effective concentrations (EC50) of 0.47 and 1.22 μg/mL, respectively. Molecular docking simulation suggested IVS320 ability to interact with the SARS-CoV-2 viral protein. Finally, the highest antichagasic activity, expressed as percentage of Tripanosoma cruzi growth inhibition, was observed with IVS320-βCD/KN (70%) and IVS320-MβCD/PM (72%), while IVS320 alone exhibited only approximately 48% inhibition at the highest concentration (100 μg/mL).publishersversionpublishe

Liming and Fertilization on the Growth of Eucalyptus benthamii and Eucalyptus dunnii in Brazil

The use of mineral fertilizers has shown substantial productivity gains for the vast majority of Eucalyptus forests. The objective of the present work is to evaluate the response of Eucalyptus benthamii and Eucalyptus dunnii at 48 months of age, to different doses of limestone, soluble NPK, and natural phosphates. The experiment was conducted in a plantation located in South Brazil. The experimental design was a randomized block with three replications, and the treatments consisted of combinations of doses of natural phosphate (NP) (0, 400, 600, and 800 kg ha−1 of Gafsa reactive NP P2O5), limestone (0, 3, 5, 6, and 10 Mg ha−1 of dolomitic limestone), and mineral fertilizer–NPK (0, 100, 133, and 167 kg ha−1 of mixed mineral fertilizer 6-30-6). Height and diameter measurements were taken after 48 months. There was a positive response with the increase of NPK fertilizer dose, and the dose where the highest averages were obtained was 167 kg ha−1. The doses of NP had significant effects on the increase of the variables up to 600 kg ha−1, also the averages decreased with the increase of the dosage. For liming, positive results were observed from its absence to the maximum dose, justifying its use in minimum dose in order to supply the necessary amounts of Ca and Mg in the soil. The use of NPK, natural phosphate, and limestone is recommended, causing an increase in the growth of both species of Eucalyptus studied

Tetracycline@silver ions-functionalized mesoporous silica for high bactericidal activity at ultra-low concentration

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