Reduction of Tubulin Expression in Angomonas deanei by RNAi Modifies the Ultrastructure of the Trypanosomatid Protozoan and Impairs Division of Its Endosymbiotic Bacterium

In the last two decades, RNA interference pathways have been employed as a useful tool for reverse genetics in trypanosomatids. Angomonas deanei is a non-pathogenic trypanosomatid that maintains an obligatory endosymbiosis with a bacterium related to the Alcaligenaceae family. Studies of this symbiosis can help us to understand the origin of eukaryotic organelles. The recent elucidation of both the A. deanei and the bacterium symbiont genomes revealed that the host protozoan codes for the enzymes necessary for RNAi activity in trypanosomatids. Here we tested the functionality of the RNAi machinery by transfecting cells with dsRNA to a reporter gene (green fluorescent protein), which had been previously expressed in the parasite and to α-tubulin, an endogenous gene. In both cases, protein expression was reduced by the presence of specific dsRNA, inducing, respectively, a decreased GFP fluorescence and the formation of enlarged cells with modified arrangement of subpellicular microtubules. Furthermore, symbiont division was impaired. These results indicate that the RNAi system is active in A. deanei and can be used to further explore gene function in symbiont-containing trypanosoma tids and to clarify important aspects of symbiosis and cell evolution. This article is protected by copyright. All rights reserved

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion\u2013toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain

Nuclear Structure of Trypanosoma cruzi

The presence of nucleus in living organisms characterizes the Eukaryote domain. the nucleus compartmentalizes the genetic material surrounded by a double membrane called nuclear envelope. the nucleus has been observed since the advent of the light microscope, and sub-compartments such as nucleoli, diverse nuclear bodies and condensed chromosomes have been later recognized, being part of highly organized and dynamic structure. the significance and function of such organization has increased with the understanding of transcription, replication, DNA repair, recombination processes. It is now recognized as consequence of adding complexity and regulation in more complex eukaryotic cells. Here we provide a description of the actual stage of knowledge of the nuclear structure of Trypanosoma cruzi. As an early divergent eukaryote, it presents unique and/or reduced events of DNA replication, transcription and repair as well as RNA processing and transport to the cytosol. Nevertheless, it shows peculiar structure changes accordingly to the cell cycle and stage of differentiation. T. cruzi proliferates only as epimastigote and amastigote stages, and when these forms differentiate in trypomastigote forms, their cell cycle is arrested. This arrested stage is capable of invading mammalian cells and of surviving harsh conditions, such as the gut of the insect vector and mammalian macrophages. Transcription and replication decrease during transformation in trypomastigotes implicating large alterations in the nuclear structure. Recent evidences also suggest that T. cruzi nucleus respond to oxidative and nutritional stresses. Due to the phylogenetic proximity with other well-known trypanosomes, such as Tryponosoma brucei and Leishmania major, they are expected to have similar nuclear organization, although differences are noticed due to distinct life cycles, cellular organizations and the specific adaptations for surviving in different host environments. Therefore, the general features of T. cruzi nuclear structure regarding unique characteristics of this protozoan parasite will be described.Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Georgia, Ctr Trop & Emerging Dis, Athens, GA 30602 USAUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Expression of non-acetylatable lysines 10 and 14 of histone H4 impairs transcription and replication in Trypanosoma cruzi

Submitted by Luciane Willcox ([email protected]) on 2016-10-13T19:10:55Z No. of bitstreams: 1 Expression of non-acetylatable lysines.pdf: 2595939 bytes, checksum: 7100c540b068524e05c9893932c2aa12 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-10-13T19:17:21Z (GMT) No. of bitstreams: 1 Expression of non-acetylatable lysines.pdf: 2595939 bytes, checksum: 7100c540b068524e05c9893932c2aa12 (MD5)Made available in DSpace on 2016-10-13T19:17:21Z (GMT). No. of bitstreams: 1 Expression of non-acetylatable lysines.pdf: 2595939 bytes, checksum: 7100c540b068524e05c9893932c2aa12 (MD5) Previous issue date: 2015-11-19Fundac¸ ão de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2011/51973-3) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (445655/2014-3 and 477143/2011-3 from the Instituto Nacional de Ciência e Tecnologia de Vacinas)Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure

Introduction and objectives: Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). Materials and methods: Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. Results: Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. Conclusions: Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment

Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: a developmental study

Submitted by Adagilson Silva ([email protected]) on 2017-06-26T13:18:14Z No. of bitstreams: 1 28231241 2017 xav-hyd.oa.pdf: 3679006 bytes, checksum: f24ae366311a286279f0f0d3a9c930eb (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2017-06-26T14:23:58Z (GMT) No. of bitstreams: 1 28231241 2017 xav-hyd.oa.pdf: 3679006 bytes, checksum: f24ae366311a286279f0f0d3a9c930eb (MD5)Made available in DSpace on 2017-06-26T14:23:58Z (GMT). No. of bitstreams: 1 28231241 2017 xav-hyd.oa.pdf: 3679006 bytes, checksum: f24ae366311a286279f0f0d3a9c930eb (MD5) Previous issue date: 2017-02Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, BrasilThe teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes

Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2â-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3â4.2 μM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-β-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability

ZIKV infects female organs, placentas and embryos from wild-type immunocompetent mice (FVB/NJ and C57BL/6J), despite the absence of detectable viremia.

<p>Maternal blood samples were collected at 1h (n = 5), 12h (n = 3) and 24h (n = 3) after intravascular ZIKV injection at 5.5 days <i>post-coitum</i> (dpc), or at 12.5 dpc. Further blood samples, maternal organs, placentas and embryos/fetuses were collected at the day of harvest 10.5 dpc (n = 4) and 11.5 dpc (n = 2), or collected at 16.5 dpc (n = 4) in females injected at 12.5 dpc. Samples were assessed for viral load using a tissue culture infectious dose (TCID₅₀) assay in Vero cells. Embryos and respective placentas (n = 8) and maternal organ samples from two 16.5 dpc ZIKV-injected pregnant females (injected in 12.5 dpc), as well as two 12.5 PBS-injected control females were assessed for viral content using a TCID₅₀ assay. ZIKV was detected in the spleen of both females and in the liver of one female. Notably, all placentas had significant amounts of ZIKV, whereas just half of the embryos were ZIKV positive. No malformations were observed.</p

Morphometric analyses in three litters of ZIKV-injected pregnant mouse females.

<p>(<b>A-C</b>) To better normalize the morphometric results we plotted Crown rump length (CRL) and Occipital-frontal diameter (OFD) as a function of individual embryonic/fetal stages, rather than as nominal litter stages. All conceptuses were classified according to Kauffman [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.ref020" target="_blank">20</a>] and Theiler [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.ref022" target="_blank">22</a>]. (<b>A</b>) CRL data. (<b>B</b>) OFD data. (<b>C</b>) OFD/CRL data. All data are displayed in relation to intervals defined as the average ± three standard deviations of PBS-injected (light grey), or reference controls (dark grey). Stage normalization eliminated all evidence for specific changes in cephalic proportions, but for two conceptuses. One fetus (shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.g007" target="_blank">Fig 7H</a>) displayed an abnormally increased OFD/CRL, which resulted from cephalic collapse in the cranio-caudal axis (arrowhead), while the other fetus (shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.g006" target="_blank">Fig 6D</a>) constitutes the only specific evidence for microcephaly in our study (arrow).</p