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Na elaboração deste artigo, reunimos duas pesquisas distintas que dialogaram com o teatro e a Geografia da Infância, cada uma em sua cronotopia. As reflexões sobre um teatro com bebês e crianças percorreram uma vasta revisão bibliográfica que sinalizou outras possibilidades de refletir sobre como estamos pensando a relação com os bebês e as crianças na sociedade, principalmente, no espaço institucional da creche. A teoria histórico-cultural e as inspirações da Geografia da Infância foram a base para pensarmos a espacialização da vida a partir do teatro. Paralelamente, dialogamos com o Kòtèba, o teatro tradicional dos Bámànàn (África Ocidental), um povo conhecido por brincar com seus problemas e seus mitos, cujas encenações, danças, cantos e música pontuam todos os aspectos de sua vida social. Seguimos as pegadas de Sotigui Kouyaté, que afirmava que, através do Kòtèba, a jovem África podia acessar todo o mundo e toda a experiência ancestral bámànàn. Além das palavras do djeli Sotigui Kouyaté, estruturam a presente pesquisa os trabalhos de Sada Sissoko e Osita Okagbue.

Neospora caninum, an apicomplexan parasite, is the etiological agent of neosporosis, a disease that leads to neurological symptoms in dogs and abortion in cattle. Vaccine or drug treatments for neosporosis remain to be determined. Therefore, it is of undeniable relevance to investigate new molecules involved in the parasite's successful survival within the host cell. The aim of this study was to characterize the N. caninum peroxiredoxin (NcPrx), an enzyme involved in the redox system of the parasite. The NcPrx amino acid sequence showed high identity and similarity compared to homologues representatives of Apicomplexa phylum. The recombinant NcPrx (rNcPrx) was cloned and expressed in Escherichia coli (BL21) with the predicted molecular weight (22 kDa), and the identity of monomer and dimer forms of rNcPrx was confirmed by mass spectrometry. Native and recombinant NcPrx were detected by ELISA and western blot, using the polyclonal anti-rNcPrx serum. Multiphoton analysis showed that NcPrx is localized in tachyzoite cytosol. H

Myeloproliferative neoplasms (MPN) are hematological disorders characterized by increased proliferation of precursor and mature myeloid cells. MPN patients may present driver mutations in JAK2, MPL, and CALR genes, which are essential to describe the molecular mechanisms of MPN pathogenesis. Despite all the new knowledge on MPN pathogenesis, many questions remain to be answered to develop effective therapies to cure MPN or impair its progression to acute myeloid leukemia. The present study examined the expression levels of the Hippo signaling pathway members in patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), as well as the role that they play in disease pathogenesis. The Hippo pathway is a tumor suppressor pathway that participates in the regulation of cell proliferation, differentiation, and death. Our main findings were: (i) expression of tumor suppressor genes from Hippo pathway were downregulated and seemed to be associated with cell resistance to apoptosis and increased proliferation rate; and (ii) Hippo pathway-related gene expression was associated with mutation status in ET and PMF patients. Therefore, the decreased expression of Hippo pathway-related genes may contribute to the malignant phenotype, apoptosis resistance, and cell proliferation in MPN pathogenesis.

Neospora caninum is a protozoan member of the Apicomplexa phylum and is closely connected with abortion in cattle. The development of the parasite in host cells is characterized by the active secretion of proteins, allied to the tight control of the redox status. In this sense, elucidating the mechanisms related to the role of the redox agents and enzymes during the invasion and proliferation of N. caninum may contribute to developing novel forms of neosporosis control. In this study we verified the effects of the recombinant forms of N. caninum glutathione reductase (rNcGR) and thioredoxin-dependent peroxide reductase (rNcPrx), as well as H

Chagas disease is a tropical illness caused by the protozoanTrypanosoma cruzi. The disease affects populations of the Americas and has been spread to other continents due to the migration process. The disease is partially controlled by two drugs, Benznidazole and Nifurtimox. These molecules are active in the acute phase of the infection but are usually ineffective during the symptomatic chronic phase. Several research groups have developed novel candidates to control Chagas disease; however, no novel commercial formulation is available. In this article, we described the anti-T. cruzieffects of phenothiazinium dyes in amastigote and trypomastigote forms of the parasite. Methylene Blue, New Methylene Blue, Toluidine Blue O, and 1,9-Dimethyl Methylene Blue inhibited the parasite proliferation at nanomolar concentrations and also demonstrated low toxicity in host cells. Moreover, combinations of phenothiazinium dyes indicated a synergic pattern against amastigotes compared to the Benznidazole counterparts. Phenothiazinium dyes levels of reactive oxygen species (ROS) and decreased the mitochondrial potential in trypomastigotes, indicating the mechanism of action of the dyes inT. cruzi. Our article offers a basis for future strategies for the control of Chagas disease using low-cost formulations, an important point for endemic underdeveloped regions.

Neospora caninum is an obligate intracellular parasite related to abortion in cattle, goats and sheep. The life cycle of N. caninum is characterized by the time-coordinated secretion of proteins contained in micronemes, rhoptries and dense granules, allowing the active invasion and the adaptation of the parasite in the cell environment. Thus, the proteins of the secretome have the potential to be considered as targets for N. caninum control. Despite the importance of neosporosis in the livestock-related economy, no commercial treatment is available. Furthermore, the process of invasion, propagation and immune evasion are not completely elucidated. In this study, we initiated the characterization of NCLIV_011700 of N. caninum, a protein with low sequence identity to NcROP15 or TgROP15 (15%). Our goal was the detection and molecular characterization of the NCLIV_011700, once homology (with low identity20%) was observed within the Apicomplexa. The NCLIV_011700 sequence was aligned and compared to the closer apicomplexan homologues (ROP15 from N. caninum, T. gondii, Hammondia hammondi, Cystospores suis), including the predicted domains. In general, the NCLIV_011700 demonstrated low identity with ROP15 of apicomplexan (20%) and had a ubiquitin domain. On the other side, the NCLIV_011700 homologues were composed of a non-cytoplasmic domain, suggesting different functions between NcROP15 (or homologues) and NCLIV_011700 during the parasite life cycle. Moreover, the NCLIV_011700 was amplified by PCR, ligated to a pET28a plasmid and expressed in Escherichia coli. The recombinant form of NCLIV_011700 was purified in a nickel-Sepharose resin and applied for polyclonal antibody production in mice. The antiserum against NCLIV_011700 (anti-rNCLIV_011700) was used to localize the native form of the protein using Western blot and confocal microscopy. Also, the NCLIV_011700 antiserum partially inhibited the parasite adhesion/invasion process, indicating an active role of the protein in the N. caninum cycle. Thus, the initial NCLIV_011700 characterization will contribute to enlarging the comprehension of N. caninum, aiming at the future development of tools to control the parasite infection/propagation.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 μM, 11 ± 2 μM, 0.7 ± 0.1 μM, and 0.9 ± 0.2 μM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.

Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds. Resumo Neospora caninum é um parasita Apicomplexa relacionado a abortos no gado bovino, que resultam em impactos econômicos. Não há tratamento comercial para neosporosis e o reposicionamento de drogas indica uma estratégia rápida para testar candidatos anti-N. caninum. Neste artigo, são descritos os efeitos da atovaquona, cloroquina, quinino, primaquine e tetraciclina na proliferação de N. caninum. As concentrações IC50 em N. caninum foram comparadas com a informação disponível, baseada em estudos publicados previamente para Plasmodium e Toxoplasma gondii, incluindo a correlação com os mecanismos de ação descritos para cada droga testada. Os padrões de inibição indicam pontos de similaridades e diferenças entre N. caninum, Plasmodium e T. gondii. Por exemplo, a atovaquona demonstra uma alta atividade antiparasitária em todos os modelos testados, enquanto a cloroquina não inibe N. caninum. Por outro lado, a tetraciclina é efetiva contra Plasmodium e N. caninum, em contraste com a baixa atividade em modelos de T. gondii. O reposicionamento de drogas antimaláricas em N. caninum é uma forma rápida e de baixo custo para o desenvolvimento de novas formulações que usam compostos bem estabelecidos.

Gold(III) complexes have been studied for the past years due to their anticancer properties and great affinity to biotargets, such as enzymes and proteins, which support their pharmacological applications. Within this scope, in this work the antiproliferative activities of two Au(III)-thiosemicarbazonate complexes, [AuClL1] (1, L1: (E,Z)-N-ethyl-N'-(3-nitroso-kN)butan-2-ylidene)carbamohydrazonothioato-k2N2,S) and [Au(Hdamp)L2]Cl (2, L2: N-(N'',N''-diethylaminothiocarbonyl)-N'(N''', N'''-dimethylcarbothioamide)benzamidineto-kN,k2S and Hdamp: 2-(N,N-dimethylaminomethyl)-phenyl-C1), and their affinities to possible biological targets were investigated. Three different tumor cell lines were used to perform the cytotoxicity assays, including one cisplatin-resistant model, and the results showed lower EC50 for 1 over 2 in every case: B16F10 (4.1 μM and 15.6 μM), A431 (4.0 μM and >50 μM) and OVCAR3 (4.2 μM and 24.5 μM). However, a lower toxicity to fibroblast 3T3 cell line was observed for 2 (30.58 μM) when compared to 1 (7.17 μM), resulting in comparable therapeutic indexes. Both complexes presented strong affinity to HSA: they distorted the secondary structure of the protein, as verified by circular dichroism, but 1 additionally presented the apparent fluorescence quenching constant (Kapp) ten times greater than 2, which was probably due to the fact of 1 being able to denature HSA. The ethidium bromide displacement assay showed that neither 1 nor 2 are strong DNA intercalators, which is in agreement with what was observed through the UV-vis titration. In both cases, the 260 nm band presented hyperchromism, which can indicate ionic interactions or DNA damage. In fact, 1 was able to damage the pGEM plasmid, similarly to cisplatin, as verified by agarose gel electrophoresis and Atomic Force Microscopy. Biophysical studies in cancer cells model membranes were also performed in order to investigate the interaction of the gold complexes to lipid bilayers and revealed that the compounds interact with the membranes by exhibiting partition coefficients of 103 order of magnitude. Overall, both complexes were found to be promising candidates for the development of a future anticancer drug against low sensitive or cisplatin resistant tumors.

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9–Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019–1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9–Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9–Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control.

Leishmaniasis, malaria, and neosporosis are parasitic diseases that affect humans and animals, causing public health problems and billions in economic losses. Despite the advances in the development of new drugs, the severe side effects of available leishmaniasis treatments, the Plasmodium spp. resistance to antimalarial drugs, and the lack of a specific treatment against neosporosis lead us to the search for new anti-protozoan molecules from underexplored sources such as the Antarctic marine environment. Herein, we describe for the first time the chemical profile of Desmarestia antarctica crude extract and fractions using GC-MS and LC-MS/MS (molecular networking) approaches, and evaluate their antiparasitic activity against Leishmania amazonensis, Neospora caninum, and multi-drug-resistant Plasmodium falciparum. Furthermore, the cytotoxicity in 3T3 BALB/c fibroblasts and Vero cells was evaluated. D. antarctica fraction E ( IC50 of 53.8±4.4 μg mL− 1 and selectivity index of 3.3) exhibited anti-promastigote activity and was fourfold more selective to L. amazonensis rather than to the host cells. D. antarctica fraction D (IC50 of 1.6±1.3 μg mL− 1 and selectivity index of 27.8), D. antarctica fraction F (IC50 of 3.1±2.1 μg mL− 1 and selectivity index of 23.1), and D. antarctica fraction H (IC50 of 3.1±2.0 μg mL− 1 and selectivity index of 12.9) presented the highest antiparasitic effects against N. caninum with no cytotoxic effects. Also, D. antarctica fraction D presented a significant antiplasmodial inhibitory effect (IC50 of 19.1±3.9 μg mL− 1 and selectivity index of 6.0). GC-MS analysis indicated palmitic acid, myristic acid, fucosterol, phthalic acid, di(2-methylbutyl) ester, loliolide, and neophytadiene as the main components in the active fractions. In addition, this is the first report of a biological screening of macroalgae secondary metabolites against N. caninum parasites.

Plants are an important source of molecules that can control acute and chronic diseases. Malaria treatment represents a successful strategy in which an infection is controlled with compounds obtained from plants (for instance, artemisinin and quinine). Natural sources are an interesting reservoir of potential molecules that can act against protozoans, especially Trypanosoma cruzi. This parasite is the causative agent of Chagas disease, whose symptomatic chronic phase leads to cardiac and digestive complications. Despite the impacting complications of this disease, only two drugs are commercially available to treat it, nifurtimox and benznidazole. Thus, novel candidates for Trypanosoma cruzi control are desirable. Among the recent advances in this field, the diterpene copalic acid and the sesquiterpene β-caryophyllene oxide purified from copaiba plants have demonstrated antipathogenic properties, including trypanocidal activity. Here, we have studied some biological properties of copalic acid and performed several chemical modifications on β-caryophyllene oxide, which afforded six derivatives. These terpenes exerted different effects on amastigotes, epimastigotes, and trypomastigotes. In addition, they also inhibited the activity of horseradish peroxidase, a heme enzyme.

Neospora caninum is a member of Apicomplexa phylum, the causative agent of neosporosis. The neosporosis combat is not well established and several strategies related to vaccine, chemotherapy and immune modulation are under development. In this work, we evaluated the effects of artemisinin (Art), methylene blue (MB) and pyrimethamine (Pyr) alone or associated, on N. caninum proliferation and elimination using LacZ tagged tachyzoites. The reactive oxygen species (ROS) production after incubation with Art were also performed. Our results indicate that combinations of classical antimalarial drugs improve the parasite control, allowing the use of three drugs in a single dose. Additionally, artemisinin demonstrated distinct ROS production patterns in intra and extracellular N. caninum forms. The drug repurposing appears as a suitable approach, allowing a fast and safe method to evaluate old drugs but novel candidates against neosporosis.

Chagas disease or American trypanosomiasis is caused by the protozoan Trypanosoma cruzi and is endemic of the Americas. The control of the disease is restricted to toxic and potentially teratogenic drugs, which limit the use during pregnancy. The use of food supplementation offers a safe and low-cost form to alleviate Chagas disease symptoms, mostly in areas with alimentary risk. For example, zinc demonstrates positive effects in immune response, including in Chagas disease during pregnancy. This study describes the innate response in pregnant rats chronically infected with T. cruzi and supplemented with zinc. Pregnant female Wistar rats, infected with T. cruzi, were treated with 20 mg/kg/day zinc sulfate and euthanized on the 18th day. Samples (plasma, splenocytes, and peritoneal exudate) were collected and several immune parameters (nitric oxide, RT1B, CD80/CD86, MCP-1, CD11b/c, NK/NKT, IL-2, IL-10, INF-cc, and apoptosis) evaluated. Under Zinc supplementation and/or T. cruzi infection, the gestation developed normally. Several innate immune parameters such as RT1B, CD80/CD86, MCP-1 expressing lymphocytes, IL-2, and IL-17 were positively altered, whereas nitric oxide, CD11b/c, NK/NKT, apoptosis, INF-γ, and corticosterone demonstrated a pro-pregnancy pattern. Our results indicated that zinc has diverse effects on immune response during pregnancy. An anti-T. cruzi immunity, as well as a pro-gestation response, were observed after zinc supplementation. The complete comprehension of zinc supplementation in pregnancy will base an adequate strategy to alleviate Chagas disease symptoms and propagation, especially for populations from endemic areas.

Chagas disease is a tropical illness caused by the protozoan Trypanosoma cruzi. The disease affects populations of the Americas and has been spread to other continents due to the migration process. The disease is partially controlled by two drugs, Benznidazole and Nifurtimox. These molecules are active in the acute phase of the infection but are usually ineffective during the symptomatic chronic phase. Several research groups have developed novel candidates to control Chagas disease; however, no novel commercial formulation is available. In this article, we described the anti-T. cruzi effects of phenothiazinium dyes in amastigote and trypomastigote forms of the parasite. Methylene Blue, New Methylene Blue, Toluidine Blue O, and 1,9-Dimethyl Methylene Blue inhibited the parasite proliferation at nanomolar concentrations and also demonstrated low toxicity in host cells. Moreover, combinations of phenothiazinium dyes indicated a synergic pattern against amastigotes compared to the Benznidazole counterparts. Phenothiazinium dyes levels of reactive oxygen species (ROS) and decreased the mitochondrial potential in trypomastigotes, indicating the mechanism of action of the dyes in T. cruzi. Our article offers a basis for future strategies for the control of Chagas disease using low-cost formulations, an important point for endemic underdeveloped regions.

Background Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic to subtropical and tropical Americas. The disease treatment remains partially ineffective, involving therapies directed to the parasite as well as palliative strategies for the clinical manifestations. Therefore, novel candidates for disease control are necessary. Additionally, strategies based on parasite inhibition via specific targets and application of compounds which improve the immune response against the disease is welcomed. Ghrelin is a peptide hormone pointed as a substance with important cardioprotective, vasodilatory, anti-apoptotic, anti-oxidative and immune modulatory functions. The aims of this study were to evaluate the immunomodulatory effects of ghrelin in male Wistar rats infected with the Y strain of T. cruzi. Methods In order to delineate an immune response against T. cruzi mediated by ghrelin, we evaluated the following parameters: quantification of blood and cardiac parasites; analysis of cell markers (CD3+, CD8+, NK, NKT, CD45RA+, macrophage and RT1B+); nitric oxide (NO) production; lymphoproliferation assays; splenocyte apoptosis; and INF-γ, IL-12 and IL-6 quantification in sera. Results The animals infected with T. cruzi and supplemented with ghrelin demonstrated an upregulated pattern in macrophage and NO production, whereas an anti-inflammatory response was observed in T cells and cytokines. The low response against T. cruzi mediated by T cells probably contributed to a higher colonization of the cardiac tissue, when compared to infected groups. On the other side, the peptide decreased the inflammatory infiltration in cardiac tissue infected with T. cruzi. Conclusions Ghrelin demonstrated a dual function in animals infected with T. cruzi. Further studies, especially related to the decrease of cardiac tissue inflammation, are needed in order to determine the advantages of ghrelin supplementation in Chagas disease, mostly for populations from endemic areas.

Neospora caninum is an apicomplexan parasite that causes infectious abortion in cows. As an obligate intracellular parasite, N. caninum requires a host cell environment to survive and replicate. The locomotion and invasion mechanisms of apicomplexan parasites are centred on the actin-myosin system to propel the parasite forwards and into the host cell. The functions of actin, an intrinsically dynamic protein, are modulated by actin-binding proteins (ABPs). Actin-depolymerising factor (ADF) is a ubiquitous ABP responsible for accelerating actin turnover in eukaryotic cells and is one of the few known conserved ABPs from apicomplexan parasites. Apicomplexan ADFs have nonconventional properties compared with ADF/cofilins from higher eukaryotes. In the present paper, we characterised the ADF from N. caninum (NcADF) using computational and in vitro biochemical approaches to investigate its function in rabbit muscle actin dynamics. Our predicted computational tertiary structure of NcADF demonstrated a conserved structure and phylogeny with respect to other ADF/cofilins, although certain differences in filamentous actin (F-actin) binding sites were present. The activity of recombinant NcADF on heterologous actin was regulated in part by pH and the presence of inorganic phosphate. In addition, our data suggest a comparatively weak disassembly of F-actin by NcADF. Taken together, the data presented herein represent a contribution to the field towards the understanding of the role of ADF in N. caninum and a comparative analysis of ABPs in the phylum Apicomplexa.

Selenium (Se) is an essential micronutrient in the diet of mammals and has an important role in the immune function. Selenium is a key element in selenoproteins involved in the in the maintenance of the antioxidant defense. Diet with selenium is beneficial for the treatment of diseases correlated with high levels of oxidative stress, also observed in the Chagas disease. Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi and several research groups are focused on the illness treatment. Immunomodulation of the infection using microelements is an important tool to avoid deleterious effects of the Chagas disease. Therefore, our objective was to evaluate the effects of selenium supplementation on pregnant Wistar rats infected with T. cruzi. Selenium treatment stimulated the weight and length of fetuses and placentas allied to the decrease of blood parasitemia. However, selenium demonstrated a low influence on T cells, diminishing the B cell population (CD45RA+). Moreover, the production of pro-inflammatory cytokines was downregulated under selenium administration. Low pro-inflammatory cytokines levels probably are related to the increase in the number of amastigote nests in infected and treated animals. Thus, selenium supplementation during pregnancy could impair the local placental immune response. Further studies are necessary to assess the interaction between selenium and the acute Chagas’ disease during pregnancy, which will base future supplementation strategies.

Chagas disease afflicts 7 to 8 million people worldwide and congenital Chagas' disease usually leads to changes in the maternal environment, culminating in fetal adaptations. Several articles have described the importance of micronutrients on pregnancy, which is sensitive to infections. In Trypanosoma cruzi endemic regions, the Chagas disease is aggravated by the lack of micronutrients in an average diet, to which pregnant women are more susceptible. The aim of this study was to evaluate distinct T cells phenotypes and intracellular cytokines by flow cytometry in pregnant Wistar rats under zinc therapy during experimental Chagas' disease. Twenty female Wistar rats were infected with 1×105 blood trypomastigotes (Y strain) and 30days after infection the animals were mated and grouped: pregnant infected (PI-n=5), pregnant infected/zinc supplied (PIZ-n=5), pregnant control (PC-n=5), control/zinc supplied (PCZ-n=5). Zinc supplementation: 20mg of zinc/Kg/day (gavage) for 18days followed by euthanasia. The immune parameters showed: decreased percentages of CD62LlowCD44high surface marker for infected and treated group (PIZ) when compared to PI (p

SUMMARYNeospora caninumis an apicomplexan parasite strongly related to reproductive problems in cattle. The neosporosis control is not well established and several fronts are under development, predominantly based on immune protection, immunomodulation and chemotherapy. The use of anti-malarial drugs as therapeutic sources has, in theory, considerable potential for any apicomplexan. Drugs such as methylene blue (MB) and pyrimethamine (Pyr) represent therapeutic options for malaria; thus, their use for neosporosis should be assessed. In this work, we tested the effects of MB and Pyr onN. caninumproliferation and clearance, using LacZ-tagged tachyzoites. The drugs inhibited at nanomolar dosages and its combination demonstrated an antagonistic interaction in proliferation assays, according to the Chou and Talalay method for drug combination index. However, the drug combination significantly improved the parasitein vitroclearance. The repositioning of well-established drugs opens a short-term strategy to obtain low-cost therapeutics approaches against neosporosis.

Este artigo tem como objetivo refletir sobre os estudos contemporâneos no campo da infância, tendo como recorte central a pesquisa com bebês. Conceitualmente, assumimos por bebês o desenho estabelecido nas legislações e na área acadêmica que envolve a primeira infância, ou seja, do nascimento até 36 meses. O trabalho situou-se na interface das vivências dos bebês e suas enunciações, possibilitadas pelas atividades cênicas que assumimos e denominamos como teatro com bebês, que consistiu na oferta de um ambiente pensado para recebê-los, criando uma situação social em que se potencializaram suas atividades criadoras. Metodologicamente, a pesquisa com os bebês foi feita no espaço da educação infantil da Escola Nossa, em Niterói, Rio de Janeiro. Como principais conclusões mencionamos a potência que o teatro com bebês promove quando em interação com os adereços cênicos; suas enunciações enquanto, ao mesmo tempo, atores e plateia a partir da oferta cênica vinculada diretamente ao ato de brincar apresentam acontecimentos irrepetíveis e portadores de vivências que possibilitam seus protagonismos.

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.

Neospora caninum is a parasite of the Apicomplexa phylum responsible for abortion and losses of fertility in cattle. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed with the surface proteins known as the SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SAG related or SRS proteins have been a target of intense research due to its immunodominant pattern, exhibiting potential as diagnostic and/or vaccine candidates. The aim of this study was the cloning, expression and characterization of the gene NcSRS67 of N. caninum using a novel designed plasmid. The coding sequence of NcSRS67 (without the signal peptide and the GPI anchor) was cloned and expressed constitutively instead of the ccdB system of pCR-Blunt II-TOPO. The protein was purified in a nickel sepharose column and identified by mass spectrometry (MS/MS). The constitutive expression did not affect the final bacterial growth, with a similar OD 600nm compared to the non-transformed strains. The recombinant NcSRS67 was over expressed and the native form was detected by the anti-rNcSRS67 serum on 1D western blot as a single band of approximately 38kDa as predicted. On an in vitro assay, the inhibitory effect of the polyclonal antiserum anti-rNcSRS67 was nearly 20% on adhesion/invasion of host cells. The NcSRS67 native protein was localised on part of the surface of N. caninum tachyzoite when compared to the nucleus by confocal immunofluorescence.

SUMMARYNeospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue†.

Neospora caninum é um protozoário Apicomplexa, parasita intracelular obrigatório que possui o cão como hospedeiro definitivo e principalmente os bovinos como hospedeiros intermediários, causando nos primeiros encefalopatia e nos últimos abortos com perda da fertilidade, o que acarreta prejuízos significativos na pecuária mundial. O parasita necessita multiplicar de modo intra-celular, utilizando a descarga de proteínas contidas em organelas filo-específicas, como as micronemas que liberam proteínas relacionadas a adesão e invasão ativa, além das roptrias e os grânulos densos. Este trabalho foi realizado sobre um tipo de proteína micronêmica denominada proteína Anônima Relacionada à Trombospondina (TRAP), relacionada à adesão e ligação com o motor intracelular, responsável pelo processo ativo de invasão. TRAP 1 já havia sido descrita pela literatura e o objetivo deste trabalho foi a caracterização molecular do até então desconhecido homólogo TRAP 2, com expressão de suas formas recombinantes e o uso de seus respectivos anti-soros para ensaios funcionais e localização da proteína nativa. Com base em banco dados de ESTs e genômicos de N. caninum e fazendo uso de RLM-RACE (Reação mediada por RNA ligase- amplificação rápida dos términos de cDNA-RNA,) foi possível a obtenção da sequencia completa do gene NcTrap 2, que possui quatro éxons separados por três íntrons. Sua forma protéica, como seu homológo, é formado por um peptídeo sinal, uma integrina, cinco trombospondinas, uma região transmembrana e a cauda citoplasmática. A integrina e as trombospondinas são motivos relacionados à adesão para início do processo invasivo e foram os alvos deste trabalho. Foram expressos dois recombinantes a partir deste cerne funcional (integrina e trombospondinas), com 52 e 78 kDa, e seus respectivos anti-soros foram obtidos. Estes foram utilizados em western blots 1D e 2D para localização da forma nativa de NcTRAP 2 de aproximadamente 80 kDa no extrato total e de sua forma processada no ESA (extrato secretado, simulando o momento invasivo do taquizoíta) com 70 kDa. O soro contra o recombinante 1 teve a capacidade de inibir o processo invasivo em valores de 53 a 61%, dependendo do método utilizado (contagem manual ou real time PCR). NcTRAP 2 foi encontrada no complexo apical parasitário do taquizoíta por imunofluorêscencia confocal, evidenciando sua provável localização nas micronemas. A nova molécula, NcTRAP 2 possui características que a diferencia de NcTRAP 1 o suficiente para ser considerada homóloga. O soro contra sua forma recombinante detectou as formas nativas integral e secretada e demonstrou que NcTRAP 2 possui capacidade de inibir a invasão dos taquizoítas, o que a torna um interessante alvo vacinal. Neospora caninum is an Apicomplexan protozoan, obligatory intracellular parasite that has the dog as definitive host and especially cattle as intermediate hosts, causing in the first ones encephalopathy and in the last ones abortion with fertility losses. Such facts lead to significant losses to livestock worldwide. The parasite must invade the cells for its development, using the discharge of proteins contained in phylum-specific organelles, like the micronemes proteins related with adhesion and active invasion, besides the proteins from rhoptries and dense granules. This work was performed on a type of micronemic protein, a thrombospondin-related anonymous protein (TRAP), related with adhesion and connection with the intracellular motor responsible for the active process of invasion. Based on the homologue previously described in the literature, TRAP 1, the aim of this study was the characterization of the undescribed TRAP 2 homologue, the expression of its recombinant forms, the use of its anti-sera for functional assays, and detection of its native form. Based on ESTs and genomic data from N. caninum, and using the RLM-RACE (ligase mediated rapid amplification of cDNA ends) technique, the complete sequence of NcTRAP 2 was obtained, which had four exons separated by three introns. As NcTRAP 1, the protein sequence is composed of a signal peptide, an integrin, five thrombospondin, a transmembrane region and a cytoplasmatic tail. The integrin and thrombospondin motifs are related to adhesion for initiation of the invasive process and were the targets of this work. We expressed two recombinant fragments from the functional core (thrombospondin and integrin), with 52 and 78 kDa, which were utilized for antisera production. The anti-sera localized the native form NcTRAP 2 which had approximately 80 kDa, and its processed form in ESA (Excreted/Secreted Antigen) with 70 kDa, both in western blot 1D and 2D. The serum against recombinant 1 had the ability to inhibit the invasive process from 53 to 61%, depending on the experimental method used (manual counting or real time PCR). NcTRAP 2 was localized at the apical complex of the parasite of the tachyzoites by confocal immunofluorescence, pointing towards its micronemal localization. The new molecule, NcTRAP 2, holds features that differentiates it from NcTRAP 1 in a substantial way to be considered homologue. The serum against the recombinant form detected the native full length and secreted forms of NcTRAP 2 and demonstrated that NcTRAP 2 has the ability to inhibit the invasion of tachyzoites, turning it into an interesting vaccine target to be investigated.

We evaluated the color vision of mercury-contaminated patients and investigated possible retinal origins of losses using electroretinography. Participants were retired workers from a fluorescent lamp industry diagnosed with mercury contamination (n= 43) and age-matched controls (n= 21). Color discrimination was assessed with the Cambridge Colour Test (CCT). Retinal function was evaluated by using the ISCEV protocol for full-field electroretinography (full-field ERG), as well as by means of multifocal electroretinography (mfERG). Color-vision losses assessed by the CCT consisted of higher color-discrimination thresholds along the protan, deutan, and tritan axes and significantly larger discrimination ellipses in mercury-exposed patients compared to controls. Full-field ERG amplitudes from patients were smaller than those of the controls for the scotopic responseb-wave, maximum response, sum of oscillatory potentials (OPs), 30-Hz flicker response, and light-adapted cone response. OP amplitudes measured in patients were smaller than those of controls for O2 and O3. Multifocal ERGs recorded from ten randomly selected patients showed smaller N1–P1 amplitudes and longer latencies throughout the 25-deg central field. Full-field ERGs showed that scotopic, photopic, peripheral, and midperipheral retinal functions were affected, and the mfERGs indicated that central retinal function was also significantly depressed. To our knowledge, this is the first demonstration of retinal involvement in visual losses caused by mercury toxicity.

Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii.

Neospora caninum é um protozoário Apicomplexa, parasita intracelular obrigatório que possui o cão e outros canídeos como hospedeiros definitivos e os bovinos como principais hospedeiros intermediários. Causa nos primeiros encefalopatia e nos últimos abortos com perda da fertilidade, gerando prejuízos significativos na pecuária mundial. Como Toxoplasma gondii e Plasmodium, N. caninum possui um sistema específico de secreção que permite a invasão ativa do parasita na célula hospedeira, formação do vacúolo parasitóforo e replicação. Devido ao seu ciclo intracelular, várias estratégias foram desenvolvidas para caracterizar o processo de invasão no filo, como deleção ou controle da expressão de genes envolvidos. Entre os genes com papel importante no processo invasivo, NcMic2-like1 possui destaque já que o soro contra essa proteína recombinante inibiu 60% da invasão de N. caninum in-vitro. Essa proteína micronêmica possui domínios adesivos (uma integrina e seis trombospondinas) que atuam na ligação entre os receptores da célula hospedeira e o motor de actina e miosina do parasita, possibilitando a invasão ativa. O objetivo central do trabalho foi o desenvolvimento de ferramentas genéticas para a deleção e hiperexpressão de NcMic2-like1 e outras proteínas em N. caninum para a compreensão profunda desse mecanismo, além da caracterização, localização e expressão de NcMic2-like1. Para isso dois modelos baseados na resistência a drogas e integração ao genoma do parasita foram construídos. Um se baseia na resistência contra cloranfenicol (pelo gene Cloranfenicol Acetil Transferase - CAT) e o outro contra pirimetamina (pela mutação da Diidrofolato Redutase-Timidilato Sintase - DHFRM2M3). As sequências que conferem resistência foram ligadas ao promotor e região 3\' UTR de três genes de N. caninum, NcSAG1 (ou SAG1-like); NcDHFR ou NcHXGPRT conferindo resistência às drogas após transfecção. A esses vetores de resistência foram ligadas duas sequências do controle de expressão por tetraciclina, o sistema TetR/tetO. Adaptando o sistema de T. gondii para N. caninum, TetR foi expresso sob controle do promotor RPS13 ou Tubulina de N. caninum e tetO controlou a expressão de Lac-Z (?-galactosidase), ii uma enzima repórter. TetR e tetO foram transfectados de modo estável utilizando os vetores CAT e DHFRM2M3. O sistema foi responsivo à adição de tetraciclina, além de indicar um meio provavelmente independente de TetR quando apenas tetO foi avaliado em N. caninum. Foi possível expressar Lac-Z em N. caninum, possibilitando o desenvolvimento e padronização de ensaios de invasão, crescimento e detecção do taquizoíta. Também foi realizada a detecção de YFP (proteína amarelo fluorescente) nos taquizoítas transfectados com YFP fundida em TetR. Essas ferramentas de inserção e deleção de genes possibilitaram a construção de vetores para a hiper expressão ou deleção de NcMic2-like1. Para hiper expressão o vetor foi obtido pela substituição de Lac-Z pelo gene NcMic2-like1. Para deleção de NcMic2-like1 o vetor foi desenvolvido pela ligação do seu promotor e região 3\' UTR ä CAT ou NcDHFRM2M3, que promove a substituição do gene alvo pelos de resistência por recombinação homóloga. Adicionalmente foram desenvolvidas formas para a expressão e purificação de proteínas em N. caninum. Também foi obtida a solubilização de NcMIC2-like1 recombinante pelo sistema pET28 após a adição de uma cauda solúvel amplificada do genoma de N. caninum. O desenvolvimento de métodos de manipulação genética é um fato inédito para N. caninum e possibilitará estudos moleculares mais profundos sobre os mecanismos de invasão e replicação, onde NcMic2-like1 aparece como primeira candidata para esses ensaios. The Apicomplexa protozoan Neospora caninum is an obligate and intracellular parasite that has the dog and other canides as definitive host and especially cattle as intermediate hosts. It causes respectively encephalopathy. abortions and loss of fertility causing deep economic impact at the world livestock. As Plasmodium and Toxoplasma gondii, N. caninum has a specific system of active secretion, which allows the invasion of the parasite into the host cell, the establishment of parasitophorous vacuole and replication. Due to the intracellular cycle of parasite, several strategies have been developed to characterize the invasion process of the phylum, such as deletion or control of expression of related genes. Among the genes with an important role in the invasive process, NcMic2-like1 stands out, once the serum against the recombinant form inhibited the in vitro invasion of N. caninum up to 69%. This microneme protein has adhesive domains (one integrin and six thrombospondins) acting between the host cell receptors and the actin/myosin motor of the parasite, allowing the active invasion. The main aim of this work was the development of genetic tools for deletion and overexpression of NcMic2-like1 and other proteins in N. caninum for a deep understanding of this mechanism, in addition to the characterization, localization and expression of NcMic2-like1. Therefore two models based on the drug resistance and integration into the genome of the parasite were constructed. One was based on the resistance against chloramphenicol (chloramphenicol acetyl transferase gene by - CAT) and the other against pyrimethamine (by mutation of dihydrofolate reductase-thymidylate synthase - DHFRM2M3). The sequences that confer these two drug resistance patterns have been ligated to the promoter and the 3\' UTR region of three genes from N. caninum, NcSAG1 (or SAG1-like); NcDHFR or NcHXGPRT, conferring resistance to the drugs after transfection. These vectors were ligated to two sequences that confer resistance controlled by tetracycline, the TetR / tetO system. The T. gondii system was adapted for N. caninum. TetR was expressed under the control of N. caninum tubulin promoter and tetO controlled the expression of Lac-Z (?-galactosidase), a reporter enzyme. TetR and tetO were stably inserted in N. caninum after transfection iv with the vectors based on CAT and DHFRM2M3.The system was responsive following tetracycline presence, in addition to a TetR independent mechanism in N. caninum . Moreover, it was possible to express Lac-Z in N. caninum enabling the development and standardization of invasion, growth and detection tachyzoite assays. It was also possible to detect through fluorescence microscopy the YFP (yellow fluorescent protein) in tachyzoites transfected with YFP fused to TetR.. The tools for gene insertion and deletion allowed the construction of vectors for hiperexpression or deletion of NcMic2-like1. For hiperexpression the vector was obtained by the replacement of the Lac-Z by the NcMic2-like1 gene. For the NcMic2-like1 deletion the vector was achieved after the ligation of its promoter and 3 \'UTR region to CAT or NcDHFRM2M3. This approach leads to the replacement of the target gene by the resistance gene through homologous recombination. Furthermore approaches for expression and purification of proteins in N. caninum were developed. Also, a soluble recombinant NcMIC2-like1 form was obtained with pET28 system added with a soluble tail amplified from a genome region of N. caninum. The development of genetic manipulation methods is an unprecedented event for N. caninum and will enable deeper molecular studies over mechanisms of invasion and replication, where NcMic2-like1 is the first candidate for these assays.

Pain induced by calcineurin inhibitors is a rare complication of unknown pathogenesis. We have reported herein a 7-year-old child who presented with abdominal pain, vomiting, and weight loss showing no significant findings after an extensive laboratory and imaging workup. After conversion from tacrolimus to sirolimus, there was complete resolution of the gastrointestinal symptoms and pain; the patient displays excellent renal function. Calcineurin inhibitor-induced pain syndrome is diagnosis of exclusion but must be considered because the withdrawal of this immunosuppressive agent is associated with improvement in symptoms and quality of life.

(unpublished). It is known that the piriform cortex and amygdala are also activated during epileptic seizures. Because of TMT biological relevance when compared with toluene, coupled to the common circuitry activation in fear and seizures, TMT may represent an interesting alternative to study if the mechanisms involved in fear generation are capable of inhibiting seizures. In the present study, our goal and first step toward seizure inhibition protocol was to verify the appropriate concentration of TMT nanoemulsion that would produce behavioral effects in rats. Methods: Nine male Wistar rats were handled for 3 consecutive days. On the third day, animals received directly into the right nasal epithelium 10 μl of 0.9% saline (SAL), and on the fourth day, animals received 10 μl of vehicle (VEH) or TMT nanoemulsion — 38.7 nmol or 300 nmol. After the olfactory exposure, animals were recorded for 2 min for behavioral analyses — ethology assessment. All behaviors and their interactions were evaluated using ETHOMATIC software (chi-square analyses) [Ethics Committee Approval (172/2010)]. Results: There was an increase of interactions between exploratory behaviors as well as between grooming behaviors in VEH when compared with SAL (p b 0.05). The TMT 38.7 nmol promoted interactions between grooming behaviors that were not detected when SAL was applied, but exploratory items showed only slight differences. The TMT 300 nmol induced an expressive reduction in all interactions between behavioral items in relation to SAL. Discussion/conclusions: The results showed that the higher concentration of TMT was the most effective because there was a reduction of the behavioral activity when compared with VEH and TMT 38.7 nmol. This is in accordance with what is expected as part of fear reactions. On the other hand, VEH raised these activities, suggesting that the VEH is not innocuous to the animal but promotes a different response than the one induced by TMT. The next step is to increase the number of animals per group to confirm behavioral data and evaluate the higher TMT concentration in the WAR strain and amygdala rapid electrical kindling.

Neospora caninum belongs to the phylum Apicomplexa, the causative agent of neosporosis, which leads to economic impacts on cattle production. A common feature among apicomplexan parasites is the invasive process driven mostly by the parasite. As a first evaluation of candidate molecules that play a possible role by interfering in this invasive process, the in vitro invasion assay is a fast and direct way to screen future agonists or antagonists. This work involved the development of a new cell culture ELISA and transient β-galactosidase activity applied to the semi-quantitative detection of N. caninum in Vero cell culture. Cell culture ELISA is based on histochemistry and immunology, resulting in a colorimetric reaction. The β-galactosidase activity was obtained by the transient transfection of the lacZ gene under control of RPS13 promoter of N. caninum. These methods were used to evaluate the effects of temperature (37°C and 85°C) on the invasion and adhesion of tachyzoites. The three tested methods (real time PCR, β-galactosidase activity and ELISA) showed a similar pattern, indicating that different methods may be complementary.