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Cardiac fibrosis is a severe outcome of Chagas disease (CD), caused by the protozoan Trypanosoma cruzi. Clinical evidence revealed a correlation between fibrosis levels with impaired cardiac performance in CD patients. Therefore, we sought to analyze the effect of inhibitors of TGF-β (pirfenidone), p38-MAPK (losmapimod) and c-Jun (SP600125) on the modulation of collagen deposition in cardiac fibroblasts (CF) and in vivo models of T. cruzi chronic infection. Sirius Red/Fast Green dye was used to quantify both collagen expression and total protein amount, assessing cytotoxicity. The compounds were also used to treat C57/Bl6 mice chronically infected with T. cruzi, Brazil strain. We identified an anti-fibrotic effect in vitro for pirfenidone (TGF-β inhibitor, IC50 114.3 μM), losmapimod (p38 inhibitor, IC50 17.6 μM) and SP600125 (c-Jun inhibitor, IC50 3.9 μM). This effect was independent of CF proliferation since these compounds do not affect T. cruzi-induced host cell multiplication as measured by BrdU incorporation. Assays of chronic infection of mice with T. cruzi have shown a reduction in heart collagen by pirfenidone. These results propose a novel approach to fibrosis therapy in CD, with the prospect of repurposing pirfenidone to prevent the onset of ECM accumulation in the hearts of the patients.

Transforming growth factor beta (TGF-β) is a determinant for inflammation and fibrosis in cardiac and skeletal muscle in Chagas disease. To determine its regulatory mechanisms, we investigated the response of Trypanosoma cruzi-infected cardiomyocytes (CM), cardiac fibroblasts (CF), and L6E9 skeletal myoblasts to TGF-β. Cultures of CM, CF, and L6E9 were infected with T. cruzi (Y strain) and treated with TGF-β (1–10 ng/mL, 1 h or 48 h). Fibronectin (FN) distribution was analyzed by immunofluorescence and Western blot (WB). Phosphorylated SMAD2 (PS2), phospho-p38 (p-p38), and phospho-c-Jun (p-c-Jun) signaling were evaluated by WB. CF and L6E9 showed an increase in FN from 1 ng/mL of TGF-β, while CM displayed FN modulation only after 10 ng/mL treatment. CF and L6E9 showed higher PS2 levels than CM, while p38 was less stimulated in CF than CM and L6E9. T. cruzi infection resulted in localized FN disorganization in CF and L6E9. T. cruzi induced an increase in FN in CF cultures, mainly in uninfected cells. Infected CF cultures treated with TGF-β showed a reduction in PS2 and an increase in p-p38 and p-c-Jun levels. Our data suggest that p38 and c-Jun pathways may be participating in the fibrosis regulatory process mediated by TGF-β after T. cruzi infection.

Chagas' Disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for up to 41% of the heart failures in endemic areas in South America and is an emerging infection in regions of North America, Europe, and Asia. Treatment is suboptimal due to two factors. First, the lack of an adequate biomarker to predict disease severity and response to therapy; and second, up to 120-days treatment course coupled with a significant incidence of adverse effects from the drug currently used. Because the disease can manifest itself clinically a few years to decades after infection, controversy remains concerning the suitability of current drug treatment (benznidazole), and the efficacy of alternative drugs (e.g. posaconazole). We therefore followed the clinical course, and PCR detection of parasite burden, in a mouse model of infection for a full year following treatment with benznidazole or posaconazole. Efficacy of the two drugs depended on whether the treatment was performed during the acute model or the chronic model of infection. Posaconazole was clearly superior in treatment of acute disease whereas only benznidazole had efficacy in the chronic model. These results have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings.

Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.

Transforming growth factor beta (TGF-β) cytokine is involved in Chagas disease establishment and progression. Since Trypanosoma cruzi can modulate host cell receptors, we analysed the TGF-β receptor type II (TβRII) expression and distribution during T. cruzi – cardiomyocyte interaction. TβRII immunofluorescent staining revealed a striated organization in cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-β treatment. Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting TβRII striations, demonstrating an association of TβRII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibited Smad 2 phosphorylation and fibronectin stimulation after TGF-β treatment in cardiomyocytes. Trypanosoma cruzi infection elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting TβRII striations, but did not interfere significantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-β, showing no enhancement of TβRII striations and a reduction of phosphorylated Smad 2, with no significant difference in TβRII expression when compared to uninfected cells. Together, these results suggest that the co-localization of TβRII with costameres is important in activating the TGF-β signalling cascade, and that T. cruzi-derived cytoskeleton disorganization could result in altered or low TGF-β response in infected cardiomyocytes.

Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell cycle is therefore key to their pathogenesis. Phosphorylation is a crucial posttranslational modification that regulates many aspects of the eukaryotic cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii . Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the proper assembly of the daughter cells’ inner membrane complex (IMC), the segregation of organelles, and nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is usually found in the latent bradyzoite form of the parasite. Using transcriptomics and phospho-proteomics, we show that TgPP1 mainly acts through posttranslational mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylates a protein bearing a starch-binding domain. Mutagenesis analysis reveals that the targeted phospho-sites are linked to the ability of the parasite to regulate amylopectin steady-state levels. Therefore, we show that TgPP1 has pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.

ABSTRACT The rate of swine wastewater application (SW) in agricultural production could result in the replacement of chemical fertilizers. However, SW destroys soil physical properties by decreasing pore bulk, which negatively affects both crop yield and development. In this context, this study aimed at monitoring the influence of swine wastewater as a cover fertilizer in maize and black oats in sequence on soil physical properties. Five application rates (0, 100, 200, 300 and 537 m3ha - 1 equivalent to 0, 11.2, 22.3, 33.5 and 60 kg ha-1 N, respectively, based on the average nitrogen concentration in SW) were tested with four replications each. In the studied area, soil porosity, density, and water content, before maize sowing and at the end of the cycles of maize and black oats, were determined by the volumetric ring method. Data were submitted for regression analyses. There was a reduction in the macroporosity and total porosity of the soil when the SW application rate, before maize cultivation, was higher. The introduction of black oats helped to improve the physical quality of the soil and reduced the compaction of the surface layer from 0 to 15 cm and 100 to 300 m3 ha-1 SW rates. RESUMO As taxas de aplicação de águas residuárias de suinocultura (ARS) utilizadas na produção agrícola podem substituir a adubação convencional, mas também podem prejudicar atributos físicos do solo pela diminuição do volume de poros, e consequentemente o rendimento e desenvolvimento das culturas. Neste contexto, o objetivo deste trabalho foi monitorar a influência do uso da água residuária de suinocultura como adubação de cobertura na cultura do milho e aveia preta em sequência sobre atributos físicos do solo. Cinco taxas de aplicação (0; 100; 200; 300 e 537 m3ha-1 sendo equivalentes a 0; 11,2; 22,3; 33,5 e 60 kg ha-1 de N, respectivamente, baseado na concentração média de N na ARS) foram testadas, com quatro repetições cada. No solo, foram determinados porosidade do solo, densidade, teor de água antes da semeadura do milho e ao final dos ciclos do milho e aveia preta, pelo método do anel volumétrico. Os dados foram submetidos a análises de regressão. Houve redução da macroporosidade e porosidade total do solo quanto maior a taxa de aplicação na cultura do milho. A introdução da aveia preta contribuiu para melhorar a qualidade física do solo e reduziu a compactação na camada superficial de 0-15 cm, para taxas de ARS de100 a 300 m3 ha-1.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life.

Among healthcare workers, nurses are at exceptionally considerable risk for contracting COVID-19. Regardless of professionals’ level of education, Brazilian nursing is one of the healthcare occupations shouldering the highest levels of responsibility and workload. Social support networks to health and nursing can be a strategy to reduce workload and stress and may contribute to implementing the activities and protecting workers’ health. This study aimed to map and analyze social support networks at workplaces as informed by frontline nursing professionals working in healthcare units in the Brazilian cities of Rio de Janeiro and Fortaleza, capitals of the States of Rio de Janeiro and Ceará, respectively. This observational and cross-sectional study used an online data collection instrument based on social network analysis methodology. We recruited 163 participants in two reference services for health professionals suspected or with COVID-19 symptoms. The research question was: “Which category or categories of health professionals have supported you or other colleagues the most in the event of diagnosis or suspicion of COVID-19 among nursing categories? Data were organized by nursing category and city and analyzed through social network analysis using Ucinet©, generating graphs and centrality metrics. Results point to the central relevance of nursing categories in the workplace social support in the pandemic, followed by other health professional categories.

SUMMARY Transforming growth factor beta (TGF-[...]) cytokine is involved in Chagas disease establishment and progression. Since Trypanosoma cruzi can modulate host cell receptors, we analysed the TGF-[...]receptor type II (T[...]RII) expression and distribution during T. cruzi - cardiomyocyte interaction. T[...]RII immunofluorescent staining revealed a striated organization in cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-[...]treatment. Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting T[...]RII striations, demonstrating an association of T[...]RII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibited Smad 2 phosphorylation and fibronectin stimulation after TGF-[...]treatment in cardiomyocytes. Trypanosoma cruzi infection elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting T[...]RII striations, but did not interfere significantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-[...], showing no enhancement of T[...]RII striations and a reduction of phosphorylated Smad 2, with no significant difference in T[...]RII expression when compared to uninfected cells. Together, these results suggest that the co-localization of T[...]RII with costameres is important in activating the TGF-[...]signalling cascade, and that T. cruzi-derived cytoskeleton disorganization could result in altered or low TGF-[...]response in infected cardiomyocytes.