Hidrólise extracelular de nucleotídeos em trichomonas vaginalis: modulação enzimática pelo ferro e propriedades estruturais

Trichomonas vaginalis é o agente etiológico da tricomonose, a DST não viral mais comum no mundo. Os mecanismos de patogenicidade são complexos e o ferro desempenha um papel fundamental na regulação destes. O ferro modula a atividade de enzimas envolvidas na hidrólise de nucleotídeos extracelulares, como o ATP, os quais atuam como moléculas sinalizadoras na inflamação e resposta imune. Entretanto, pouco se sabe acerca desta modulação, especialmente considerando as particularidades dos isolados de T. vaginalis provenientes de pacientes de diferentes sexos. Neste contexto, o objetivo deste estudo foi investigar o efeito do ferro na atividade das enzimas NTPDase e ecto-5’-nucleotidase. Isolados provenientes de pacientes do sexo feminino (padrões e frescos) e masculinos (somente frescos) foram tratados com quelante bipiridil, sulfato ferroso, hemoglobina e hemina. Técnicas de modelagem molecular foram utilizadas para a construção do modelo 3D da NTPDase. O ferro proveniente da hemoglobina e hemina ativaram a enzima NTPDase de isolados de pacientes do sexo feminino e a inibiram em isolados de pacientes do sexo masculino. Verificou-se um perfil de hidrólise de ATP, ADP e AMP distinto entre os isolados de pacientes do sexo feminino e masculino influenciado pela hemoglobina e hemina, indicando maior remoção do ATP citotóxico e eficiência no estabelecimento da infecção em mulheres, corroborando com a exacerbação dos sintomas observada no período pós-menstrual, onde essas fontes de ferro estão disponíveis. Os resultados revelam a participação do sistema purinérgico no estabelecimento da infecção por T. vaginalis através da degradação do ATP e produção de adenosina modulada pelo ferro. Modelos 3D para a NTPDase de T. vaginalis foram construídos. Neste contexto, a NTPDase e a ecto-5’-nucleotidase podem ser consideradas potenciais alvos terapêuticos para o desenvolvimento de novos fármacos anti-T. vaginalis.Trichomonas vaginalis is the etiologic agent of trichomonosis, the most prevalent non-viral sexual transmitted disease. The pathogenesis mechanisms are complex and iron plays an important role in this regulation. Iron modulates the enzymes involved in extracellular nucleotide hydrolysis, as ATP, which act as signaling molecule in inflammation and immune response. However, few is known regarding this modulation, mainly considering the differences between T. vaginalis isolates from female and male patients. In this context, the aim of this study was to investigate the iron effect on NTPDase and ecto-5’-nucleotidase activities. T. vaginalis isolates from female (long-term grown and fresh) and male (only fresh) patients were treated with bipiridil chelator, ferrous sulfate, hemoglobin and hemin. Molecular modeling techniques were applied to build NTPDase tridimensional model. NTPDase activity was activated by hemoglobin- and hemin-treated isolates from female patients and, conversely, it was inhibited in isolates from male patients. A distinct ATP, ADP and AMP hydrolysis profile was observed between isolates from female and male patients, indicating a removal of ATP and infection establishment success in women. These findings corroborate with symptoms exacerbation observed after menstrual period, with hemoglobin and hemin availability. The results reveal the purinergic system involvement on T. vaginalis infection through ATP degradation and adenosine production modulated by iron. NTPDase 3D models were constructed. In this context, NTPDase and ecto-5’-nculeotidase may be considered potential therapeutic targets to anti-T. vaginalis new drugs development

Specific pathogenic mechanisms of mucosal protozoans : Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis

Entamoeba histolytica e Giardia lamblia são protozoários que podem parasitar a mucosa intestinal, causando principalmente diarreia. Trichomonas vaginalis coloniza a mucosa vaginal causando tricomonose, a doença sexualmente transmissível não viral mais comum no mundo. Embora coletivamente estes parasitos infectem mais de um bilhão de pessoas a cada ano, seus mecanismos de patogenicidade ainda não estão totalmente esclarecidos. Assim, esta revisão reúne os principais mecanismos envolvidos na patogenicidade destes protozoários, bem como os fatores do microambiente que podem interferir no sucesso da colonização. A patogênese da E. histolytica envolve adesão, lise, fagocitose de células epiteliais e bactérias, invasão tecidual por ação de enzimas e evasão da resposta imune do hospedeiro. A lectina Gal/GalNAc, os amebaporos e as cisteína proteases são as principais moléculas envolvidas nesses processos. O estabelecimento da giardiose depende de diversos mecanismos patogênicos e de virulência desenvolvidos pela G. lamblia, tais como as moléculas envolvidas na adesão, encistamento e variação antigênica. Para o sucesso da colonização da mucosa vaginal, o T. vaginalis expressa moléculas como as adesinas de superfície, lipofosfoglicanos e galectina, envolvidas na adesão às células epiteliais vaginais e alteração da expressão gênica, tanto do parasito como do hospedeiro.Entamoeba histolytica and Giardia lamblia are protozoans that may parasitize the intestinal mucosa, mainly causing diarrhea. Trichomonas vaginalis colonizes the vaginal mucosa causing trichomonosis, the most common non-viral sexually transmitted disease in the world. Although collectively these parasites infect over a billion people each year, their pathogenic mechanisms have not been completely understood so far. Hence, this review of the literature demonstrates the main mechanisms involved in the pathogenicity of these protozoans, as well as the microenvironmental factors that can interfere with successful colonization. The pathogenesis of E. histolytica involves adhesion, lysis, phagocytosis of epithelial cells and bacteria, tissue invasion by enzymatic action, and evasion of host immune response. Lectin Gal/GalNac, amoebapores, and cysteine proteases are the main molecules involved in these processes. The establishment of giardiosis depends on several pathogenic mechanisms and virulence developed by G. lamblia, such as molecules involved in adhesion, encystation and antigenic variation. For successful colonization of vaginal mucosa, T. vaginalis express molecules like adhesins on the surface and galectin and lipophosphoglycan, involved in the adherence to vaginal epithelial cells and altered gene expression of both the parasite and the host

Mecanismos específicos de patogenicidade de protozoários de mucosa: Entamoeba histolytica, Giardia lamblia e Trichomonas vaginalis

Entamoeba histolytica e Giardia lamblia são protozoários que podem parasitar a mucosa intestinal, causando principalmente diarreia. Trichomonas vaginalis coloniza a mucosa vaginal causando tricomonose, a doença sexualmente transmissível não viral mais comum no mundo. Embora coletivamente estes parasitos infectem mais de um bilhão de pessoas a cada ano, seus mecanismos de patogenicidade ainda não estão totalmente esclarecidos. Assim, esta revisão reúne os principais mecanismos envolvidos na patogenicidade destes protozoários, bem como os fatores do microambiente que podem interferir no sucesso da colonização. A patogênese da E. histolytica envolve adesão, lise, fagocitose de células epiteliais e bactérias, invasão tecidual por ação de enzimas e evasão da resposta imune do hospedeiro. A lectina Gal/GalNAc, os amebaporos e as cisteína proteases são as principais moléculas envolvidas nesses processos. O estabelecimento da giardiose depende de diversos mecanismos patogênicos e de virulência desenvolvidos pela G. lamblia, tais como as moléculas envolvidas na adesão, encistamento e variação antigênica. Para o sucesso da colonização da mucosa vaginal, o T. vaginalis expressa moléculas como as adesinas de superfície, lipofosfoglicanos e galectina, envolvidas na adesão às células epiteliais vaginais e alteração da expressão gênica, tanto do parasito como do hospedeiro

Callogénesis in vitro para la inducción de embriogénesis somática y producción de antioxidantes en Eugenia uniflora

Eugenia uniflora is an American tree species with ecological, agronomical and medicinal importance. However, just few studies have focused on the in vitro propagation and production of secondary metabolites. This study investigated the explant sources and culture conditions for the in vitro callogenesis in E. uniflora towards induction of somatic embryogenesis and production of antioxidant compounds. Nodal segments, leaf sections and root segments from in vitro germinated seeds were used as explants and eight different combinations of auxins (2,4-D or NAA) and cytokinins (BAP or TDZ) were tested for the callus induction. The best callogenic response was observed in nodal segments, followed by leaf sections. Root segments presented comparatively poorer callogenic performance. Calli from nodal segments cultivated in MS medium with NAA (10 mM) + TDZ (5 mM) originated putative pro-embryogenic structures, while the culture in liquid medium using MS supplemented with NAA (10 mM) + TDZ (5 mM) revealed significantly higher content of phenols and flavonoids, as well as higher reducing capacity than the tested treatments and the control (fresh leaves). In summary, the calli obtained from nodal segments revealed competence for somatic embryogenesis induction and development as well as the production of secondary metabolites with pharmaceutical potential.Eugenia uniflora es una especie arbórea americana de importancia ecológica, agronómica y medicinal. Sin embargo, pocos estudios se han centrado en la propagación y producción in vitro de metabolitos secundarios. Este estudio investigó las fuentes de explantes y las condiciones de cultivo para la callogénesis in vitro en E. uniflora para la inducción de embriogénesis somática y la producción de compuestos antioxidantes. Se utilizaron segmentos nodales, secciones de hojas y segmentos de raíz de semillas germinadas in vitro como explantes y se probaron ocho combinaciones diferentes de auxinas (2,4-D o ANA) y citoquininas (BAP o TDZ) para la formación de callos. La mejor respuesta callogénica se observó en segmentos nodales, seguidos de secciones foliares. Los segmentos de raíz presentaron un rendimiento callogénico comparativamente menor. Los callos de segmentos nodales cultivados en medio de cultivo MS con ANA (10 mM) + TDZ (5 mM) originaron estructuras proembriogénicas putativas, mientras que el cultivo en MS medio líquido con ANA (10 mM) + TDZ (5 mM) reveló un contenido significativamente mayor de fenoles y flavonoides, así como una mayor capacidad reductora que los tratamientos probados y el control (hojas frescas). En resumen, los callos obtenidos de segmentos nodales revelaron competencia para la inducción y el desarrollo de embriogénesis somática, así como para la producción de metabolitos secundarios con potencial farmacéutico

Trichomonicidal and parasite membrane damaging activity of bidesmosic saponins from Manilkara rufula

The infection caused by Trichomonas vaginalis is the most common but overlooked nonviral sexually transmitted disease worldwide. Treatment relies on one class of drugs, the 5- nitroimidazoles, but resistance is widespread. New drugs are urgently needed. We reported the effect of crude and purified saponin fractions of Manilkara rufula against Trichomonas vaginalis. The compound responsible for antitrichomonal activity was isolated and identified as an uncommon bidesmosic saponin, Mi-saponin C. This saponin eliminated parasite viability without toxicity against the human vaginal epithelial line (HMVII). In addition, the isolated saponin fraction improved the metronidazole effect against a metronidazole-resistant isolate and dramatically reduced the cytoadherence of T. vaginalis to human cells. Investigation of the mechanism of death showed that the saponin fraction induced the parasite death due to profound membrane damage, inducing a disturbance of intracellular content without nuclear damage. To the best of our knowledge, this is the first report of antitrichomonal activity in the bidesmosic saponins of Manilkara rufula

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio