The dispersant Corexit 9500 and (dispersed) oil are lethal to coral endosymbionts.

Endosymbionts (Symbiodiniaceae) play a vital role in the health of corals. Seawater pollution can harm these endosymbionts and dispersants used during oil spill cleanup can be extremely toxic to these organisms. Here, we examined the impact of oil and a specific dispersant, Corexit-9500, on two representative endosymbionts - Symbiodinium and Cladocopium - from the Southwestern endemic coral Mussismilia braziliensis. The survival and photosynthetic potential of the endosymbionts decreased dramatically after exposure to the dispersant and oil by ~25 % after 2 h and ~50 % after 7 days. Low concentrations of dispersant (0.005 ml/l) and dispersed oil (Polycyclic Aromatic Hydrocarbons, 1132 µg/l; Total Petroleum Hydrocarbons, 595 µg/l) proved highly toxic to both Symbiodinium and Cladocopium. These levels triggered a reduction in growth rate, cell size, and cell wall thickness. After a few hours of exposure, cellular organelles were damaged or destroyed. These acute toxic effects underline the fragile nature of coral endosymbionts.

Mpox outbreak in Rio de Janeiro, Brazil: A translational approach.

Mpox is a zoonotic disease historically reported in Africa. Since 2003, limited outbreaks have occurred outside Africa. In 2022, the global spread of cases with sustained interhuman transmission and unusual disease features raised public health concerns. We explore the mpox outbreak in Rio de Janeiro (RJ) state, Brazil, in an observational study of mpox-suspected cases from June to December 2022. Data collection relied on a public healthcare notification form. Diagnosis was determined by MPXV-PCR. In 46 confirmed cases, anti-OPXV IgG was determined by ELISA, and seven MPXV genomes were sequenced. A total of 3095 cases were included, 816 (26.3%) with positive MPXV-PCR results. Most positive cases were men in their 30 s and MSM. A total of 285 (34.9%) MPXV-PCR+ patients live with HIV. Eight were coinfected with varicella-zoster virus. Anogenital lesions and adenomegaly were associated with the diagnosis of mpox. Females and individuals under 18 represented 9.4% and 5.4% of all confirmed cases, respectively, showing higher PCR cycle threshold (Ct) values and fewer anogenital lesions compared to adult men. Anti-OPXV IgG was detected in 29/46 (63.0%) patients. All analyzed sequences belonged to clade IIb. In RJ state, mpox presented a diverse clinical picture, represented mainly by mild cases with low complication rates and prominent genital involvement. The incidence in females and children was higher than usually reported. The observation of a bimodal distribution of Ct values, with few positive results, may suggest the need to review the diagnostic criteria in these groups.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

Como melhorar a mobilidade ativa em São Paulo, Brasil? Inquérito com lideranças de organizações não governamentais e com gestores públicos e privados / How to improve active mobility in São Paulo, Brazil? Survey with leaders of nongovernmental organizations and public and private sector managers / ¿Cómo mejorar la movilidad activa en São Paulo, Brasil? Encuesta con líderes de organizaciones no gubernamentales y gestores públicos y privados

Resumo: Este estudo teve como objetivo descrever um inquérito quantitativo realizado com lideranças para investigar ações efetivas, viáveis e que podem ser testadas em modelos computacionais para informar políticas de promoção da mobilidade ativa, tendo como base a cidade de São Paulo, Brasil. Em 2022, foi realizado um inquérito online no contexto da pesquisa de Atividade Física e Ambiente do Inquérito de Saúde de São Paulo, acompanhada por representantes de organizações não governamentais, gestores públicos e de entidades privadas. Foi elaborado questionário com três perguntas com 13 alternativas de respostas sobre ações para promoção da caminhada ou uso de bicicleta. As lideranças deveriam selecionar até três alternativas a partir de seu potencial em termos de (1) efetividade; (2) viabilidade ou facilidade de implementação; e (3) desejo de realizar testes em modelos computacionais para informar políticas. O inquérito foi respondido por 18 lideranças de 16 instituições, sendo 13 (72%) mulheres e 12 (67%) representantes do terceiro setor, cuja média de idade era 48 anos, todos com nível superior de escolaridade. A redução da velocidade dos veículos motorizados foi a opção mais citada nas três questões. Outras ações citadas referem-se ao controle de circulação de veículos em regiões centrais, à segurança de pedestres, à diminuição das distâncias entre residências e locais de emprego, às campanhas educativas e à ampliação e melhoria de estruturas como ciclovias e calçadas. Os resultados são relevantes para apoiar a tomada de decisões baseadas em evidências na gestão pública e oferecer subsídios para a elaboração de modelos computacionais com vistas à promoção da mobilidade ativa.

Abstract: This study aimed to describe a quantitative survey conducted with leaders to investigate effective and feasible actions that can be evaluated in computational models to inform policies to promote active mobility based in the city of São Paulo, Brazil. In 2022, an online survey was conducted during the Health Survey in São Paulo (Physical Activity and Environment study), which is monitored by representatives of nongovernmental organizations and public and private sector managers. A questionnaire was elaborated with three questions with 13 alternative answers about actions to promote walking and/or cycling. Leaders should select up to three alternatives based on their potential regarding: (1) effectiveness; (2) feasibility or ease of implementation; and (3) desire to verify tests in computational models to inform policies. The survey was answered by 18 leaders from 16 institutions, comprising 13 (72%) women and 12 (67%) representatives of the third sector, whose average age was 48 years and all had complete higher education. Reducing the speed of motor vehicles was the most cited option in all three questions. Other actions mentioned refer to controlling the traffic of vehicles in central areas, improving pedestrian safety, reducing the distances between homes and places of employment, conducting educational campaigns, and expanding and enhancing structures such as bicycle lanes and sidewalks. The results are relevant to support evidence-based decision-making in public management and to provide subsidies for the development of computational models with a view to promoting active mobility.

Resumen: Este estudio tuvo como objetivo presentar una encuesta cuantitativa realizada con líderes para investigar las acciones efectivas, viables y que puedan probarse en modelos informáticos para orientar las políticas que promuevan la movilidad activa en la ciudad de São Paulo, Brasil. En 2022 se realizó una encuesta en línea en el contexto de la Encuesta de Salud de São Paulo (Actividad Física y Medio Ambiente), que es monitoreada por representantes de organizaciones no gubernamentales, gestores públicos y entidades privadas. Se elaboró un cuestionario de tres preguntas con 13 respuestas alternativas sobre acciones para promover la caminata o el uso de la bicicleta. Los líderes podían seleccionar hasta tres alternativas en función de su potencial en términos de (1) efectividad; (2) viabilidad o facilidad de implementación; y (3) deseo de verificar las pruebas en modelos informáticos para orientar las políticas. La encuesta fue respondida por 18 líderes de 16 instituciones; de los cuales 13 (72%) eran mujeres y 12 (67%) representaban el tercer sector; la edad promedio de ellos fue de 48 años y todos contaban con educación superior. Reducir la velocidad de los vehículos de motor fue la opción más citada en las tres preguntas. Otras acciones mencionadas se refieren al control de la circulación de vehículos en las regiones centrales, la seguridad de los peatones, la reducción de las distancias entre los hogares y los lugares de trabajo, las campañas educativas y la expansión y mejora de estructuras como carriles bici y aceras. Los resultados son relevantes para apoyar la toma de decisiones basada en la evidencia en la gestión pública y ofrecer subsidios para la elaboración de modelos computacionales destinados a promover la movilidad activa.

Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Giardia intestinalis and its endomembrane system.

Giardia intestinalis has unique characteristics, even in the absence of certain organelles. For instance, Golgi and mitochondria are not found. On the other hand, there is a network of peripheral vacuoles (PVs) and mitosomes. The endoplasmic reticulum (ER), nuclear membrane, peroxisomes, and lipid bodies are present. The peripheral vacuole system seems to play several simultaneous roles. It is involved in the endocytic activity of the trophozoite but also has characteristics of early and late endosomes and even lysosomes, establishing a connection with the ER. Some of the PVs contain small vesicles, acting as multivesicular bodies, including the release of exosomes. The mitosomes are surrounded by two membranes, divide during mitosis, and are distributed throughout the cell. They do not contain DNA, enzymes involved in the citric acid cycle, respiratory chain, or ATP synthesis. However, they contain the iron-sulfur complex and transporters as TOM and TIM. Some mitosomes are linked to flagellar axonemes through a fibrillar connection. During encystation, two types of larger cytoplasmic vesicles appear. One originating from the ER contains the cyst wall proteins. Another contains carbohydrates. Both migrate to the cell periphery and fuse with plasma membrane secreting their contents to give rise to the cell wall.

Morphological and biochemical repercussions of Toxoplasma gondii infection in a 3D human brain neurospheres model.

BACKGROUND: Toxoplasmosis is caused by the parasite Toxoplasma gondii that can infect the central nervous system (CNS), promoting neuroinflammation, neuronal loss, neurotransmitter imbalance and behavioral alterations. T. gondii infection is also related to neuropsychiatric disorders such as schizophrenia. The pathogenicity and inflammatory response in rodents are different to the case of humans, compromising the correlation between the behavioral alterations and physiological modifications observed in the disease. In the present work we used BrainSpheres, a 3D CNS model derived from human pluripotent stem cells (iPSC), to investigate the morphological and biochemical repercussions of T. gondii infection in human neural cells. METHODS: We evaluated T. gondii ME49 strain proliferation and cyst formation in both 2D cultured human neural cells and BrainSpheres. Aspects of cell morphology, ultrastructure, viability, gene expression of neural phenotype markers, as well as secretion of inflammatory mediators were evaluated for 2 and 4 weeks post infection in BrainSpheres. RESULTS: T. gondii can infect BrainSpheres, proliferating and inducing cysts formation, neural cell death, alteration in neural gene expression and triggering the release of several inflammatory mediators. CONCLUSIONS: BrainSpheres reproduce many aspects of T. gondii infection in human CNS, constituting a useful model to study the neurotoxicity and neuroinflammation mediated by the parasite. In addition, these data could be important for future studies aiming at better understanding possible correlations between psychiatric disorders and human CNS infection with T. gondii.

Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids.

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure-activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 µM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

Tubastatin A, a deacetylase inhibitor, as a tool to study the division, cell cycle and microtubule cytoskeleton of trypanosomatids.

Trypanosoma cruzi is a protozoan of great medical interest since it is the causative agent of Chagas disease, an endemic condition in Latin America. This parasite undergoes epigenetic events, such as phosphorylation, methylation and acetylation, which play a role in several cellular processes including replication, transcription and gene expression. Histone deacetylases (HDAC) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. Tubastatin A (TST) is a specific HDAC6 inhibitor that affects cell growth and promotes structural modifications in cancer cells and parasites. In the present study, we demonstrated that T. cruzi epimastigote cell proliferation and viability are reduced after 72 h of TST treatment. The results obtained through different microscopy methodologies suggest that this inhibitor impairs the polymerization dynamics of cytoskeleton microtubules, generating protozoa displaying atypical morphology and cellular patterns that include polynucleated parasites. Furthermore, the microtubules of treated protozoa were more intensely acetylated, especially at the anterior portion of the cell body. A cell cycle analysis demonstrated an increase in the number of trypanosomatids in the G2/M phase. Together, our results suggest that TST should be explored as a tool to study trypanosomatid cell biology, including microtubule cytoskeleton dynamics, and as an antiparasitic drug.

In vitro study of the trypanocidal activity of anilinophenanthrolines against Trypanosoma cruzi.

Chagas disease is present in Latin America, North America, Europe, and Asia, where between 6 and 7 million people are infected. This illness is transmitted mainly by the insect vector during blood feeding and by oral transmission. Chagas disease is treated with benznidazole and its effectiveness depends on which phase of the disease the treatment starts. Therefore, the identification of new compounds with anti-Chagas activities is important. Protozoan parasites present cysteine proteases, important for host cell infection and differentiation, which have been explored as valid targets against pathogenic parasites. In the present study, the effects of 10 new 1,10-phenanthroline derivatives were evaluated on T. cruzi. Three of them were effective against amastigotes (IC50 from 0.5 to 3 µM), epimastigotes (IC50 from 0.5 to at least 10 µM) and trypomastigotes (and LD50 from 1 to 10 µM), and they were not toxic to mammalian cells (CC50 ≥ 20 µM). These compounds also promoted the formation of autophagosomes, alter the level of heterochromatin condensation, caused the loss of kDNA topology, and the elongated cell body shape. Apart from ultrastructural alterations, an increased generation of ROS and decreased mitochondrial membrane potential were observed. Therefore, these drugs revealed potential trypanocidal effects and warrant further antiparasitic studies against Chagas disease.

Furan derivatives impair proliferation and affect ultrastructural organization of Trypanosoma cruzi and Leishmania amazonensis.

Chagas disease and leishmaniasis are neglected diseases caused by parasites of the Trypanosomatidae family and together they affect millions of people in the five continents. The treatment of Chagas disease is based on benznidazole, whereas for leishmaniasis few drugs are available, such as amphotericin B and miltefosine. In both cases, the current treatment is not entirely efficient due to toxicity or side effects. Encouraged by the need to discover valid targets and new treatment options, we evaluated 8 furan compounds against Trypanosoma cruzi and Leishmania amazonensis, considering their effects against proliferation, infection, and ultrastructure. Many of them were able to impair T. cruzi and L. amazonensis proliferation, as well as cause ultrastructural alterations, such as Golgi apparatus disorganization, autophagosome formation, and mitochondrial swelling. Taken together, the results obtained so far make these compounds eligible for further steps of chemotherapy study.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain.

BACKGROUND: Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-ß signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES: To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS: Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS: Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS: These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Antinociceptive and anti-inflammatory activities of Copaifera pubiflora Benth oleoresin and its major metabolite ent-hardwickiic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera species folkloric names are "copaíbas, copaibeiras, copaívas or oil stick", which are widely used in Brazilian folk medicine. Among all ethnopharmacological applications described for Copaifera spp oleoresins, their anti-inflammatory effect stands out. However, the knowledge of anti-inflammatory and antinociceptive properties of Copaifera pubiflora Benth is scarce. AIM OF THE STUDY: To investigate the cytotoxic, anti-inflammatory, and antinociceptive activities of C. pubiflora oleoresin (CPO), and its major compound ent-hardwickiic acid (HA). MATERIAL AND METHODS: The phosphatase assay was used to evaluate the cytotoxicity of CPO and HA in three different cell lines. CPO and HA doses of 1, 3, and 10 mg/kg were employed in the biological assays. The assessment of motor activity was performed using open-field and rotarod tests. Anti-inflammatory activity of CPO and HA was assessed through luciferase assay, measurement of INF-γ, IL-1ß, IL-6, IL-10, and TNF-α in a multi-spot system with the immortalized cell line THP-1, zymosan-induced arthritis, and carrageenan-induced paw edema. Acetic acid-induced abdominal writhing and formalin tests were undertaken to evaluate the antinociceptive potential of CPO and HA. In addition, the evaluation using carrageenan was performed to investigate the effect of CPO in pain intensity to a mechanical stimulus (mechanical hyperalgesia), using the von Frey filaments. A tail-flick test was used to evaluate possible central CPO and HA actions. RESULTS: In the cytotoxicity evaluation, CPO and HA were not cytotoxic to the cell lines tested. CPO and HA (10 mg/kg) did not affect animals' locomotor capacity in both open-field and rotarod tests. In the luciferase assay, CPO and HA significantly reduced luciferase activity (p < 0.05). This reduction indicates a decrease in NF-κB activity. HA and CPO decreased INF-γ, IL-1ß, IL-6, IL-10, and TNF-α at 24 and 72 h in the multi-spot system. In zymosan-induced arthritis, CPO and HA decreased the number of neutrophils in the joint of arthritic mice and the number of total leukocytes (p < 0.05). In experimental arthritis HA significantly decreased joint swelling (p < 0.05). CPO and HA also increased the mechanical threshold during experimental arthritis. HA and CPO significantly inhibited the carrageenan-induced paw edema, being the doses of 10 mg/kg the most effective, registering maximum inhibitions of 58 ± 8% and 76 ± 6% respectively, p < 0.05. CPO and HA reduced the nociceptive behavior in both phases of formalin at all tested doses. The highest doses tested displayed inhibitions of 87 ± 1% and 72 ± 4%, respectively, p < 0.001, in the first phase, and 87 ± 1% and 81 ± 2%, respectively, p < 0.001, in the second phase. Oral treatment of CPO and HA (1, 3, 10 mg/kg) significantly reduced the nociceptive response in acetic acid-induced abdominal writhings, and the 10 mg/kg dose was the most effective with maximum inhibitions of 86 ± 2% and 82 ± 1%, respectively, p < 0.001. Both HA and CPO significantly decreased the intensity of mechanical inflammatory hyper-nociception on carrageenan-induced hyperalgesia at all tested doses, and 10 mg/kg was the most effective dose with maximum inhibitions of 73 ± 5% and 74 ± 7%, respectively, p < 0.05.CPO increased the tail-flick latencies in mice, and concomitant administration of naloxone partially reduced its effect. CONCLUSIONS: CPO and HA may inhibit the production of inflammatory cytokines by suppressing the NF-κB signaling pathway, resulting in anti-inflammatory and antinociceptive activities.

Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

HDAC inhibitors Tubastatin A and SAHA affect parasite cell division and are potential anti-Toxoplasma gondii chemotherapeutics.

The redirectioning of drugs in the pharmaceutical market is a well-known practice to identify new therapies for parasitic diseases. The histone deacetylase inhibitors Tubastatin A (TST) and Suberoylanilide Hydroxamic Acid (SAHA), firstly developed for cancer treatment, are effective against protozoa parasites. In this work, we aimed to demonstrate the activity of these drugs as potential agents against Toxoplasma gondii, the causative agent of toxoplasmosis. TST and SAHA were active against different genotypes of Toxoplasma gondii, such as, RH (type I), EGS (I/III) and ME49 (type II) strains. The IC50 values for the RH strain were 19 ± 1 nM and 520 ± 386 nM for TST and 41 ± 3 nM and 67 ± 36 nM for SAHA, for 24 and 48 h, respectively. Both compounds were highly selective for T. gondii and their anti-proliferative effect was irreversible for 8 days. The calculated selectivity indexes (39 for TST and 30 for SAHA) make them lead compounds for the future development of anti-Toxoplasma molecules. Western blotting showed TST led to a significant increase of the nuclear histone H4 and a decrease of H3 acetylation levels. Treatment with 1 µM TST and 0.1 µM SAHA for 48 h decreased the amount of global α-tubulin. Fluorescence and electron microscopy showed that both drugs affected the endodyogeny process impairing the budding of daughter cells. The drugs led to the formation of large, rounded masses of damaged parasites with several centrosomes randomly dispersed and incorrect apicoplast division and positioning. TST-treated parasites showed a rupture of the mitochondrial membrane potential and led to a failure of the IMC assembling of new daughter cells. SAHA and TST possibly inhibit HDAC3 and other cytoplasmic or organelle targeted HDACs involved in the modification of proteins other than histones.

Cellular toxicology and mechanism of the response to silver-based nanoparticle exposure in Ewing's sarcoma cells.

Ewing's sarcoma is the most aggressive connective tissue tumor, mainly affecting children and adolescents; the 5 year survival rate is only 50%. Current treatments have poor effectiveness, and more efficient treatments are being sought. Silver-based nanoparticles, such as silver chloride nanoparticles (AgCl-NPs) and silver/silver chloride (Ag/AgCl-NPs) nanoparticles, can be biologically produced and can release Ag+ ions into solution; however, their antitumor activity has been minimally investigated. The aim of this study was to evaluate the antitumor potential of AgCl-NPs and Ag/AgCl-NPs against Ewing's sarcoma cells. A673 cells (Ewing's sarcoma) were treated for 72 h with 0-12.5 µg ml-1 of Ag/AgCl-NPs or 0-40 µg ml-1 of AgCl-NPs. Human cells from the RPE-1 cell line (pigmented retinal epithelium) were used as a model of nontumor cells. The RPE-1 cells were less affected by the administration of AgCl-NPs or Ag/AgCl-NPs, with small reductions in the number of cells and viability and a small increase in apoptosis rates, while lysosomal damage, changes in reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and alterations in microfilaments or cell areas were not observed. A673 tumor cells had significantly reduced number and viability levels when treated with AgCl-NPs, with reductions of 65.05% and 99.17%, respectively, whereas with Ag/AgCl-NP treatment, reductions of 65.53% and 92.51% were observed, respectively. When treated with silver-based nanoparticles, A673 cells also showed a significant increase in ROS production and loss of mitochondrial membrane potential, which culminated in an increase in the percentage of apoptosis among the population. Lysosomal damage was also observed when A673 cells were treated with the highest concentration of AgCl-NPs. In conclusion, the results showed that both AgCl-NPs and Ag/AgCl-NPs had some antitumor activity with minimal effects against healthy cells, which demonstrated the possibility of their use in cancer therapy.

Treatment with melatonin induces a reduction of Toxoplasma gondii development in LLC-MK2 cells.

It is known that the current treatment for toxoplasmosis causes side effects. Thus, it is essential to develop new therapies with reduced adverse effects while concurrently maintaining broad coverage and prophylactic therapy. Melatonin is a hormone that participates in the circadian cycle in vertebrates and has antioxidant, immunomodulatory, and antitumoral functions. In addition, it has been shown that melatonin can modulate immune responses and parasitic development during infection by Trypanosoma cruzi and Leishmania spp. Furthermore, studies indicate that melatonin increases the number of lymphocytes in rats infected by Toxoplasma gondii. However, there is no information on the possible effects of melatonin in T. gondii-infected host cells in vitro. This study analyzed the effects of melatonin treatment in the monkey kidney cell epithelial cell line, LLC-MK2, after infection with T. gondii. LLC-MK2 cells were infected and treated/not treated with melatonin, and the infection index was then quantified. Melatonin treatment did not alter host cell viability and was able to reduce parasite proliferation in LLC-MK2 cells at 24 and 48 h and at 6 days. Analysis by scanning electron microscopy confirmed reduction of parasite proliferation and alterations of tachyzoite shapes. Transmission electron microscopy images showed parasites with ruptured plasma membranes and cytoplasmic leakage. After treatment, parasites showed positive staining for apoptotic-like cell death. These results suggest that the use of melatonin as the lead compound for the synthesis of new compounds may constitute an alternative treatment for toxoplasmosis.

Microscopy analysis of Zika virus morphogenesis in mammalian cells.

Zika virus (ZIKV) is an arbovirus that recently emerged in the Americas as an important pathogen mainly because of its expanded pathogenesis, and elevated tropism for neuronal cells, transposition across the placental barrier, and replication in reproductive tract cells. Thus, transmission modes are eventually independent of an invertebrate vector, which is an atypical behavior for the flavivirus genus and indicates the need to study the replication of this virus in different cell types. Although ZIKV became a target for public health programs, the interaction of this flavivirus with the infected cell is still poorly understood. Herein, we analyzed the main stages of virus morphogenesis in mammalian cells, from establishment of the viroplasm-like zone to viral release from infected cells, using super-resolution fluorescence microscopy and electron microscopy. In addition, we compared this with other host cell types and other members of the Flaviviridae family that present a similar dynamic.

Silencing of RpATG8 impairs the biogenesis of maternal autophagosomes in vitellogenic oocytes, but does not interrupt follicular atresia in the insect vector Rhodnius prolixus.

Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia-induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus.

In vitro activities of adamantylidene-substituted alkylphosphocholine TCAN26 against Trypanosoma cruzi: Antiproliferative and ultrastructural effects.

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.