Trypanocidal and Anti-Inflammatory Effects of Three ent-Kaurane Diterpenoids from Gymnocoronis spilanthoides var. subcordata (Asteraceae).

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects 6-7 million people worldwide. The dichloromethane extract obtained from the aerial parts of Gymnocoronis spilanthoides var subcordata showed trypanocidal activity in vitro. The fractionation of the dewaxed organic extract via column chromatography led to the isolation of three diterpenoids: ent-9α,11α-dihydroxy-15-oxo-kaur-16-en-19-oic acid or adenostemmoic acid B, (16R)-ent-11α-hydroxy-15-oxokauran-19-oic acid and ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid. These compounds showed IC50 values of 10.6, 15.9 and 4.8 µM against T. cruzi epimastigotes, respectively. When tested against amastigotes, the diterpenoids afforded IC50 values of 6.1, 19.5 and 60.6 µM, respectively. The cytotoxicity of the compounds was tested on mammalian cells using an MTT assay, resulting in CC50s of 321.8, 23.3 and 14.8 µM, respectively. The effect of adenostemmoic acid B on T. cruzi was examined at the ultrastructural level using transmission microscopy. Treatment with 20 µM for 48 h stimulated the formation of abnormal cytosolic membranous structures in the parasite. This compound also showed an anti-inflammatory effect in murine macrophages stimulated with LPS and other TLR agonists. Treatment of macrophages with adenostemmoic acid B was able to reduce TNF secretion and nitric oxide production, while increasing IL-10 production. The combination of adenostemmoic acid B with benznidazole resulted in greater inhibition of NF-kB and a decrease in nitrite concentration. The administration of adenostemmoic acid B to mice infected with trypomastigotes of T. cruzi at the dose of 1 mg/kg/day for five days produced a significant decrease in parasitemia levels and weight loss. Treatment with the association with benznidazole increased the survival time of the animals. In view of these results, adenostemmoic acid B could be considered a promising candidate for further studies in the search for new treatments for Chagas disease.

High-Resolution Ultrasound for Diagnosis and Treatment of Filler-Related Septal Necrosis.

Nonsurgical rhinoplasty using filler injections is a popular procedure; however, it carries risks of significant complications, including vascular occlusion, skin necrosis, blindness, and cerebral ischemia. We present a case of septal necrosis after nonsurgical rhinoplasty and describe management using ultrasound guidance. Awareness of this complication and high clinical index of suspicion are necessary for early diagnosis and treatment, crucial to minimizing permanent sequelae. We show that high-definition ultrasound can help to guide the management and improve the treatment outcomes.

Prácticas de crianza en alimentación, conducta alimentaria y riesgo de diabetes: Una revisión sistemática / Parenting practices in feeding, eating behavior and risk of diabetes: A systematic review

INTRODUCCIÓN: Las prácticas de crianza en alimentación y la conducta alimentaria han sido ampliamente estudiadas en niños, sin embargo, es necesario proporcionar información sobre su impacto en adultos. La evidencia sugiere que las prácticas de crianza en alimentación pueden tener un impacto en la conducta alimentarias emocional, descontrolada, restrictiva y desordenada, las cuales están asociadas con el incremento del Índice de masa corporal (IMC), el sobrepeso y la obesidad, mismos que se encuentran como principal factor de riesgo para el desarrollo de DT2. OBJETIVO: analizar través de una revisión sistemática la evidencia existente acerca de la relación entre las prácticas de crianza en alimentación, la conducta alimentaria y el riesgo de diabetes en adultos. METODOLOGÍA: Se realizó una búsqueda de literatura publicada de 2013 a 2023 en las bases de datos Pubmed, Scopus, EBSCOhost, Clarivate Science Citation Index Expanded y SpringerLink. Se identificaron 459 estudios, luego de aplicar los criterios de exclusión, se revisaron 15 estudios en total. RESULTADOS: Las subescalas de las prácticas de crianza en alimentación más empleadas fueron: preocupación, control, presión para comer y restricción. Se encontró asociación entre las prácticas de crianza en alimentación, la conducta alimentaria y el incremento del IMC. CONCLUSIÓN: Se consideran insuficientes las investigaciones que muestran el impacto de las prácticas de crianza en alimentación sobre la conducta alimentaria y si estas a su vez tienen efectos en el riesgo de diabetes en la etapa adulta.

INTRODUCTION: Parenting practices in feeding and eating behavior have been widely studied in children, however, it is necessary to provide information on their impact on adults. The evidence suggests that parenting practices in feeding can have an impact on emotional, uncontrolled, restrictive and disordered eating behavior, which are associated with an increase in BMI, overweight and obesity, which are found as the main factor of risk for the development of T2D. OBJECTIVE: To analyze, through a systematic review, the existing evidence about the relationship between parenting feeding practices, eating behavior and the risk of diabetes in adults. METHODOLOGY: A search of literature published from 2013 to 2023 was carried out in the Pubmed, Scopus, EBSCOhost, Clarivate Science Citation Index Expanded and SpringerLink databases. 459 studies were identified, after applying the exclusion criteria, 15 studies in total were reviewed. RESULTS: The subscales of the most frequently used parenting practices in feeding were: concern, control, pressure to eat and restriction. An association was found between parenting feeding practices, eating behavior and increased BMI. CONCLUSION: Research showing the impact of parenting feeding practices on eating behavior and whether these in turn have effects on the risk of diabetes in adulthood are considered insufficient.

Direct mosquito feedings on dengue-2 virus-infected people reveal dynamics of human infectiousness.

Dengue virus (DENV) transmission from humans to mosquitoes is a poorly documented, but critical component of DENV epidemiology. Magnitude of viremia is the primary determinant of successful human-to-mosquito DENV transmission. People with the same level of viremia, however, can vary in their infectiousness to mosquitoes as a function of other factors that remain to be elucidated. Here, we report on a field-based study in the city of Iquitos, Peru, where we conducted direct mosquito feedings on people naturally infected with DENV and that experienced mild illness. We also enrolled people naturally infected with Zika virus (ZIKV) after the introduction of ZIKV in Iquitos during the study period. Of the 54 study participants involved in direct mosquito feedings, 43 were infected with DENV-2, two with DENV-3, and nine with ZIKV. Our analysis excluded participants whose viremia was detectable at enrollment but undetectable at the time of mosquito feeding, which was the case for all participants with DENV-3 and ZIKV infections. We analyzed the probability of onward transmission during 50 feeding events involving 27 participants infected with DENV-2 based on the presence of infectious virus in mosquito saliva 7-16 days post blood meal. Transmission probability was positively associated with the level of viremia and duration of extrinsic incubation in the mosquito. In addition, transmission probability was influenced by the day of illness in a non-monotonic fashion; i.e., transmission probability increased until 2 days after symptom onset and decreased thereafter. We conclude that mildly ill DENV-infected humans with similar levels of viremia during the first two days after symptom onset will be most infectious to mosquitoes on the second day of their illness. Quantifying variation within and between people in their contribution to DENV transmission is essential to better understand the biological determinants of human infectiousness, parametrize epidemiological models, and improve disease surveillance and prevention strategies.

Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses (lineage 2.3.4.4b) are rapidly invading the Americas, threatening wildlife, poultry, and potentially evolving into the next global pandemic. In November 2022 HPAI arrived in Peru, triggering massive pelican and sea lion die-offs. We report genomic characterization of HPAI/H5N1 in five species of marine mammals and seabirds (dolphins, sea lions, sanderlings, pelicans and cormorants). Peruvian viruses belong to lineage 2.3.4.4b, but they are 4:4 reassortants where 4 genomic segments (PA, HA, NA and MP) position within the Eurasian lineage that initially entered North America from Eurasia, while the other 4 genomic segments (PB2, PB1, NP and NS) position within the American lineage (clade C) that circulated in North America. These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.

Candimine from Hippeastrum escoipense (Amaryllidaceae): Anti-Trypanosoma cruzi activity and synergistic effect with benznidazole.

BACKGROUND: Chagas disease (CD), caused by Trypanosoma cruzi, represents a health threat to around 20 million people worldwide. Side effects of benznidazole (Bzn) cause 15-20% of patients to discontinue their treatment. Evidence has increased in favor of the use of drug combinations to improve the efficacy and tolerance of the treatment. Natural products are well known to provide structures that could serve as new drugs or scaffolds for CD treatment. Spp of the Amaryllidoideae sub family of Amaryllidaceae family are known by their bioactives alkaloids, which have been reported by their antiparasitic activities. PURPOSE: To evaluate the anti-T. cruzi activity of the isolated alkaloid candimine (Cnd) from Hippeastrum escoipense Slanis & Huaylla; and to assess the combination effect between Cnd and Bzn against different life stages of T. cruzi parasites. METHODS: The chemical profile of H. escoipense alkaloids extract (AE-H. escoipense), including quantitation of Cnd was performed through GC/MS and UPLC-MS/MS techniques. Subsequently, Cnd was isolated using Shephadex LH-20. Then, the AE-H. escoipense and Cnd were tested against T. cruzi, (epimastigotes, trypomastigotes, and amastigotes) by in vitro proliferation and viability assays. The cytotoxicity was evaluated against Vero and HepG2 mammalian cells. The ultrastructural analysis was perform by transmission electron microscopy (TEM) and mitochondrial activity was carried out by MTT assay. Drug combination assay between Cnd and Bzn was evaluated using the Chou-Talalay method. RESULTS: The AE-H. escoipense and Cnd showed high and specific anti-T. cruzi activity, comparable to Bzn. Cnd induces ultrastructural changes in T. cruzi, such as vacuolization, membrane blebs, and increased mitochondrial activity. Regarding the interaction between Cnd and Bzn, it generates synergism in the combinations of 0.25×IC50 in epimastigotes, 2×IC50 in trypomastigotes+amastigotes, and 0.25, 2, and 4×IC50 in amastigotes. CONCLUSION: The synergism between Cnd and Bzn indicates that the combination at the concentration of 4×IC50 could be useful as an effective new therapy against CD in the chronic stage. Thus, Cnd isolated from the leaves of H. escoipense emerges as potential candidate for the development of a new drug for the treatment of CD.

Anti-Trypanosoma cruzi Properties of Sesquiterpene Lactones Isolated from Stevia spp.: In Vitro and In Silico Studies.

Stevia species (Asteraceae) have been a rich source of terpenoid compounds, mainly sesquiterpene lactones, several of which show antiprotozoal activity. In the search for new trypanocidal compounds, S. satureiifolia var. satureiifolia and S. alpina were studied. Two sesquiterpene lactones, santhemoidin C and 2-oxo-8-deoxyligustrin, respectively, were isolated. These compounds were assessed in vitro against Trypanosoma cruzi stages, showing IC50 values of 11.80 and 4.98 on epimastigotes, 56.08 and 26.19 on trypomastigotes and 4.88 and 20.20 µM on amastigotes, respectively. Cytotoxicity was evaluated on Vero cells by the MTT assay. The effect of the compounds on trypanothyone reductase (TcTR), Trans-sialidase (TcTS) and the prolyl oligopeptidase of 80 kDa (Tc80) as potential molecular targets of T. cruzi was investigated. Santhemoidin C inhibited oligopeptidase activity when tested against recombinant Tc80 using a fluorometric assay, reaching an IC50 of 34.9 µM. Molecular docking was performed to study the interaction between santhemoidin C and the Tc80 protein, reaching high docking energy levels. Plasma membrane shedding and cytoplasmic vacuoles, resembling autophagosomes, were detected by transmission microscopy in parasites treated with santhemoidin C. Based on these results, santhemoidin C represents a promising candidate for further studies in the search for new molecules for the development of trypanocidal drugs.

Quantifying heterogeneities in arbovirus transmission: Description of the rationale and methodology for a prospective longitudinal study of dengue and Zika virus transmission in Iquitos, Peru (2014-2019).

Current knowledge of dengue virus (DENV) transmission provides only a partial understanding of a complex and dynamic system yielding a public health track record that has more failures than successes. An important part of the problem is that the foundation for contemporary interventions includes a series of longstanding, but untested, assumptions based on a relatively small portion of the human population; i.e., people who are convenient to study because they manifest clinically apparent disease. Approaching dengue from the perspective of people with overt illness has produced an extensive body of useful literature. It has not, however, fully embraced heterogeneities in virus transmission dynamics that are increasingly recognized as key information still missing in the struggle to control the most important insect-transmitted viral infection of humans. Only in the last 20 years have there been significant efforts to carry out comprehensive longitudinal dengue studies. This manuscript provides the rationale and comprehensive, integrated description of the methodology for a five-year longitudinal cohort study based in the tropical city of Iquitos, in the heart of the Peruvian Amazon. Primary data collection for this study was completed in 2019. Although some manuscripts have been published to date, our principal objective here is to support subsequent publications by describing in detail the structure, methodology, and significance of a specific research program. Our project was designed to study people across the entire continuum of disease, with the ultimate goal of quantifying heterogeneities in human variables that affect DENV transmission dynamics and prevention. Because our study design is applicable to other Aedes transmitted viruses, we used it to gain insights into Zika virus (ZIKV) transmission when during the project period ZIKV was introduced and circulated in Iquitos. Our prospective contact cluster investigation design was initiated by detecttion of a person with a symptomatic DENV infection and then followed that person's immediate contacts. This allowed us to monitor individuals at high risk of DENV infection, including people with clinically inapparent and mild infections that are otherwise difficult to detect. We aimed to fill knowledge gaps by defining the contribution to DENV transmission dynamics of (1) the understudied majority of DENV-infected people with inapparent and mild infections and (2) epidemiological, entomological, and socio-behavioral sources of heterogeneity. By accounting for factors underlying variation in each person's contribution to transmission we sought to better determine the type and extent of effort needed to better prevent virus transmission and disease.

Antiparasitic Activity of Hippeastrum Species and Synergistic Interaction between Montanine and Benznidazole against Trypanosoma cruzi.

BACKGROUND: Hippeastrum species have a wide range of biological properties. In Argentina, this genus comprises ten widely distributed species. PURPOSE: To evaluate the antiparasitic and anticholinesterase activities and chemical profiles of seven Argentinean Hippeastrum species and determine the synergism between the major isolated alkaloid-montanine-and benznidazole in anti-Trypanosoma cruzi activity. METHODS: The antiparasitic activity was evaluated through antiproliferative and viability assays against T. cruzi epimastigotes. Synergism assays were performed using the Chou-Talalay method. AChE and BuChE inhibitory activities were also assessed. The alkaloid composition was obtained using GC-MS analysis. RESULTS: All extracts showed strong growth inhibition of T. cruzi epimastigote proliferation. The extracts from H. aglaiae, H. aulicum, and H. hybrid stand out for their potent and total growth inhibition, which was comparable to benznidazole. The H. reticulatum extract showed strong Acetylcholinesterase (AChE) inhibitory activities, while five species showed moderate Butyrylcholinesterase (BuChE) inhibition. Fifteen alkaloids were identified by means of GC-MS. Regarding the synergism assessment, the highest synergistic effect was obtained from the combination of montanine and benznidazole. CONCLUSION: Hippeastrum species bulb extracts from Argentina were shown to be a good source of antiparasitic alkaloids and cholinesterase inhibitors. The synergism between montanine and benznidazole emerges as a potential combination for future studies to treat Chagas disease.

Self-regulation of TNF-α Induces Dysfunction of Endothelial Colony-forming Cells from Patients with Venous Thromboembolic Disease.

BACKGROUND: Endothelial colony-forming cells (ECFCs) contribute to postnatal vasculogenesis. In venous thromboembolic disease (VTD), they are functionally abnormal and produce high concentrations of TNF-α. OBJECTIVE: To analyze the TNF-α signaling pathway and its relationship with the expression of cell-cycle regulators. METHODS: Mononuclear cells (MNCs) were collected from the peripheral blood of 20 healthy human volunteers (controls) and 30 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We analyzed the relative quantification of the gene transcripts of TNF, NFkB1, PLAU, HMOX1, GSS, eNOS, CDKN1A, and CDKN1B through quantitative RT-PCR (qRT-PCR assays). Identification of NF-κB and activated targets of each pathway: NF-κB (Ser536); IκBα (Ser32/Ser36); p38 (Thr180/Tyr182) JNK (Thr183/Tyr185), p53 and cell-cycle regulators: p16, p18, p21, p27, p57, Cyclin D, Cyclin E, Cyclin A, Cyclin B, CDK2, CDK4; cell-cycle status was determined by KI-67 and 7-AAD. Cells were analyzed with flow cytometry and the FlowJo vX software. RESULTS: In ECFCs from VTD patients, TNF-α receptor and NFkB were overexpressed and hyper-phosphorylated; eNOS and HMOX1 were down-regulated; cell-cycle regulators (p53, p18, p21) were elevated. In addition, the cell cycle was locked in the G2 phase. CONCLUSIONS: Our results strongly suggest that these molecular alterations in the pathway of TNF-α and cell cycle regulation induce endothelial dysfunction, reduced proliferation potential and vascular regeneration, and consequently, the occurrence of new thrombotic events.

Cell Contact with Endothelial Cells Favors the In Vitro Maintenance of Human Chronic Myeloid Leukemia Stem and Progenitor Cells.

Chronic Myeloid Leukemia (CML) originates in a leukemic stem cell that resides in the bone marrow microenvironment, where they coexist with cellular and non-cellular elements. The vascular microenvironment has been identified as an important element in CML development since an increase in the vascularization has been suggested to be related with poor prognosis; also, using murine models, it has been reported that bone marrow endothelium can regulate the quiescence and proliferation of leukemic stem and progenitor cells. This observation, however, has not been evaluated in primary human cells. In this report, we used a co-culture of primitive (progenitor and stem) CML cells with endothelial colony forming cells (ECFC) as an in vitro model to evaluate the effects of the vascular microenvironment in the leukemic hematopoiesis. Our results show that this interaction allows the in vitro maintenance of primitive CML cells through an inflammatory microenvironment able to regulate the proliferation of progenitor cells and the permanence in a quiescent state of leukemic stem cells.

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos, Peru.

Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Mode of action of the sesquiterpene lactones eupatoriopicrin and estafietin on Trypanosoma cruzi.

BACKGROUND: The sesquiterpene lactones (STLs) eupatoriopicrin (EP) and estafietin (ES), isolated from Stevia alpina Griseb. (Asteraceae) and Stevia maimarensis (Hieron.) Cabrera (Asteraceae) respectively, have previously showed promising trypanocidal activity, both in vitro and in vivo. PURPOSE: In this work, using biochemical studies and electron microscopy, we aimed at characterizing the mode of action of both STLs on Trypanosoma cruzi. METHODS: The interaction of STLs with hemin was examined by measuring modifications in the Soret absorption band of hemin; the thiol groups interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate; the effect on cruzipain activity was also assayed by spectrophotometry. The synthesis of sterols were qualitatively and quantitatively tested by TLC. Mitochondrial functionality was assessed by measuring mitochondrial membrane potential and the activity of NADH-cytochrome c reductase and succinate-cytochrome c reductase enzymes. The status of the antioxidant system was assessed by quantifying the level of free thiols by spectrophotometry, together with the intracellular oxidative state by flow cytometry. Ultrastructural changes were analyzed by transmission electron microscopy. RESULTS: EP and ES were found to impair the functionality and the redox status of the parasite. ES produced a greater decrease in the activity of succinate dehydrogenase than eupatoriopicrin, affecting the functioning of the respiratory chain and the Krebs cycle. EP increased the formation of triglycerides leading to the presence of cytoplasmic lipid droplets. By electron microscopy, alterations in the kinetoplast and the appearance of large translucent vacuoles in the cytoplasm were observed for both compounds. CONCLUSIONS: Both sesquiterpenelactones proved to act additively on T. cruzi, supporting the hypothesis that each compound would be acting on different primary targets.. The treatment combining eupatoriopicrin and estafietin could be considered a promising alternative for the treatment of Chagas' disease.

Corrigendum: A Novel Calcium-Activated Potassium Channel Controls Membrane Potential and Intracellular pH in Trypanosoma cruzi.

[This corrects the article DOI: 10.3389/fcimb.2019.00464.].

A novel mechanosensitive channel controls osmoregulation, differentiation, and infectivity in Trypanosoma cruzi.

The causative agent of Chagas disease undergoes drastic morphological and biochemical modifications as it passes between hosts and transitions from extracellular to intracellular stages. The osmotic and mechanical aspects of these cellular transformations are not understood. Here we identify and characterize a novel mechanosensitive channel in Trypanosoma cruzi (TcMscS) belonging to the superfamily of small-conductance mechanosensitive channels (MscS). TcMscS is activated by membrane tension and forms a large pore permeable to anions, cations, and small osmolytes. The channel changes its location from the contractile vacuole complex in epimastigotes to the plasma membrane as the parasites develop into intracellular amastigotes. TcMscS knockout parasites show significant fitness defects, including increased cell volume, calcium dysregulation, impaired differentiation, and a dramatic decrease in infectivity. Our work provides mechanistic insights into components supporting pathogen adaptation inside the host, thus opening the exploration of mechanosensation as a prerequisite for protozoan infectivity.

Assessment of Cell Cycle in Primitive Chronic Myeloid Leukemia Cells by Flow Cytometry After Coculture with Endothelial Cells.

From the knowledge that hematopoiesis does not occur randomly in the bone marrow but is regulated by the different components of the microenvironment, the use of in vitro coculture systems has been used as a powerful tool in the analysis of different processes that are involved in the maintenance of blood cells. In this chapter, we describe a methodological strategy to perform a coculture between primitive hematopoietic cells and endothelial cells to evaluate cell cycle, an aspect of relevant importance in the permanence of primitive leukemic cells.

A Novel Calcium-Activated Potassium Channel Controls Membrane Potential and Intracellular pH in Trypanosoma cruzi.

Trypanosoma cruzi develops in environments where nutrient availability, osmolarity, ionic concentrations, and pH undergo significant changes. The ability to adapt and respond to such conditions determines the survival and successful transmission of T. cruzi. Ion channels play fundamental roles in controlling physiological parameters that ensure cell homeostasis by rapidly triggering compensatory mechanisms. Combining molecular, cellular and electrophysiological approaches we have identified and characterized the expression and function of a novel calcium-activated potassium channel (TcCAKC). This channel resides in the plasma membrane of all 3 life stages of T. cruzi and shares structural features with other potassium channels. We expressed TcCAKC in Xenopus laevis oocytes and established its biophysical properties by two-electrode voltage clamp. Oocytes expressing TcCAKC showed a significant increase in inward currents after addition of calcium ionophore ionomycin or thapsigargin. These responses were abolished by EGTA suggesting that TcCAKC activation is dependent of extracellular calcium. This activation causes an increase in current and a negative shift in reversal potential that is blocked by barium. As predicted, a single point mutation in the selectivity filter (Y313A) completely abolished the activity of the channels, confirming its potassium selective nature. We have generated knockout parasites deleting one or both alleles of TcCAKC. These parasite strains showed impaired growth, decreased production of trypomastigotes and slower intracellular replication, pointing to an important role of TcCAKC in regulating infectivity. To understand the cellular mechanisms underlying these phenotypic defects, we used fluorescent probes to evaluate intracellular membrane potential, pH, and intracellular calcium. Epimastigotes lacking the channel had significantly lower cytosolic calcium, hyperpolarization, changes in intracellular pH, and increased rate of proton extrusion. These results are in agreement with previous reports indicating that, in trypanosomatids, membrane potential and intracellular pH maintenance are linked. Our work shows TcCAKC is a novel potassium channel that contributes to homeostatic regulation of important physiological processes in T. cruzi and provides new avenues to explore the potential of ion channels as targets for drug development against protozoan parasites.

Antiproliferative effect and ultrastructural alterations induced by 5-O-methylembelin on Trypanosoma cruzi.

BACKGROUND: Embelin (EMB), obtained from Oxalis erythrorhiza Gillies ex Hooker et Arnott (Oxalidaceae), was reported against Trypanosoma cruzi and Leishmania spp. Additionally, antiprotozoan activity against Plasmodium falciparum was reported for its methylated derivative (ME). PURPOSE: To evaluate the potential anti-Trypanosoma cruzi activity of EMB, ME and 2,5-di-O-methylembelin (DME) and analyze the possible mechanism of action. STUDY DESIGN/METHODS: EMB was isolated by a chromatographic method from the air-dried ground whole plant. To evaluate the effects of methylation, ME and DME were synthesized and tested against T. cruzi epimastigotes and trypomastigotes. The most active compound ME was evaluated against amastigotes. Ultrastructural alterations, ROS generation and the effect on mitochondrial activity of ME were measured. RESULTS: Compounds inhibited the proliferation of epimastigotes. ME was also active against intracellular amastigotes. Mitochondrial alterations were observed by TEM. Additionally, ME modified the mitochondrial activity, and induced an increase in ROS levels. These evidences postulate the mitochondrion as a possible target of ME. CONCLUSION: ME inhibited amastigotes proliferation, thus being a potential lead compound for the treatment of Chagas' disease.

Guide for Selection of Relevant Cell Lines During the Evaluation of new Anti-Cancer Compounds.

BACKGROUND: Human cancer cell lines are valuable models for anti-cancer drug development. Although all cancer cells share common biological features, each cancer cell line has unique genotypic/ phenotypic characteristics that affect drug response. Thus, the information obtained with a specific cancer cell line cannot be easily extrapolated to other cancer cells. Consequently, cell line selection during experimental design is critical for providing proper and clinically relevant structure-activity analysis. METHODS: Herein, we critically review the use of cancer cell lines as tools for activity analysis by comparing two different scenarios: i) the use of multiple cancer cell lines, with the NCI-60 Program as the most representative example; and, ii) the selection of a single cell line with specific biological characteristics that match the rationale of compound design. RESULTS: Considering that most laboratories evaluate the activity of new compounds using few cell lines, we provide a systematic strategy for selection based on the expression levels and genetic status of the target and the effectiveness of target inhibition or silencing. We exemplify the use of public databases for data retrieval and analysis as well as the critical comparison of such information with published results. CONCLUSION: This approach refines cell line selection, avoiding the perpetuation of published poor selection and enhancing the relevance of the results.

Reduced proliferation of endothelial colony-forming cells in unprovoked venous thromboembolic disease as a consequence of endothelial dysfunction.

BACKGROUND: Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). METHODS AND RESULTS: Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated ß-galactosidase (SA-ß-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). CONCLUSIONS: As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events.