Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance in Toxoplasma gondii.

The single mitochondrion of the obligate intracellular parasite Toxoplasma gondii is highly dynamic. Toxoplasma's mitochondrion changes morphology as the parasite moves from the intracellular to the extracellular environment and during division. Toxoplasma's mitochondrial dynamic is dependent on an outer mitochondrion membrane-associated protein LMF1 and its interaction with IMC10, a protein localized at the inner membrane complex (IMC). In the absence of either LMF1 or IMC10, parasites have defective mitochondrial morphology and inheritance defects. As little is known about mitochondrial inheritance in Toxoplasma, we have used the LMF1/IMC10 tethering complex as an entry point to dissect the machinery behind this process. Using a yeast two-hybrid screen, we previously identified Myosin A (MyoA) as a putative interactor of LMF1. Although MyoA is known to be located at the parasite's pellicle, we now show through ultrastructure expansion microscopy (U-ExM) that this protein accumulates around the mitochondrion in the late stages of parasite division. Parasites lacking MyoA show defective mitochondrial morphology and a delay in mitochondrion delivery to the daughter parasite buds during division, indicating that this protein is involved in organellar inheritance. Disruption of the parasite's actin network also affects mitochondrion morphology. We also show that parasite-extracted mitochondrion vesicles interact with actin filaments. Interestingly, mitochondrion vesicles extracted out of parasites lacking LMF1 pulled down less actin, showing that LMF1 might be important for mitochondrion and actin interaction. Accordingly, we are showing for the first time that actin and Myosin A are important for Toxoplasma mitochondrial morphology and inheritance.

Correction: How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

[This corrects the article DOI: 10.1371/journal.ppat.1011522.].

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

Comparison of the Permeability of Potential Ballast Rocks from Northern Rio de Janeiro State under Different Fouling Rates after Sodium Sulfate Attack.

This paper aimed to analyze the reduction in the ballast layer permeability simulated in a laboratory in saturated conditions by the presence of rock dust as a contaminant of three types of rocks explored in different deposits in the northern region of the state of Rio de Janeiro, Brazil, through laboratory testing relating the physical properties of rock particles before and after sodium sulfate attack. Sodium sulfate attack is justified by the proximity of some sections of the planned EF-118 Vitória-Rio railway line to the coast and of the sulfated water table to the ballast bed, which could degrade the material used and compromise the railway track. Granulometry and permeability tests were performed to compare ballast samples with fouling rates of 0, 10, 20, and 40% rock dust by volume. A constant head permeameter was used to analyze hydraulic conductivity and establish correlations between the petrography and mercury intrusion porosimetry of the rocks, namely two types of metagranite (Mg1 and Mg3) and a gneisse (Gn2). Rocks, such as Mg1 and Mg3, with a larger composition of minerals susceptible to weathering according to petrography analyses, tend to be more sensitive to weathering tests. This, in conjunction with the climate in the region studied, with average annual temperature and rainfall of 27 °C and 1200 mm, could compromise track safety and user comfort. Additionally, the Mg1 and Mg3 samples showed greater percentage variation in wear after the Micro-Deval test, which could damage the ballast due to the considerable changeability of the material. The mass loss caused by abrasion due to the passage of rail vehicles was assessed by the Micro-Deval test, with Mg3 (intact rock) declining from 8.50 ± 1.5 to 11.04 ± 0.5% after chemical attack. However, Gn2, which exhibited the greatest mass loss among the samples, showed no significant variation in average wear, and its mineralogical characteristics remained almost unchanged after 60 sodium sulfate cycles. These aspects, combined with its satisfactory hydraulic conductivity rate, indicate that Gn2 is suitable for use as railway ballast in the EF-118 railway line.

Previously Unidentified Histone H1-Like Protein Is Involved in Cell Division and Ribosome Biosynthesis in Toxoplasma gondii.

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii ("TgH1-like") that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. IMPORTANCE Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

IMC10 and LMF1 mediate mitochondrial morphology through mitochondrion-pellicle contact sites in Toxoplasma gondii.

The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular parasites. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion. Intracellular parasites lacking LMF1 fail to form the lasso-shaped mitochondrion. To identify other proteins that tether the mitochondrion of the parasite to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors localized in different cellular compartments, such as the apical end of the parasite, mitochondrial membrane and the inner membrane complex (IMC), including with the pellicle protein IMC10. Using protein-protein interaction assays, we confirmed the interaction of LMF1 with IMC10. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between the mitochondrion and the IMC in Toxoplasma. This article has an associated First Person interview with the first author of the paper.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains.

BACKGROUND: Leishmaniases are diseases caused by Leishmania protozoans that affect around 12 million people. Leishmania promastigotes are transmitted to vertebrates by female phlebotomine flies during their blood meal. Parasites attach to phagocytic cells, are phagocytosed and differentiate into amastigotes. We previously showed that PH8 and LV79 strains of Leishmania amazonensis have different virulence in mice and that their amastigotes differ in their proteomes. In this work, we compare promastigotes' infectivity in macrophages, their proteomes and morphologies. METHODS/PRINCIPAL FINDINGS: Phagocytosis assays showed that promastigotes adhesion to and phagocytosis by macrophages is higher in PH8 than LV79. To identify proteins that differ between the two strains and that may eventually contribute for these differences we used a label-free proteomic approach to compare promastigote´s membrane-enriched fractions. Proteomic analysis enabled precise discrimination of PH8 and LV79 protein profiles and the identification of several differentially abundant proteins. The proteins more abundant in LV79 promastigotes participate mainly in translation and amino acid and nucleotide metabolism, while the more abundant in PH8 are involved in carbohydrate metabolism, cytoskeleton composition and vesicle/membrane trafficking. Interestingly, although the virulence factor GP63 was more abundant in the less virulent LV79 strain, zymography suggests a higher protease activity in PH8. Enolase, which may be related to virulence, was more abundant in PH8 promastigotes. Unexpectedly, flow cytometry and morphometric analysis indicate higher abundance of metacyclics in LV79. CONCLUSIONS/SIGNIFICANCE: Proteome comparison of PH8 and LV79 promastigotes generated a list of differential proteins, some of which may be further prospected to affect the infectivity of promastigotes. Although proteomic profile of PH8 includes more proteins characteristic of metacyclics, flow cytometry and morphometric analysis indicate a higher abundance of metacyclics in LV79 cultures. These results shed light to the gaps in our knowledge of metacyclogenesis in L. amazonensis, and to proteins that should be studied in the context of infection by this species.

Protein control of membrane and organelle dynamics: Insights from the divergent eukaryote Toxoplasma gondii.

Integral membrane protein complexes control key cellular functions in eukaryotes by defining membrane-bound spaces within organelles and mediating inter-organelles contacts. Despite the critical role of membrane complexes in cell biology, most of our knowledge is from a handful of model systems, primarily yeast and mammals, while a full functional and evolutionary understanding remains incomplete without the perspective from a broad range of divergent organisms. Apicomplexan parasites are single-cell eukaryotes whose survival depends on organelle compartmentalisation and communication. Studies of a model apicomplexan, Toxoplasma gondii, reveal unexpected divergence in the composition and function of complexes previously considered broadly conserved, such as the mitochondrial ATP synthase and the tethers mediating ER-mitochondria membrane contact sites. Thus, Toxoplasma joins the repertoire of divergent model eukaryotes whose research completes our understanding of fundamental cell biology.

Previously unidentified histone H1-like protein is involved in cell division and ribosome biosynthesis in toxoplasma gondii

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii (“TgH1-like”) that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

Depressão entre idosos portadores de doença de Parkinson: opinião dos membros da Associação Capixaba de Parkinson / Depression among elderly people with Parkinson disease: opinion of the members of the Capixaba Parkinson Associaton

INTRODUÇÃO: A doença de Parkinson (DP) e a segunda enfermidade neurodegeneratva mais comum no idoso, enquanto a depressão e uma síndrome com transtorno do humor. Os sintomas depressivos são as manifestações não motoras mais comuns entre os acometdos pela DP. OBJETIVO: Demonstrar a percepção que os portadores de DP possuem em relação a depressão e seus sintomas, sendo, para isso, necessário caracterizar as mudanças ocorridas nos hábitos de vida dos portadores de Parkinson, identfcar a percepção dos membros da associação sobre os sintomas de depressão e sobre a relação entre DP e depressão, alem de caracterizar o convívio social dos portadores de Parkinson com depressão. METODOLOGIA: Estudo descritvo qualitatvo, no qual foram entrevistados membros idosos da Associação Capixaba de Parkinson. As entrevistas foram gravadas, transcritas, categorizadas, armazenadas e submetdas a análise de discurso. Este estudo foi aprovado pelo Comitê de Étca da Faculdade Brasileira - Multvix sob número 47289215.1.10000.5066. RESULTADOS: O estudo revela que os diferentes quadros clínicos do Parkinson são defnidores dos hábitos de vida desses pacientes, já que causam limitação motora, transtornos do sono e memória, perda da autonomia e desmotvação. Na visão dos membros da Associação Capixaba de Parkinson, os sintomas depressivos são reconhecidos pela maioria dos portadores de Parkinson, que acreditam que há relação entre ambas as doenças. Dessa forma, indivíduos diagnostcados com as duas enfermidades podem ter alterações em seu convívio social, como o isolamento e a busca por grupos de apoio. CONCLUSÃO: O Parkinson gera prejuízos em vários âmbitos da vida do portador, principalmente quando está associado a depressão. Os parkinsonianos reconhecem os sintomas depressivos e percebem a relação com a DP. Aspectos fsicos e mentais devem ser levados em consideração no tratamento da doença, necessitando de uma abordagem multprofssional e interdisciplinar.

INTRODUCTION: Parkinson's disease (PD) is the second most common neurodegeneratve disease in the elderly while depression is a mood disorder syndrome. Depressive symptoms are non-motor manifestatons more common among those afected by Parkinson's disease. OBJECTIVE: To demonstrate the PD patents' percepton in regard to depression and its symptoms and, for that, it was necessary to characterize the changes in lifestyle of those afected by Parkinson; to identfy the percepton of members of the Associaton about the symptoms of depression and the relatonship between PD and depression, in additon, to characterize the social life of Parkinson's patents with depression. METHODOLOGY: Qualitatve descriptve study, in which senior members of Parkinson Capixaba Associaton were interviewed. Interviews were recorded, transcribed, categorized, stored and submited to discourse analysis. This study was approved by the Faculdade Brasileira- Multvix Ethics Commitee No. 47289215.1.10000.5066. RESULTS: The study revealed that Parkinson's diferent clinical manifestatons are defners of the lifestyle of these patents. In the view of members of the Parkinson Associaton Capixaba, depressive symptoms are recognized by the majority of people with Parkinson's, who believe that there is a relatonship between both diseases. Thus, individuals diagnosed with both diseases may have changes in their social life, as isolaton and search for support groups. CONCLUSION: Parkinson generates losses in various areas of the person's life, especially when it is associated with depression. Parkinsonians recognize depressive symptoms and perceive its relatonship with PD. Physical and mental aspects should be taken into consideraton in the treatment of the disease, requiring a multdisciplinary and interdisciplinary approach.

Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi.

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline.

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

Genome-scale metabolic models highlight stage-specific differences in essential metabolic pathways in Trypanosoma cruzi.

Chagas disease is a neglected tropical disease and a leading cause of heart failure in Latin America caused by a protozoan called Trypanosoma cruzi. This parasite presents a complex multi-stage life cycle. Anti-Chagas drugs currently available are limited to benznidazole and nifurtimox, both with severe side effects. Thus, there is a need for alternative and more efficient drugs. Genome-scale metabolic models (GEMs) can accurately predict metabolic capabilities and aid in drug discovery in metabolic genes. This work developed an extended GEM, hereafter referred to as iIS312, of the published and validated T. cruzi core metabolism model. From iIS312, we then built three stage-specific models through transcriptomics data integration, and showed that epimastigotes present the most active metabolism among the stages (see S1-S4 GEMs). Stage-specific models predicted significant metabolic differences among stages, including variations in flux distribution in core metabolism. Moreover, the gene essentiality predictions suggest potential drug targets, among which some have been previously proven lethal, including glutamate dehydrogenase, glucokinase and hexokinase. To validate the models, we measured the activity of enzymes in the core metabolism of the parasite at different stages, and showed the results were consistent with model predictions. Our results represent a potential step forward towards the improvement of Chagas disease treatment. To our knowledge, these stage-specific models are the first GEMs built for the stages Amastigote and Trypomastigote. This work is also the first to present an in silico GEM comparison among different stages in the T. cruzi life cycle.

Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 µM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

Glutamine Analogues Impair Cell Proliferation, the Intracellular Cycle and Metacyclogenesis in Trypanosoma cruzi.

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Dual inhibition of glutaminase and carnitine palmitoyltransferase decreases growth and migration of glutaminase inhibition-resistant triple-negative breast cancer cells.

Triple-negative breast cancers (TNBCs) lack progesterone and estrogen receptors and do not have amplified human epidermal growth factor receptor 2, the main therapeutic targets for managing breast cancer. TNBCs have an altered metabolism, including an increased Warburg effect and glutamine dependence, making the glutaminase inhibitor CB-839 therapeutically promising for this tumor type. Accordingly, CB-839 is currently in phase I/II clinical trials. However, not all TNBCs respond to CB-839 treatment, and the tumor resistance mechanism is not yet fully understood. Here we classified cell lines as CB-839-sensitive or -resistant according to their growth responses to CB-839. Compared with sensitive cells, resistant cells were less glutaminolytic and, upon CB-839 treatment, exhibited a smaller decrease in ATP content and less mitochondrial fragmentation, an indicator of poor mitochondrial health. Transcriptional analyses revealed that the expression levels of genes linked to lipid metabolism were altered between sensitive and resistant cells and between breast cancer tissues (available from The Cancer Genome Atlas project) with low versus high glutaminase (GLS) gene expression. Of note, CB-839-resistant TNBC cells had increased carnitine palmitoyltransferase 2 (CPT2) protein and CPT1 activity levels. In agreement, CB-839-resistant TNBC cells mobilized more fatty acids into mitochondria for oxidation, which responded to AMP-activated protein kinase and acetyl-CoA carboxylase signaling. Moreover, chemical inhibition of both glutaminase and CPT1 decreased cell proliferation and migration of CB-839-resistant cells compared with single inhibition of each enzyme. We propose that dual targeting of glutaminase and CPT1 activities may have therapeutic relevance for managing CB-839-resistant tumors.

L-Glutamine uptake is developmentally regulated and is involved in metacyclogenesis in Trypanosoma cruzi.

Trypanosoma cruzi, the aetiological agent of Chagas disease, can obtain L-glutamine (Gln) through the enzyme glutamine synthetase (GS) using glutamate (Glu) and ammonia as substrates. In this work, we show additional non-canonical roles for this amino acid: its involvement in ATP maintenance and parasite survival under severe metabolic stress conditions and its participation in the differentiation process occurring in the insect vector (metacyclogenesis). These roles are dependent on the supply of Gln from an extracellular source. We show that T. cruzi incorporates Gln through a saturable and specific transport system, which results in unusual stability at elevated temperatures. The activity was moderately higher at pH values between 6 and 7 and was sensitive to the dissipation of the H+ gradient at the plasma membrane. When analysed in the different life cycle stages, we found that Gln transport is developmentally regulated. In fact, Gln uptake and GS activity seem to be finely regulated at most stages: when GS activity is increased, transport is decreased and vice versa, with the exception of trypomastigotes, where both sources of Gln are diminished. This metabolic adaptation reflects the relevance of Gln in T. cruzi biology and the plasticity of these parasites to adjust their metabolism to changing environments.

Ventral medial prefrontal cortex (vmPFC) as a target of the dorsolateral prefrontal modulation by transcranial direct current stimulation (tDCS) in drug addiction.

Here, we report some electrophysiologic and imaging effects of the transcranial direct current stimulation (tDCS) over the dorsolateral prefrontal cortex (dlPFC) in drug addiction, notably in alcohol and crack-cocaine dependence. The low resolution electromagnetic tomography (LORETA) analysis obtained through event-related potentials (ERPs) under drug-related cues, more specifically in its P3 segment (300-500 ms) in both, alcoholics and crack-cocaine users, showed that the ventral medial prefrontal cortex (vmPFC) was the brain area with the largest change towards increasing activation under drug-related cues in those subjects that kept abstinence during and after the treatment with bilateral tDCS (2 mA, 35 cm(2), cathodal left and anodal right) over dlPFC, applied repetitively (five daily sessions). In an additional study in crack-cocaine, which showed craving decreases after repetitive bilateral tDCS, we examined data originating from diffusion tensor imaging (DTI), and we found increased DTI parameters in the left connection between vmPFC and nucleus accumbens (NAcc), such as the number of voxels, fractional anisotropy (FA) and apparent diffusion coefficient (ADC), in tDCS-treated crack-cocaine users when compared to the sham-tDCS group. This increasing of DTI parameters was significantly correlated with craving decreasing after the repetitive tDCS. The vmPFC relates to the control of drug seeking, possibly by extinguishing this behavior. In our studies, the bilateral dlPFC tDCS reduced relapses and craving to the drug use, and increased the vmPFC activation under drug cues, which may be of a great importance in the control of drug use in drug addiction.