Gisele A. B. Canuto

Caracterização físico-química de polpas de frutos da Amazônia e sua correlação com a atividade anti-radical livre

Physical and chemical characteristics (color, pH, titratable acidity, total soluble solids, lipid content, moisture) and levels of bioactive compounds (ascorbic acid, total phenolics) were determined in fifteen samples of fruit pulps from Amazonia (abiu, acerola, acai, araca-boi, bacaba, bacuri, buriti, caja, cajarana, caju, cupuacu, graviola, murici, noni e tamarindo). The free radical scavenger activity was evaluated by the ABTS assay. Some pulps presented high antioxidant potential, associated with the free radical scavenger activity measured and the content of bioactive components, such as phenolic compounds and ascorbic acid, especialy in acerola and acai. The total phenolic content was correlated to antioxidant capacity of pulps.

CE‐ESI‐MS metabolic fingerprinting of Leishmania resistance to antimony treatment

Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical‐chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE‐ESI‐TOF‐MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC50 = 20.9 μM) and its variation when treated with 120 μM of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step‐wise manner up to 180 μM. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol‐dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania

AbstractMiltefosine (MT) (hexadecylphosphocholine) was implemented to cope with resistance against antimonials, the classical treatment in Leishmaniasis. Given the scarcity of anti- Leishmania (L) drugs and the increasing appearance of resistance, there is an obvious need for understanding the mechanism of action and development of such resistance. Metabolomics is an increasingly popular tool in the life sciences due to it being a relatively fast and accurate technique that can be applied either with a particular focus or in a global manner to reveal new knowledge about biological systems. Three analytical platforms, gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) have been coupled to mass spectrometry (MS) to obtain a broad picture of metabolic changes in the parasite. Impairment of the polyamine metabolism from arginine (Arg) to trypanothione in susceptible parasites treated with MT was in some way expected, considering the reactive oxygen species (ROS) production described for MT. Importantly, in resistant parasites an increase in the levels of amino acids was the most outstanding feature, probably related to the adaptation of the resistant strain for its survival inside the parasitophorous vacuole. Online Abstract Figureᅟ

Development and validation of a liquid chromatography method for anthocyanins in strawberry (Fragaria spp.) and complementary studies on stability, kinetics and antioxidant power

A RPLC-DAD method for the analysis of eight anthocyanins was developed, validated and applied to strawberry extracts. The chromatographic method was conducted under gradient elution in acidulated water-methanol mobile phase and octadecyl-silica columns. An ultrasound extraction procedure was optimized by a 3(2) factorial design (%HCl in methanol, temperature, and time) and response surface methodology. Method validation was performed according to the following parameters: linearity (R(2)>0.99, p-value<10(-4), F>725), LOD (3-7 μmol L(-1)) and LOQ (9-22 μmol L(-1)), selectivity/specificity (baseline separation of all analytes and peak purity), instrumental precision (<6.4%CV), repeatability (<6.3%CV) and intermediate precision (<9.9%CV), recovery (83-99%), robustness (mobile phase pH, column temperature and flow rate) and stability (high temperatures and storage; 1st order kinetics). The antioxidant power of anthocyanins was measured on-line (ABTS(+) reaction; Trolox as reference). Ten strawberry extracts were quantified (average values: 24.2 μg/g for cyanidin-3-glucoside and 49.1 μg/g for pelargonidin-3-glucoside).

A Multiplatform Metabolomic Approach to the Basis of Antimonial Action and Resistance in Leishmania infantum

There is a rising resistance against antimony drugs, the gold-standard for treatment until some years ago. That is a serious problem due to the paucity of drugs in current clinical use. In a research to reveal how these drugs affect the parasite during treatment and to unravel the underlying basis for their resistance, we have employed metabolomics to study treatment in Leishmania infantum promastigotes. This was accomplished first through the untargeted analysis of metabolic snapshots of treated and untreated parasites both resistant and responders, utilizing a multiplatform approach to give the widest as possible coverage of the metabolome, and additionally through novel monitoring of the origin of the detected alterations through a 13C traceability experiment. Our data stress a multi-target metabolic alteration with treatment, affecting in particular the cell redox system that is essential to cope with detoxification and biosynthetic processes. Additionally, relevant changes were noted in amino acid metabolism. Our results are in agreement with other authors studying other Leishmania species.

Capillary electrophoresis reveals polyamine metabolism modulation in Leishmania (Leishmania) amazonensis wild-type and arginase-knockout mutants under arginine starvation

l‐Arginine is an essential amino acid in Leishmania (Leishmania) amazonensis metabolism. A key enzyme for parasite l‐arginine metabolism is arginase (ARG) that uses arginine to produce urea and ornithine, a precursor of polyamine pathway guaranteeing parasite replication in both insect and mammal hosts. There is an alternative pathway to produce ornithine via l‐proline and glutamate, but this mechanism is not described in Leishmania. In the mammal host, two enzymes can use l‐arginine as substrate, the host ARG and the induced nitric oxide synthase that produces nitric oxide. The competition between induced nitric oxide synthase and both parasite and host ARG can favor the success of the infection or its control. Here, we established the metabolomics profile of the polyamine pathway of wild type (WT) L. (L.) amazonensis, submitted or not to l‐arginine starvation, and compared to the ARG‐knockout mutant (arg−). Our results indicated that arginine starvation induces a decrease in arginine, ornithine, and putrescine, but we could not detect the significative level changes of spermidine, spermine, or agmatine. However, the absence of ARG on the arg− induced an increase of arginine and citrulline levels, but decreased the levels of ornithine and putrescine. Similarly to the WT arginine‐starved parasites, the arg− parasites presented lower levels of proline when compared to the WT ones. This could be indicative of an alternative pathway to surpass the enzyme or its substrate absence.

Metabolomics: Definitions and Significance in Systems Biology

Nowadays, there is a growing interest in deeply understanding biological mechanisms not only at the molecular level (biological components) but also the effects of an ongoing biological process in the organism as a whole (biological functionality), as established by the concept of systems biology. Within this context, metabolomics is one of the most powerful bioanalytical strategies that allow obtaining a picture of the metabolites of an organism in the course of a biological process, being considered as a phenotyping tool. Briefly, metabolomics approach consists in identifying and determining the set of metabolites (or specific metabolites) in biological samples (tissues, cells, fluids, or organisms) under normal conditions in comparison with altered states promoted by disease, drug treatment, dietary intervention, or environmental modulation. The aim of this chapter is to review the fundamentals and definitions used in the metabolomics field, as well as to emphasize its importance in systems biology and clinical studies.

Neglected diseases prioritized in Brazil under the perspective of metabolomics: A review

This review article compiles in a critical manner literature publications regarding seven neglected diseases (ND) prioritized in Brazil (Chagas disease, dengue, leishmaniasis, leprosy, malaria, schistosomiasis, and tuberculosis) under the perspective of metabolomics. Both strategies, targeted and untargeted metabolomics, were considered in the compilation. The majority of studies focused on biomarker discovery for diagnostic purposes, and on the search of novel or alternative therapies against the ND under consideration, although temporal progression of the infection at metabolic level was also addressed. Tuberculosis, followed by schistosomiasis, malaria and leishmaniasis are the diseases that received larger attention in terms of number of publications. Dengue and leprosy were the least studied and Chagas disease received intermediate attention. NMR and HPLC–MS technologies continue to predominate among the analytical platforms of choice in the metabolomic studies of ND. A plethora of metabolites were identified in the compiled studies, with expressive predominancy of amino acids, organic acids, carbohydrates, nucleosides, lipids, fatty acids, and derivatives.