Desarrollo de métodos analíticos para la determinación de seis especies químicas de arsénico en muestras medioambientales y orina mediante acoplamiento : cromatografía iónica de alta resolución-horno microondas-generación de hidruros-espectroscopía de absorción atómica

Se ha abordado el desarrollo del procedimiento analítico por acoplamiento de técnicas instrumentales para la determinación de seis especies de as (as(III), as(v), dma, mma, asb y asc). El acoplamiento se realiza con una técnica de separación de especies, como es la cromatografía liquida de alta eficacia (hplc), una interfase de termooxidación de las especies orgánicas (no formadoras de hidruros) y el sistema detector hg-aas. Asimismo, se han evaluado las prestaciones de diferentes columnas cromatográficas en función de los parámetros fisicoquímicos que influyen en la separación. El acoplamiento hplc-horno microondas-hg-aas se ha utilizado para la especiación de as en diversas muestras medioambientales y orina. Por otro lado, su aplicación en muestras de orina de individuos expuestos a las distintas especies de as, a través de la alimentación, ha permitido conocer la evolución de dichas especies en el organismo y distinguir entre las formas tóxicas y no tóxicas del as, así como abordar el problema de la estabilidad de las especies de as en aguas y orinas.Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUEpu

Metabolic clustering analysis as a strategy for compound selection in the drug discovery pipeline for leishmaniasis

A lack of viable hits, increasing resistance, and limited knowledge on mode of action is hindering drug discovery for many diseases. To optimize prioritization and accelerate the discovery process, a strategy to cluster compounds based on more than chemical structure is required. We show the power of metabolomics in comparing effects on metabolism of 28 different candidate treatments for Leishmaniasis (25 from the GSK Leishmania box, two analogues of Leishmania box series, and amphotericin B as a gold standard treatment), tested in the axenic amastigote form of Leishmania donovani. Capillary electrophoresis–mass spectrometry was applied to identify the metabolic profile of Leishmania donovani, and principal components analysis was used to cluster compounds on potential mode of action, offering a medium throughput screening approach in drug selection/prioritization. The comprehensive and sensitive nature of the data has also made detailed effects of each compound obtainable, providing a resource to assist in further mechanistic studies and prioritization of these compounds for the development of new antileishmanial drugs

Desarrollo de métodos analíticos para la determinación de seis especies químicas de arsénico en muestras medioambientales y orina mediante acoplamiento : cromatografía iónica de alta resolución-horno microondas-generación de hidruros-espectroscopía de absorción atómica

Se ha abordado el desarrollo del procedimiento analítico por acoplamiento de técnicas instrumentales para la determinación de seis especies de as (as(III), as(v), dma, mma, asb y asc). El acoplamiento se realiza con una técnica de separación de especies, como es la cromatografía liquida de alta eficacia (hplc), una interfase de termooxidación de las especies orgánicas (no formadoras de hidruros) y el sistema detector hg-aas. Asimismo, se han evaluado las prestaciones de diferentes columnas cromatográficas en función de los parámetros fisicoquímicos que influyen en la separación. El acoplamiento hplc-horno microondas-hg-aas se ha utilizado para la especiación de as en diversas muestras medioambientales y orina. Por otro lado, su aplicación en muestras de orina de individuos expuestos a las distintas especies de as, a través de la alimentación, ha permitido conocer la evolución de dichas especies en el organismo y distinguir entre las formas tóxicas y no tóxicas del as, así como abordar el problema de la estabilidad de las especies de as en aguas y orinas

A host-specific diaminobutyrate aminotransferase contributes to symbiotic performance, homoserine metabolism, and competitiveness in the Rhizobium leguminosarum/Pisum sativum system

16 Pág.Rhizobium leguminosarum bv. viciae (Rlv) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a diaminobutyrate-2-oxoglutarate aminotransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor β-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere.This work was funded by a grant from Agencia Española de Investigacion, Ministerio de Ciencia e Innovación (PID2021-124344OB-I00). MB-G was recipient of a fellowship from Programa Propio from Universidad Politécnica de Madrid.Peer reviewe

Metabolomic Reprogramming of C57BL/6-Macrophages during Early Infection with <i>L. amazonensis</i>

Leishmania survival inside macrophages depends on factors that lead to the immune response evasion during the infection. In this context, the metabolic scenario of the host cell–parasite relationship can be crucial to understanding how this parasite can survive inside host cells due to the host’s metabolic pathways reprogramming. In this work, we aimed to analyze metabolic networks of bone marrow-derived macrophages from C57BL/6 mice infected with Leishmania amazonensis wild type (La-WT) or arginase knocked out (La-arg−), using the untargeted Capillary Electrophoresis-Mass Spectrometry (CE-MS) approach to assess metabolomic profile. Macrophages showed specific changes in metabolite abundance upon Leishmania infection, as well as in the absence of parasite-arginase. The absence of L. amazonensis-arginase promoted the regulation of both host and parasite urea cycle, glycine and serine metabolism, ammonia recycling, metabolism of arginine, proline, aspartate, glutamate, spermidine, spermine, methylhistidine, and glutathione metabolism. The increased L-arginine, L-citrulline, L-glutamine, oxidized glutathione, S-adenosylmethionine, N-acetylspermidine, trypanothione disulfide, and trypanothione levels were observed in La-WT-infected C57BL/6-macrophage compared to uninfected. The absence of parasite arginase increased L-arginine, argininic acid, and citrulline levels and reduced ornithine, putrescine, S-adenosylmethionine, glutamic acid, proline, N-glutamyl-alanine, glutamyl-arginine, trypanothione disulfide, and trypanothione when compared to La-WT infected macrophage. Moreover, the absence of parasite arginase leads to an increase in NO production levels and a higher infectivity rate at 4 h of infection. The data presented here show a host-dependent regulation of metabolomic profiles of C57BL/6 macrophages compared to the previously observed BALB/c macrophages infected with L. amazonensis, an important fact due to the dual and contrasting macrophage phenotypes of those mice. In addition, the Leishmania-arginase showed interference with the urea cycle, glycine, and glutathione metabolism during host–pathogen interactions

Plasma Metabolic Signature of Atherosclerosis Progression and Colchicine Treatment in Rabbits.

Balloon catheter endothelial denudation in New Zealand white rabbits fed high cholesterol diet is a validated atherosclerosis model. Well-characterized in terms of atherosclerosis induction and progression, the metabolic changes associated with the atherosclerosis progression remain indeterminate. Non-targeted metabolomics permits to develop such elucidation and allows to evaluate the metabolic consequences of colchicine treatment, an anti-inflammatory drug that could revert these changes. 16 rabbits underwent 18 weeks of atherosclerosis induction by diet and aortic denudation. Thereafter animals were randomly assigned to colchicine treatment or placebo for 18 weeks while on diet. Plasma samples were obtained before randomization and at 36 weeks. Multiplatform (GC/MS, CE/MS, RP-HPLC/MS) metabolomics was applied. Plasma fingerprints were pre-processed, and the resulting matrixes analyzed to unveil differentially expressed features. Different chemical annotation strategies were accomplished for those significant features. We found metabolites associated with either atherosclerosis progression, or colchicine treatment, or both. Atherosclerosis was profoundly associated with an increase in circulating bile acids. Most of the changes associated with sterol metabolism could not be reverted by colchicine treatment. However, the variations in lysine, tryptophan and cysteine metabolism among others, have shown new potential mechanisms of action of the drug, also related to atherosclerosis progression, but not previously described.M.A.I. received funding from Airbus Defense and Space through the CLX-2 program developed with Comando da Aeronáutica (COMAER) and the Brazilian Government. This work has been partially funded by the Ministerio de Ciencia, Innovación y Universidades of Spain (MICINN RTI2018-095166-B-I00), the Spanish Society of Cardiology (“Proyecto investigación traslacional” to BI), the Carlos III Institute of Health (ISCiii FIS-FEDER PI14-01427 to JM), the Ministerio de Economía, Industria y Competitividad (MINECO SAF2017-84494-C2-1R to JR-C), a project granted by the BBVA Foundation to JR-C. This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (MDM-2017-0720). The CNIC is supported by the ISCiii, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.This work was supported by grants of Sao Paulo State Research Foundation (FAPESP 2019/10434-4 to S.E.T.B. and 2021/04464-8 and 2017/50333-7 to A.G.T.); Subdirección General de Redes y Centros de Investigación Cooperativa - FEDER (RICET RD16/0027/0010, and Ministerio de Ciencia e Innovación España. Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020-FEDER PID2019- 108166GB-100 /AEI/10.13039/501100011033 to L. R.), EADS-CASA/Brazilian Air Force (FAB) mobility program to M.L.L., and Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to M. L.L.Peer reviewe

A host-specific diaminobutyrate aminotransferase contributes to symbiotic performance, homoserine metabolism, and competitiveness in the Rhizobium leguminosarum/Pisum sativum system

Rhizobium leguminosarum bv. viciae (Rlv) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a diaminobutyrate-2-oxoglutarate aminotransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor β-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere

Leishmanicidal mechanism of sertraline

42 p.-8 fig.Drug repurposing affords the implementation of new treatments at a moderate cost and under a faster time-scale. Most of the clinical drugs against Leishmania share this origin. The antidepressant sertraline has been successfully assayed in a murine model of visceral leishmaniasis. Nevertheless, sertraline targets in Leishmania were poorly defined. In order to get a detailed insight into the leishmanicidal mechanism of sertraline on Leishmania infantum, unbiased multiplatform metabolomics and transmission electron microscopy were combined with a focused insight into the sertraline effects on bioenergetics metabolism of the parasite. Sertraline induced respiration uncoupling, a significant decrease of intracellular ATP level, and oxidative stress in L. infantum promastigotes. Metabolomics evidenced an extended metabolic disarray caused by sertraline. This encompasses a remarkable variation of the levels of thiol-redox and polyamine biosynthetic intermediates, as well as shortage of intracellular amino acids used as metabolic fuel by Leishmania Sertraline killed Leishmania through a multitarget mechanism of action, tackling essential metabolic pathways of the parasite. As such, sertraline is a valuable candidate for visceral leishmaniasis treatment under a drug repurposing strategy.This work was supported by grants of the Fondo de Investigaciones Sanitarias-ISCIII-FEDER (PI12-02706), and (RD16/0027/0010, Red de Enfermedades Tropicales, subprogram RETICS del Plan Estatal de I+D+i (2013-2016) co-financed with FEDER funds, SAF2015-65740-R, and CSIC PIE 201620E038 to L. R., São Paulo State Research Foundation (FAPESP 2015/23403-9 to A. G.T.); EADS-CASA/ Brazilian Air Force (FAB) mobility program to M.L.L., and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to M.L.L.Peer reviewe