1. Investigación

Short-chain fatty acids (SCFAs) are organic saturated monocarboxylic acids with fewer than six carbon atoms. Their recently described relevance in health and disease has brought to the table the need for a convenient method for their analysis. The current research presents an optimized and validated LC-QqQ-MS method for the absolute quantification of SCFAs in plasma and feces. This method has proven to be accurate and precise in a wide range of concentrations (0.2 – 200 µM). It has been applied to more than 500 samples showing good precision, reproducibility, and linearity results. The method was applied to study samples from anorexia nervosa (AN) patients and matched healthy controls (HC). The results showed significant differences in the concentration of plasmatic and fecal SCFAs between both groups whose implications in AN are yet to be determined. Nonetheless, these findings denote important alterations in the SCFAs homeostasis in AN which can be linked to the metabolic alterations and the intestinal dysbiosis already characterized in the disease. More research is still required to identify the function of these metabolites in the pathophysiology of AN, but the present work provides a reliable methodology and valuable information to continue delving into the potential role of the microbiota as a therapeutic target via the gut-brain axis.

Metabolomics has become an essential discipline in the study of microbiome, emerging gas chromatography coupled to mass spectrometry as the most mature, robust, and reproducible analytical technique. Silylation is the most widely used chemical derivatization strategy, although it has some limitations. In this regard, alkylation by alkyl chloroformate offers some advantages, such as a rapid reaction, milder conditions, better reproducibility, and the generation of more stable derivatives. However, commercial spectral libraries do not include many of the alkyl derivatives, mainly for polyfunctional metabolites, which can form multiple derivatives. That introduces a huge bias in untargeted metabolomics leading to common errors such as duplicates, unknowns, misidentifications, wrong assignations, and incomplete results from which non-reliable findings and conclusions will be retrieved. For this reason, the purpose of this study is to overcome these shortcomings and to expand the knowledge of metabolites in general and especially those closely related to the gut microbiota through the thorough study of the reactivity of the different functional groups in real matrix derivatized by methyl chloroformate, a common representative alkylation reagent. To this end, a systematic workflow has been developed based on exhaustive structural elucidation, along with computational simulation, and taking advantage of the high sensitivity and high-resolution gas chromatography-mass spectrometry. Several empirical rules have been established according to chemically different entities (free fatty acids, amino acids, polyols, sugars, amines, and polyfunctional groups, etc.) to predict the number of derivatives formed from a single metabolite, as well as their elution order and structure. In this work, some methyl chloroformate derivatives not previously reported as well as the mechanisms to explain them are given. Extremely important is the interconversion of E- and Z- geometric isomers of unsaturated dicarboxylic acids (case of fumaric-maleic and case of citraconic-mesaconic acids), or the formation of cycled derivatives for amino acids, as well as common metabolites, as in the case of serine and cysteine, and many others.

GC-MS for untargeted metabolomics is a well-established technique. Small molecules and molecules made volatile by derivatization can be measured and those compounds are key players in main biological pathways. This tutorial provides ready-to-use protocols for GC-MS-based metabolomics, using either the well-known low-resolution approach (GC-Q-MS) with nominal mass or the more recent high-resolution approach (GC-QTOF-MS) with accurate mass, discussing their corresponding strengths and limitations. Analytical procedures are covered for different types of biofluids (plasma/serum, bronchoalveolar lavage, urine, amniotic fluid) tissue samples (brain/hippocampus, optic nerve, lung, kidney, liver, pancreas) and samples obtained from cell cultures (adipocytes, macrophages, Leishmania promastigotes, mitochondria, culture media). Together with the sample preparation and data acquisition, data processing strategies are described specially focused on Agilent equipments, including deconvolution software and database annotation using spectral libraries. Manual curation strategies and quality control are also deemed. Finally, considerations to obtain a semiquantitative value for the metabolites are also described. As a case study, an illustrative example from one of our experiments at CEMBIO Research Centre, is described and findings discussed.

Plasmid-mediated antimicrobial resistance is one of the major threats to public health worldwide. The mechanisms involved in the plasmid/host coadaptation are still poorly characterized, and their understanding is crucial to comprehend the genesis and evolution of multidrug-resistant bacteria. With this purpose, we designed an experimental evolution using Haemophilus influenzae RdKW20 as the model strain carrying the ColE1-like plasmid pB1000. Five H. influenzae populations adapted previously to the culture conditions were transformed with pB1000 and subsequently evolved to compensate for the plasmid-associated fitness cost. Afterward, we performed an integrative multiomic analysis combining genomics, transcriptomics, and metabolomics to explore the molecular mechanisms involved in the compensatory evolution of the plasmid. Our results demonstrate that minimal modifications in the host are responsible for plasmid adaptation. Among all of them, the most enriched process was amino acid metabolism, especially those pathways related to serine, tryptophan, and arginine, eventually related to the genesis and resolution of plasmid dimers. Additional rearrangements occurred during the plasmid adaptation, such as an overexpression of the ribonucleotide reductases and metabolic modifications within specific membrane phospholipids. All these findings demonstrate that the plasmid compensation occurs through the combination of diverse host-mediated mechanisms, of which some are beyond genomic and transcriptomic modifications. IMPORTANCE The ability of bacteria to horizontally transfer genetic material has turned antimicrobial resistance into one of the major sanitary crises of the 21st century. Plasmid conjugation is considered the main mechanism responsible for the mobilization of resistance genes, and its understanding is crucial to tackle this crisis. It is generally accepted that the acquisition and maintenance of mobile genetic elements entail a fitness cost to its host, which is susceptible to be alleviated through a coadaptation process or compensatory evolution. Notwithstanding, despite recent major efforts, the underlying mechanisms involved in this adaptation remain poorly characterized. Analyzing the plasmid/host coadaptation from a multiomic perspective sheds light on the physiological processes involved in the compensation, providing a new understanding on the genesis and evolution of plasmid-mediated antimicrobial-resistant bacteria.

Leishmania donovani infection of macrophages drives profound changes in the metabolism of both the host macrophage and the parasite, which undergoes different phases of development culminating in replication and propagation. However, the dynamics of this parasite-macrophage cometabolome are poorly understood. In this study, a multiplatform metabolomics pipeline combining untargeted, high-resolution CE-TOF/MS and LC-QTOF/MS with targeted LC-QqQ/MS was followed to characterize the metabolome alterations induced in L. donovani-infected human monocyte-derived macrophages from different donors at 12, 36, and 72 h post-infection. The set of alterations known to occur during Leishmania infection of macrophages, substantially expanded in this investigation, characterized the dynamics of the glycerophospholipid, sphingolipid, purine, pentose phosphate, glycolytic, TCA, and amino acid metabolism. Our results showed that only citrulline, arginine, and glutamine exhibited constant trends across all studied infection time points, while most metabolite alterations underwent a partial recovery during amastigote maturation. We determined a major metabolite response pointing to an early induction of sphingomyelinase and phospholipase activities and correlated with amino acid depletion. These data represent a comprehensive overview of the metabolome alterations occurring during promastigote-to-amastigote differentiation and maturation of L. donovani inside macrophages that contributes to our understanding of the relationship between L. donovani pathogenesis and metabolic dysregulation.

Haemophilus influenzae is a gram-negative bacterium of relevant clinical interest. H. influenzae Rd KW20 was the first organism to be sequenced and for which a genome-scale metabolic model (GEM) was developed. However, current H. influenzae GEMs are unable to capture several aspects of metabolome nature related to metabolite pools. To directly and comprehensively characterize the endometabolome of H. influenzae Rd KW20, we performed a multiplatform MS-based metabolomics approach combining LC-MS, GC-MS and CE-MS. We obtained direct evidence of 15-20% of the endometabolome present in current H. influenzae GEMs and showed that polar metabolite pools are interconnected through correlating metabolite islands. Notably, we obtained high-quality evidence of 18 metabolites not previously included in H. influenzae GEMs, including the antimicrobial metabolite cyclo(Leu-Pro). Additionally, we comprehensively characterized and evaluated the quantitative composition of the phospholipidome of H. influenzae, revealing that the fatty acyl chain composition is largely independent of the lipid class, as well as that the probability distribution of phospholipids is mostly related to the conditional probability distribution of individual acyl chains. This finding enabled us to provide a rationale for the observed phospholipid profiles and estimate the abundance of low-level species, permitting the expansion of the phospholipidome characterization through predictive probabilistic modelling.

The mechanisms whereby Mycobacterium tuberculosis (Mtb) rewires the host metabolism in vivo are surprisingly unexplored. Here, we used three high-resolution mass spectrometry platforms to track altered lung metabolic changes associated with Mtb infection of mice. The multiplatform data sets were merged using consensus orthogonal partial least squaresdiscriminant analysis (cOPLS-DA), an algorithm that allows for the joint interpretation of the results from a single multivariate analysis. We show that Mtb infection triggers a temporal and progressive catabolic state to satisfy the continuously changing energy demand to control infection. This causes dysregulation of metabolic and oxido-reductive pathways culminating in Mtbassociated wasting. Notably, high abundances of trimethylamine-N-oxide (TMAO), produced by the host from the bacterial metabolite trimethylamine upon infection, suggest that Mtb could exploit TMAO as an electron acceptor under anaerobic conditions. Overall, these new pathway alterations advance our understanding of the link between Mtb pathogenesis and metabolic dysregulation and could serve as a foundation for new therapeutic intervention strategies. Mass spectrometry data has been deposited in the Metabolomics Workbench repository (data-set identifier: ST001328).

Glioma tumors are one of the most devastating cancer types. Glioblastoma is the most advanced stage with the worst prognosis. Current therapies are still unable to provide an effective cure. Recent advances in oncolytic immunotherapy have generated great expectations in the cancer therapy field. The use of oncolytic viruses (OVs) in cancer treatment is one such immune-related therapeutic alternative. OVs have a double oncolytic action by both directly destroying the cancer cells and stimulating a tumor specific immune response to return the ability of tumors to escape the control of the immune system. OVs are one promising alternative to conventional therapies in glioma tumor treatment. Several clinical trials have proven the feasibility of using some viruses to specifically infect tumors, eluding undesired toxic effects in the patient. Here, we revisited the literature to describe the main OVs proposed up to the present moment as therapeutic alternatives in order to destroy glioma cells in vitro and trigger tumor destruction in vivo. Oncolytic viruses were divided with respect to the genome in DNA and RNA viruses. Here, we highlight the results obtained in various clinical trials, which are exploring the use of these agents as an alternative where other approaches provide limited hope.

Inorganic materials can provide a set of tools to decontaminate solid, liquid or air containing viral particles. The use of disinfectants can be limited or not practical in scenarios where continuous cleaning is not feasible. Physicochemical differences between viruses raise the need for effective formulations for all kind of viruses. In the present work we describe two types of antimicrobial inorganic materials: i) a novel soda-lime glass (G3), and ii) kaolin containing metals nanoparticles (Ag or CuO), as materials to disable virus infectivity. Strong antiviral properties can be observed in G3 glass, and kaolin-containing nanoparticle materials showing a reduction of viral infectivity close to 99% in the first 10 ​min of contact of vesicular stomatitis virus (VSV). A potent virucidal activity is also present in G3 and kaolin containing Ag or CuO nanoparticles against all kinds of viruses tested, reducing more than 99% the amount of HSV-1, Adenovirus, VSV, Influenza virus and SARS-CoV-2 exposed to them. Virucidal properties could be explained by a direct interaction of materials with viruses as well as inactivation by the presence of virucidal elements in the material lixiviates. Kaolin-based materials guarantee a controlled release of active nanoparticles with antiviral activity. Current coronavirus crisis highlights the need for new strategies to remove viruses from contaminated areas. We propose these low-cost inorganic materials as useful disinfecting antivirals in the actual or future pandemic threats.