Una Ressonància Magnètica Nuclear més ràpida i efectiva

Conèixer la puresa d'un fàrmac, caracteritzar un compost nou, analitzar contaminants atmosfèrics... Aquestes són només algunes de les qüestions que pot resoldre la Ressonància Magnètica Nuclear (RMN). Investigadors del Servei de RMN de la UAB han realitzat una sèrie d'experiments per reduir el temps necessari per obtenir les dades, abaratint a més el sempre elevat cost relacionat amb el temps de mesura en l'espectròmetre.Conocer la pureza de un fármaco, caracterizar un compuesto nuevo, analizar contaminantes atmosféricos... Éstas son sólo algunas de las cuestiones que puede resolver la Resonancia Magnética Nuclear (RMN). Investigadores del Servicio de RMN de la UAB han realizado una serie de experimentos para reducir el tiempo necesario para obtener los datos, abaratando además el siempre elevado coste relacionado con el tiempo de medida en el espectrómetro

Chloroperoxidase-catalyzed aminoalcohol oxidation : substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal

The ability of chloroperoxidase (CPO) to catalyze amino alcohol oxidations was investigated. The oxidations of compounds with different configurations with respect to the amine position towards hydroxyl - using H₂O₂ and tert-butyl hydroperoxide (t-BuOOH) - were analyzed in terms of the initial reaction rate, substrate conversion, and CPO operational stability. It was observed that the further the amino group from the hydroxyl, the lower the initial reaction rate. The effect of the amino-protecting group and other substituents (i.e., methyl and hydroxyl) was also examined, revealing an increase in steric hindrance due to the effect of bulky substituents. The observed reaction rates were higher with t-BuOOH, whereas CPO was more stable with H₂O₂. Moreover, CPO stability had to be determined case by case as the enzyme activity was modulated by the substrate. The oxidation of N-Cbz-3-aminopropanol (Cbz, carboxybenzyl) to N-Cbz-3-aminopropanal was investigated. Main operational conditions such as the reaction medium, initial amino alcohol concentration, and peroxide nature were studied. The reaction kinetics was determined, and no substrate inhibition was observed. By-products from a chemical reaction between the formed amino aldehyde and the peroxide were identified, and a novel reaction mechanism was proposed. Finally, the biotransformation was achieved by reducing side reactions and identifying the key factors to be addressed to further optimize the product yield

Chiral recognition by dissolution dynamic nuclear polarization NMR spectroscopy

The recognition of enantiomeric molecules by chemical analytical techniques is still a challenge. A method based on d-DNP (dissolution dynamic nuclear polarization) NMR spectroscopy to study chiral recognition was described for the first time [1]. DNP allows NMR sensitivity to be boosted by several orders of magnitude, overcoming one of the main limitations of NMR spectroscopy [2]. A method integrating d-DNP and 13C-NMR-aided enantiodifferentiation using chiral solvating agents (CSA) was developed, in which only the chiral analyte was hyperpolarized and selectively observed by NMR. The described method enhances the sensitivity of the conventional NMR-based procedure [3] and lightens the common problem of signal overlapping between analyte and CSA. As proof of concept, racemic metabolite 13C-labeled DL-methionine was enantiodifferentiated by a single-scan 13C-NMR experiment. This method entails a step forward in the chiral recognition of small molecules by NMR spectroscopy; it opens new possibilities in situations where the sensitivity is limited, for example, when low analyte concentration is available or when measurement of an insensitive nucleus is required. The advantages and current limitations of the method, as well as future perspectives, are discussed

Hyperpolarization-Enhanced NMR Spectroscopy of Unaltered Biofluids Using Photo-CIDNP

Altres ajuts: acords transformatius de la UABThe direct and unambiguous detection and identification of individual metabolite molecules present in complex biological mixtures constitute a major challenge in (bio)analytical research. In this context, nuclear magnetic resonance (NMR) spectroscopy has proven to be particularly powerful owing to its ability to provide both qualitative and quantitative atomic-level information on multiple analytes simultaneously in a noninvasive manner. Nevertheless, NMR suffers from a low inherent sensitivity and, moreover, lacks selectivity regarding the number of individual analytes to be studied in a mixture of a myriad of structurally and chemically very different molecules, e.g., metabolites in a biofluid. Here, we describe a method that circumvents these shortcomings via performing selective, photochemically induced dynamic nuclear polarization (photo-CIDNP) enhanced NMR spectroscopy on unmodified complex biological mixtures, i.e., human urine and serum, which yields a single, background-free one-dimensional NMR spectrum. In doing this, we demonstrate that photo-CIDNP experiments on unmodified complex mixtures of biological origin are feasible, can be performed straightforwardly in the native aqueous medium at physiological metabolite concentrations, and act as a spectral filter, facilitating the analysis of NMR spectra of complex biofluids. Due to its noninvasive nature, the method is fully compatible with state-of-the-art metabolomic protocols providing direct spectroscopic information on a small, carefully selected subset of clinically relevant metabolites. We anticipate that this approach, which, in addition, can be combined with existing high-throughput/high-sensitivity NMR methodology, holds great promise for further in-depth studies and development for use in metabolomics and many other areas of analytical research

Fatty Acids and Metabolomic Composition of Follicular Fluid Collected from Environments Associated with Good and Poor Oocyte Competence in Goats

In goats, embryo oocyte competence is affected by follicle size, regardless of the age of the females. In previous studies, we found differences in blastocyst development between oocytes coming of small ( 3 mm) in prepubertal (1-2 month-old) goats. Oocyte competence and follicular fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze the fatty acids (FAs) composition and metabolomic profiles of FF recovered from small and large follicles of prepubertal goats and follicles of adult goats. FAs were analyzed by chromatography and metabolites by 1H-nuclear magnetic resonance (1H-NMR) spectrometry. The results showed important differences between adult and prepubertal follicles: a) the presence of α,β-glucose in adult and no detection in prepubertal; b) lactate, -N-(CH3)3 groups and inositol were higher in prepubertal; c) the percentage of linolenic acid, total saturated fatty acids and n-3 PUFAs were higher in adults; and d) the percentage of linoleic acid, total MUFAs, PUFAs, n-6 PUFAs and n-6 PUFAs:n-3 PUFAs ratio were higher in prepubertal goats. No significant differences were found in the follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production

Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol

The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3-hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum-based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3-HP from glycerol through the malonyl-CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14-fold increase in the production of 3-HP compared to the initial strain. The best strain (PpHP6) was tested in a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g l−1, a product yield of 0.13 g g−1 and a volumetric productivity of 0.54 g l−1 h−1, which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3-HP

Opposite metabolic responses of shoots and roots to drought

Shoots and roots are autotrophic and heterotrophic organs of plants with different physiological functions. Do they have different metabolomes? Do their metabolisms respond differently to environmental changes such as drought? We used metabolomics and elemental analyses to answer these questions. First, we show that shoots and roots have different metabolomes and nutrient and elemental stoichiometries. Second, we show that the shoot metabolome is much more variable among species and seasons than is the root metabolome. Third, we show that the metabolic response of shoots to drought contrasts with that of roots; shoots decrease their growth metabolism (lower concentrations of sugars, amino acids, nucleosides, N, P, and K), and roots increase it in a mirrored response. Shoots are metabolically deactivated during drought to reduce the consumption of water and nutrients, whereas roots are metabolically activated to enhance the uptake of water and nutrients, together buffering the effects of drought, at least at the short term

Trehalose polyphleates, external cell wall lipids in Mycobacterium abscessus, are associated with the formation of clumps with cording morphology, which have been associated with virulence

Mycobacterium abscessus is a reemerging pathogen that causes pulmonary diseases similar to tuberculosis, which is caused by Mycobacterium tuberculosis. When grown in agar medium, M. abscessus strains generate rough (R) or smooth colonies (S). R morphotypes are more virulent than S morphotypes. In searching for the virulence factors responsible for this difference, R morphotypes have been found to form large aggregates (clumps) that, after being phagocytozed, result in macrophage death. Furthermore, the aggregates released to the extracellular space by damaged macrophages grow, forming unphagocytosable structures that resemble cords. In contrast, bacilli of the S morphotype, which do not form aggregates, do not damage macrophages after phagocytosis and do not form cords. Cording has also been related to the virulence of M. tuberculosis. In this species, the presence of mycolic acids and surface-exposed cell wall lipids has been correlated with the formation of cords. The objective of this work was to study the roles of the surface-exposed cell wall lipids and mycolic acids in the formation of cords in M. abscessus. A comparative study of the pattern and structure of mycolic acids was performed on R (cording) and S (non-cording) morphotypes derived from the same parent strains, and no differences were observed between morphotypes. Furthermore, cords formed by R morphotypes were disrupted with petroleum ether (PE), and the extracted lipids were analyzed by thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry. Substantial amounts of trehalose polyphleates (TPP) were recovered as major lipids from PE extracts, and images obtained by transmission electron microscopy suggested that these lipids are localized to the external surfaces of cords and R bacilli. The structure of M. abscessus TPP was revealed to be similar to those previously described in Mycobacterium smegmatis. Although the exact role of TPP is unknown, our results demonstrated that TPP are not toxic by themselves and have a function in the formation of clumps and cords in M. abscessus, thus playing an important role in the pathogenesis of this species

Coping with iron limitation : a metabolomic study of Synechocystis sp. PCC 6803

Iron (Fe) is a key element for all living systems, especially for photosynthetic organisms because of its important role in the photosynthetic electron transport chain. Fe limitation in cyanobacteria leads to several physiological and morphological changes. However, the overall metabolic responses to Fe limitation are still poorly understood. In this study, we integrated elemental, stoichiometric, macromolecular, and metabolomic data to shed light on the responses of Synechocystis sp. PCC 6803, a non-N2-fixing freshwater cyanobacterium, to Fe limitation. Compared to Synechocystis growing at nutrient replete conditions, Fe-limited cultures had lower growth rates and amounts of chlorophyll a, RNA, RNA:DNA, C, N, and P, and higher ratios of protein:RNA, C:N, C:P, and N:P, in accordance with the growth rate hypothesis which predicts faster growing organisms will have decreased biomass RNA contents and C:P and N:P ratios. Fe-limited Synechocystis had lower amounts Fe, Mn, and Mo, and higher amount of Cu. Several changes in amino acids of cultures growing under Fe limitation suggest nitrogen limitation. In addition, we found substantial increases in stress-related metabolites in Fe-limited cyanobacteria such antioxidants. This study represents an advance in understanding the stoichiometric, macromolecular, and metabolic strategies that cyanobacteria use to cope with Fe limitation. This information, moreover, may further understanding of changes in cyanobacterial functions under scenarios of Fe limitation in aquatic ecosystems