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Item type:Publication, Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid(Nature Research, 2020-12-01)Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Morphological and metabolic profiling of a tropical-adapted potato association panel subjected to water recovery treatment reveals new insights into plant vigor(Blackwell Publishing Ltd, 2020-09-01)Potato (Solanum tuberosum L.) is one of the world's most important crops, but it is facing major challenges due to climatic changes. To investigate the effects of intermittent drought on the natural variability of plant morphology and tuber metabolism in a novel potato association panel comprising 258 varieties we performed an augmented block design field study under normal irrigation and under water-deficit and recovery conditions in Ica, Peru. All potato genotypes were profiled for 45 morphological traits and 42 central metabolites via nuclear magnetic resonance. Statistical tests and norm of reaction analysis revealed that the observed variations were trait specific, that is, genotypic versus environmental. Principal component analysis showed a separation of samples as a result of conditional changes. To explore the relational ties between morphological traits and metabolites, correlation-based network analysis was employed, constructing one network for normal irrigation and one network for water-recovery samples. Community detection and difference network analysis highlighted the differences between the two networks, revealing a significant correlational link between fumarate and plant vigor. A genome-wide association study was performed for each metabolic trait. Eleven single nucleotide polymorphism (SNP) markers were associated with fumarate. Gene Ontology analysis of quantitative trait loci regions associated with fumarate revealed an enrichment of genes regulating metabolic processes. Three of the 11 SNPs were located within genes, coding for a protein of unknown function, a RING domain protein and a zinc finger protein ZAT2. Our findings have important implications for future potato breeding regimes, especially in countries suffering from climate change. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Study of the Acetylation Pattern of Chitosan by Pure Shift NMR(American Chemical Society, 2020-09-15)Chitosan is a biodegradable, antibacterial, and nontoxic biopolymer used in a wide range of applications including biotechnology, pharmacy, and medicine. The physicochemical and biological properties of chitosan have been associated with parameters such as the degree of polymerization (DP) and the fraction of acetylation ( F A ). New methods are being developed to yield chitosans of specific acetylation patterns, and, recently, a correlation between biological activity and the distribution of the acetylated units ( P A: pattern of acetylation) has been demonstrated. Although there are numerous well-established methods for the determination of DP and F A values, this is not the case for P A . The methods available are either not straightforward or not sensitive enough, limiting their use for routine analysis. In this study, we demonstrate that by applying HOmodecoupled Band-Selective (HOBS) decoupling NMR on signals assigned by multidimensional Pure Shift NMR methods, P A can be easily and accurately determined on various chitosan samples. This novel methodology—easily implemented for routine analysis—could become a standard for chitosan P A assessment. In addition, by applying Spectral Aliased Pure Shift HSQC, the analysis was enhanced with the determination of triads. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Measuring the 3JHNHa coupling by a simple 2D-intra-HNCA IP/AP-E.COSY with simultaneous encoding of 15N chemical shift and 1JHaCa evolution(Elsevier, 2021-12-18)The 3J coupling values are commonly used in biomolecular NMR to extract structural information. Here we present a novel intra HNCA IP/AP E.COSY pulse sequence that allows to measure 3J HNHa coupling constants by a simple and rapid two-dimensional 1H-15N correlation experiment where the 15N frequency is encoded at the same time as the 1J HaCa coupling evolution. The advantage with respect to the conventional 3D HNCA E.COSY pulse sequence is the dimensionality reduction to a simple 2D experiment, which decreases acquisition time and facilitates data analysis. The performance of this new experiment is demonstrated with an ubiquitin sample at 500 MHz. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, NMR-based leaf metabolic profiling of V. planifolia and three endemic Vanilla species from the Peruvian Amazon(Elsevier, 2021-10-01)The fruit of Vanilla planifolia is broadly preferred by the agroindustry and gourmet markets due to its refined flavor and aroma. Peruvian Vanilla has been proposed as a possible source for genetic improvement of existing Vanilla cultivars, but, little has been done to facilitate comprehensive studies of these and other Vanilla. Here, a nuclear magnetic resonance (NMR) metabolomic platform was developed to profile for the first time the leaves – organ known to accumulate vanillin putative precursors – of V. planifolia and those of Peruvian V. pompona, V. palmarum, and V. ribeiroi, with the aim to determine metabolic differences among them. Analysis of the NMR spectra allowed the identification of thirty-six metabolites, twenty-five of which were quantified. One-way ANOVA and post-hoc Tukey test revealed that these metabolites changed significantly among species, whilst multivariate-analyses allowed the identification of malic and homocitric acids, together with two vanillin precursors, as relevant metabolic markers for species differentiation. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Combined H-N cross-polarization and carbonyl detection NMR spectroscopy allow to record high-resolution, high-sensitivity spectra of alpha-synuclein in bacterial cells(Springer Science+Business Media, 2022-10-30)Studies of intrinsically disordered proteins (IDPs) under physiological conditions by conventional NMR methods based on proton detection are severely limited by fast proton amide solvent exchange. Carbon detection has been proposed as a solution to the exchange problem but is hampered by low sensitivity. Here, we present a protocol combining proton–nitrogen cross-polarization and carbonyl detection to record high-resolution and high-sensitivity NMR spectra of IDPs under physiological conditions. The protocol describes a step-by-step method to register high-quality N-CO correlation spectrum of alpha-synuclein in E.coli bacterial cells at 37 °C. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Urine 1H-NMR metabolomics to discriminate neurocysticercosis patients from healthy controls: an exploratory study(American Society of Tropical Medicine and Hygiene, 2022-07-01)The diagnosis of neurocysticercosis (NCC) is principally based on neuroimaging (magnetic resonance imaging or computed tomography), instrumentation that is scarcely available in the rural regions where Taenia solium transmission, primarily occurs due to poor sanitation conditions. Immunological assays for antigen or antibody detection complement the neuroimaging approach. However, no field-applicable assays to diagnose viable NCC or to guide the referral of cases for neuroimaging or for appropriate management are available. We performed an exploratory study on urine and serum samples using 1H-nuclear magnetic resonance (NMR)-based metabolomics to discriminate NCC patients (n 5 14) from healthy control subjects (n 5 22). Metabolic profiles demonstrated a discrimination between the urines of NCC patients and noninfected control subjects with a moderate predictive accuracy (R2 5 0.999, Q2 5 0.434). NMR metabolomics analysis has been proven useful in depicting biomarkers linked to other infectious diseases, various types of cancer, and other disorders. Our results, albeit preliminary, open a door to the development of better methods for detecting NCC through the identification of biomarkers participating in disturbed metabolic pathways.3 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Quantitative 1H Nuclear Magnetic Resonance Assay for the Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis from Sputum Samples(American Society for Microbiology, 2023-05-01)Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the 10 leading killer diseases in the world. At least one-quarter of the population has been infected, and there are 1.3 million deaths annually. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains challenges TB treatments. One of the drugs widely used in first- and second-line regimens is pyrazinamide (PZA). Statistically, 50% of MDR and 90% of XDR clinical strains are resistant to PZA, and recent studies have shown that its use in patients with PZA-resistant strains is associated with higher mortality rates. Therefore, the is an urgent need for the development of an accurate and efficient PZA susceptibility assay. PZA crosses the M. tuberculosis membrane and is hydrolyzed to its active form, pyrazinoic acid (POA), by a nicotinamidase encoded by the pncA gene. Up to 99% of clinical PZA-resistant strains have mutations in this gene, suggesting that this is the most likely mechanism of resistance. However, not all pncA mutations confer PZA resistance, only the ones that lead to limited POA production. Therefore, susceptibility to PZA may be addressed simply by its ability to form, or not, POA. Here, we present a nuclear magnetic resonance method to accurately quantify POA directly in the supernatant of sputum cultures collected from TB patients. The ability of the clinical sputum culture to hydrolyze PZA was determined, and the results were correlated with the results of other biochemical and molecular PZA drug susceptibility assays. The excellent sensitivity and specificity values attained suggest that this method could become the new gold standard for the determination of PZA susceptibility. - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Unraveling structural transitions and kinetics along the fold-switching pathway of the RfaH C-terminal domain using exchange-based NMR(National Academy of Sciences, 2025-05-20)The bacterial transcriptional regulator RfaH comprises structurally and functionally distinct N- (NTD) and C- (CTD) terminal domains. The latter switches from a helical hairpin packed against the NTD to a five-stranded β-roll upon displacement by RNA polymerase binding. Here, we use exchange-based NMR to probe fold-switching intermediates sampled by the isolated CTD. In addition to the predominant (~76 to 77%), semistable β-roll conformation (state A), we identify four structurally and kinetically distinct states: A′, B, B′, and B″. State B is NMR observable with an occupancy of ~23%, exchanges slowly (τ ex ~ 300 ms) with the major A species, and comprises a largely unfolded ensemble with transient occupancy of helical (α5*) and β-hairpin (β1*/β2*) elements. Backbone chemical shift-based structure predictions using the program CS-ROSETTA suggest that the two transient structural elements within the B state may interact with one another to form a semicompact structure. A′ (~0.35%) is an off-pathway state that exchanges rapidly (τ ex ~ 1 ms) with state A and likely entails a minor localized conformational change in the β1/β2 loop. State B′ (~0.3%) exchanges rapidly (τ ex ~ 1.2 ms) with state B and exhibits downfield 15 N backbone shifts (relative to B) in the α5* region indicative of reduced helicity. Finally state B″ (~0.05%) exchanges rapidly (τ ex ~ 0.8 to 1 ms) with either B′ (linear model) or B (branched model), displays significant differences in absolute 15 N chemical shift from states B and B′, and likely represents a further intermediate with increased helicity along the fold-switching pathway.1