Some of the SeRMN staff presented our last research work about chirality at The first International Conference on Symmetry, Symmetry 2017, that took place from16th to 18th October in Barcelona. Find below a summary of our contribution.
Míriam Pérez-Trujillo presented a lecture entitled: “Chiral Recognition by Dissolution Dynamic Nuclear Polarization NMR Spectroscopy”
Abstract: 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 . DNP allows boosting NMR sensitivity by several orders of magnitude, overcoming one of the main limitations of NMR spectroscopy . 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  and lightens the common problem of signal overlapping between analyte and CSA. As proof on 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 available or when measurement of an insensitive nucleus required. The advantages and current limitations of the method, as well as future perspectives, are discussed.
“Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy” by M. Palomino-Schätzlein, Y. Wang, A. Brailsford, T. Parella, D. Cowan, C. Legido-Quigley, M. Pérez-Trujillo. Anal. Chem., 2017, 89 (16), pp 8343–8350. DOI: http://dx.doi.org/10.1021/acs.analchem.7b01567
γ-Hydroxybutyric acid (GHB) is a popular drug increasingly associated with cases of drug-facilitated sexual assault (DFSA). Currently, expanding procedures of analysis and having forensic evidence of GHB intake in a long term are mandatory. Up to now, most studies have been performed using GC/MS and LC-MS as analytical platforms, which involve significant manipulation of the sample and, often, indirect measurements. In this work, procedures used in NMR-based metabolomics were applied to a GHB clinical trial on urine and serum. Detection, identification, and briefly quantification of the drug by NMR methods were surveyed, as well as the use of NMR-based metabolomics for the search of potential surrogate biomarkers of GHB consumption. Results demonstrated the suitability of NMR spectroscopy, as a robust nondestructive technique, to fast and directly monitor exogenous GHB in almost intact body fluids and its high potential in the search for metabolites associated with GHB intake. This initial work show some strengths of NMR spectroscopy and standard methods routinely used in the NMR analysis of biological samples to approach the problem. These features could open up new interesting possibilities in future studies, complementing current procedures.
This work on media: spectroscopynow.com / phys.org / sciencedaily.com / canadafreepress.com / forensicmag.com / cbinsights.com
” Trehalose polyphleates, external cell wall lipids in Mycobacterium abcessus, are associated with the formation of clumps with cording morphology, which have been associated with virulence” by M. Llorens-Fons, M. Pérez-Trujillo, E. Julián, C. Brambilla, F. Alcaide, T. F. Byrd and M. Luquin. Frontiers in Microbiology, 2017, 8:1402. DOI: http://dx.doi.org/10.3389/fmicb.2017.01402
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. 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.
“Preliminary evaluation of Pleurotus ostreatus for the removal of selected pharmaceuticals from hospital wastewater” by L. Palli,* F. Castellet‐Rovira, M. Pérez‐Trujillo, D. Caniani, M. Sarrà‐Adroguer, R. Gori Biotechnology Progress, 2017. DOI: http://dx.doi.org/10.1002/btpr.2520
The fungus Pleurotus ostreatus was investigated to assess its ability to remove diclofenac, ketoprofen, and atenolol in hospital wastewater. The degradation test was carried out in a fluidized bed bioreactor testing both the batch and the continuous mode. In batch mode, diclofenac disappeared in less than 24 h, ketoprofen was degraded up to almost 50% in 5 days while atenolol was not removed. In continuous mode, diclofenac and ketoprofen removals were about 100% and 70% respectively; atenolol degradation was negligible during the first 20 days but it increased up to 60% after a peak of laccase production and notable biomass growth. In order to identify the enzymatic system involved, further experiments were carried out in flasks. Two intermediates of diclofenac and ketoprofen were detected by nuclear magnetic resonance (NMR) spectroscopy. Moreover P. ostreatus was able to reduce chemical oxygen demand of the hospital wastewater which is an important advantage comparing to other fungi in order to develop a wastewater treatment process.
“Long-term fertilization determines different metabolomic profiles and responses in saplings of three rainforest tree species with different adult canopy position” by A. Gargallo-Garriga, S. J. Wright, J. Sardans, M. Pérez-Trujillo, M. Oravec, K. Večeřová,O. Urban, M. Fernández-Martínez, T. Parella, J. Peñuelas.
Plos One, 2017, 1-21. DOI: 10.1371/journal.pone.0177030
Tropical rainforests are frequently limited by soil nutrient availability. However, the response of the metabolic phenotypic plasticity of trees to an increase of soil nutrient availabilities is poorly understood. We expected that increases in the ability of a nutrient that limits some plant processes should be detected by corresponding changes in plant metabolome profile related to such processes. We studied the foliar metabolome of saplings of three abundant tree species in a 15 year field NPK fertilization experiment in a Panamanian rainforest. The largest differences were among species and explained 75% of overall metabolome variation.
“Mycobacteria clumping increase their capacity to damage macrophages” by C. Brambilla, M. Llorens-Fons, E. Julián, E. Noguera-Ortega, C. Tomàs-Martínez, M. Pérez-Trujillo, T. F. Byrd, F. Alcaide and M. Luquin.
Front. Microbiol. 7:1562. DOI: 10.3389/fmicb.2016.01562
The rough morphotypes of non-tuberculous mycobacteria have been associated with the most severe illnesses in humans. This idea is consistent with the fact that Mycobacterium tuberculosis presents a stable rough morphotype. Unlike smooth morphotypes, the bacilli of rough morphotypes grow close together, leaving no spaces among them and forming large aggregates (clumps). Currently, the initial interaction of macrophages with clumps remains unclear. Thus, we infected J774 macrophages with bacterial suspensions of rough morphotypes of Mycobacterium abscessus containing clumps and suspensions of smooth morphotypes, primarily containing isolated bacilli. Using confocal laser scanning microscopy and electron microscopy, we observed clumps of at least 5 rough-morphotype bacilli inside the phagocytic vesicles of macrophages at 3 hours post-infection. These clumps grew within the phagocytic vesicles, killing 100% of the macrophages at 72 hours post-infection, whereas the proliferation of macrophages infected with smooth morphotypes remained unaltered at 96 hours post-infection. Thus, macrophages phagocytose large clumps, exceeding the bactericidal capacities of these cells. Furthermore, proinflammatory cytokines and granuloma-like structures were only produced by macrophages infected with rough morphotypes. Thus, the present study provides a foundation for further studies that consider mycobacterial clumps as virulence factors.
Figure. Content of GPL and structure of mycolic acids. (A) 1-D TLC analysis of the crude lipid extracts of M. abscessus strains. (B) 1H-NMR spectra of purified mycolic acid methyl esters from M. abscessus. (C) Relative molar ratios of molecular moieties cis-db, trans-db, cis-cp and trans-cp of mycolic acid methyl esters from M. abscessus.
The 32nd European Embryo Transfer Association Meeting of 2016 was held in Barcelona (from the 9th to the 10th of September).
We presentented the poster:
“Nuclear magnetic resonance (NMR) of goat follicular fluid shows different metabolic profiles among follicle size and female age” of S. Soto, M. Pérez-Trujillo, M.G. Catalá, M. Roura, D. Izquierdo, T. Parella, M.T. Paramio.
Abstract: Oocytes recovered from prepubertal goats are very heterogeneous in growth and grade of atresia which make them unpredictable for IVEP programs. We have observed that oocytes from prepubertal goats obtained from >3 mm follicles develop up to blastocyst stage at a similar percentage than oocytes from adult goats (18% vs 21%), suggesting that the follicle development and the follicular fluid (FF) content are more relevant to oocyte competence than the age of the donor. The aim of this study is to characterize the FF metabolomic profile from different follicular environments through a high-resolution 1H NMR-based metabolomic study. Samples of adult (n=40) and prepubertal (n=16) FF where collected by laparoscopic ovum pick-up (LOPU) and by aspiration of slaughterhouse ovaries, respectively. FF from small (< 3 mm) and large (> 3 mm) diameter follicles where pooled for each female. Multivariate ordination principal component analysis (PCA) was performed to detect patterns of sample ordination in the metabolomes. The unsupervised method clearly differed between the FF metabolomes of large and small follicles of prepubers and between the FF of preadolescent and adult individuals.
Figure. a) PCA scores plot (PC1-PC2) from 1H NMR spectral data of follicular fluid samples of preadolescent (n=16; blue dots) and adult (n=40; black dots) goats. b) PCA heat map loadings plot (PC1-PC2) with some discriminant variables assigned.
“Chloroperoxidase-catalyzed amino alcohol oxidation: Substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal” by G. Masdeu, M. Pérez-Trujillo, J. López-Santín and Gregorio Álvaro. Process Biochemistry 2016; DOI:10.1016/j.procbio.2016.05.022
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. Continue reading Chloroperoxidase-catalyzed amino alcohol oxidation: Substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal
Today we are saying goodbye to our dear Yaoyao, though we hope to see her very soon again.
Yaoyao is currently finishing her PhD on metabonomics applied to clinical biomarkers in the Legido-Quigley Lab at King’s College London (KCL).
She has been visiting us for the last two months, during which we have been working together in two metabonomics projects related to drug misuse biomarkers and chiral metabonomics. It has been a great pleasure for us to spend this time with her and continue with this collaboration from now on.
Yaoyao Wang, visiting PhD student from King’s College London, will be giving a talk on December the 18th at 10:00 h in the SeRMN. Her talk is entitled “Biomarker and metabolomics: a novel approach to detect drug misuse”.
Yaoyao is from Clinical Biomarkers Lab of the Institute of Pharmaceutical Science, King’s College London (KCL). Her PhD project focuses on developing metabolomics approach to identify biomarkers for drug misuse through the analysis of human biofluids samples and the development of data treatment methods, using R (XCMS) and other bioinformatics tools. She has been collaborating intensively with the Drug Control Centre of KCL using LC-MS.
Her talk will include the metabolomics biomarker discovery of low dose salbutamol in urine collected for anti-doping tests and preliminary longitudinal metabolomics study of “date-rape” drug GHB, as well as a glance of the other on-going projects in Clinical Biomarkers Lab.
All interested people are welcome to attend this seminar.