Category Archives: Our Research

These are short reports about the research activities carried out at the SeRMN.
In them we describe the work done in collaboration with research groups, to summarize communications presented at scientific meetings, to report visits and stages at other laboratories or facilities, and to comment the meetings and workshops we have attended.

Exopolysaccharides from olive brines could reduce the adhesion of ETEC K88 to intestinal epithelial cells

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Zhu, Yanan, Gemma González-Ortiz, Rufino Jiménez-Díaz, Míriam Pérez-Trujillo, Teodor Parella, Paola López-Colom & Susana María Martín-Orúe. 2018. Exopolysaccharides from olive brines could reduce the adhesion of ETEC K88 to intestinal epithelial cells. Food & Function 9(7). 3884–3894. DOI: https://doi.org/10.1039/c8fo00690c

This study aims to explore the biological functions of the isolated exopolysaccharides (EPSs) produced during the industrial fermentation of olives against enterotoxigenic E. coli (ETEC) K88. Exopolysaccharides were isolated from five industrial fermenters. Analysis of their monosaccharide composition by GLC revealed that the main components were glucose (27%–50%) and galactose (23%–33%) followed by rhamnose (4–23%) and arabinose (6–17%). The 1H NMR spectrum showed a very similar profile between samples, and a more in-depth analysis revealed the presence of an α-pyranose in the form of α-D-Glcp-(1→) and two different α-furanoses, with chemicals shift values, suggesting the presence of α-D-Glcf and α-D-Galf. Miniaturized in vitro tests demonstrated the ability of EPS samples to attach specifically to ETEC K88 (P < 0.05) with variable intensities. The competition test did not show the ability to block the ETEC K88 adhesion to IPEC-J2 cells; however, in the displacement test, all EPS samples were shown to effectively remove the pathogens attached to the cells (P < 0.01).

These results suggest that the EPSs produced during the fermentation of table green olives could interfere with the attachment of opportunistic pathogens onto the intestinal epithelial cells. This would open the possibility of novel functional properties for this traditional Mediterranean fermented food and for the isolated EPSs as candidates for nutraceutics to be used in human and/or animal diets in the prevention and treatment of ETEC diarrhoea.

PhD Thesis: Development of Resolution-Enhanced NMR Techniques for Improved Small Molecules Structural Analysis

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Last July 14th 2018 Núria Marcó defended her PhD Thesis entitled: Development of Resolution-Enhanced NMR Techniques for Improved Small Molecules Structural Analysis

The present doctoral thesis is framed within the field of Nuclear Magnetic Resonance (NMR) spectroscopy.

NMR spectroscopy is an analytic technique and, therefore, one of its main objectives is to unravel the correct structure of the molecules analyzed.This present doctoral thesis  is focused on this main objective. This work consists in a compendium of 7 publications, written in several prestigious scientific journals, that develop in depth the efficient and accurate determination of the constitution, configuration and conformation of small molecules thanks to the application of resolution improvements techniques.

Continue reading PhD Thesis: Development of Resolution-Enhanced NMR Techniques for Improved Small Molecules Structural Analysis →

PhD Thesis: New Applications of Covariance NMR and Experimental Development for Measurements of Homonuclear Coupling Constants in Overlapping Signals

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On 21st March 2018 I sucessfully defended my PhD Thesis entitled: “New Applications of Covariance NMR and Experimental Development for Measurements of Homonuclear Coupling Constants in Overlapping Signals” (ISBN: 9788449079252), supervised by Dr. Teodor Parella and Dr. Pau Nolis, and obtained the degree of Ph.D. in Chemistry at the Department of Chemistry, Universitat Autònoma de Barcelona.

The thesis can be downloaded in PDF format from the TDX repository and from the TESEO repository.

Accurate measurement of proton homonuclear coupling constants in overlapped signals by using a doubly-selective TOCSY G-SERF experiment.

Abstract

The experimental results obtained in this thesis are presented in the form of three papers published in NMR specialised scientific peer-reviewed journals.

  • Exploring the use of Generalized Indirect Covariance to Reconstruct Pure shift NMR Spectra: Current Pros and Cons. André Fredi, Pau Nolis, Carlos Cobas, Gary E. Martin and Teodor Parella. Journal of Magnetic Resonance, Volume 266, May 2016, Pages 16-22. DOI: 10.1016/j.jmr.2016.03.003
  • Access to experimentally infeasible spectra by pure-shift NMR covariance. André Fredi, Pau Nolis, Carlos Cobas and Teodor Parella. Journal of Magnetic Resonance, Volume 270, September 2016, Pages 161-168. DOI: 10.1016/j.jmr.2016.07.010
  • Accurate measurement of JHH in overlapped signals by a TOCSY‐edited SERF Experiment. André Fredi, Pau Nolis and Teodor Parella. Magnetic Resonance in Chemistry, Volume 55, Issue 6, June 2017, Pages 525-529. DOI: 10.1002/mrc.4572

The first two articles deal with the use of covariance NMR as a general method to generate novel psNMR spectra. The last work describes a new selTOCSY G-SERF experiment, for accurately measuring JHH in overlapped regions.

The first publication describes a novel general protocol to generate psNMR spectra by Covariance NMR. This new approach is unique in NMR spectroscopy; giving a cheap, fast an easy way to reconstruct psNMR spectra without spending time in the spectrometer. This new strategy has been referenced to as psNMR Covariance.

The concept of psNMR Covariance has been extended in the second publication by inserting Multiplicity-Edited (ME) information into 2D experiments that are difficult or even impossible to achieve experimentally. It is shown how the ME information can be efficiently transferred to a set of homonuclear and heteronuclear 2D NMR spectra by Covariance processing, reconstructing new psME spectra in a fast way. Finally, G-SERF and related methods only work for isolated 1H signals on which selective excitation can be successfully applied.

Unfortunately, as it happens in other frequency-selective experiments, this approach fails for overlapped signals. A doubly-selective TOCSY G-SERF scheme is presented in the third publication to circumvent this limitation, by measuring JHH efficiently even for protons resonating in crowded regions.

Precise characterization of mycobacterial cell wall lipid PTTM

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“Molecule confirmation and structure characterization of pentatriacontatrienyl mycolate in Mycobacterium smegmatis” by M. Llorens-Fons, E. Julián, M. Luquin and M. Pérez-Trujillo. Chemistry and Physics of Lipids, 2018, Accepted Manuscript. DOI: https://doi.org/10.1016/j.chemphyslip.2017.12.006

Mycobacterium smegmatis is often used to study the different components of mycobacterial cell wall. Mycolic acids are important components of mycobacterial cell wall that have been associated with virulence. Recently, a novel lipid containing mycolic acids has been described in M. smegmatis. However, some uncertainties regarding the structure of this molecule named mycolate ester wax have been reported. The objective of this work was to perform an in depth structural study of this molecule for its precise characterization. Using 1H and 13C NMR spectroscopy, the molecular structure of mycolate ester wax found in M. smegmatis has been elucidated. The characterization was complemented with MS analyses. This molecule is formed by a carbon chain with three methyl substituted olefinic units and a mycolate structure with trans double bonds and cis cyclopropane rings. The present molecular study will facilitate the detection and identification of pentatriacontatrienyl mycolate (PTTM) in future studies by the performance of a simple 1D 1H NMR experiment.

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Multiplicity-edited 1H-1H TOCSY experiment

Pau Nolis and Teodor Parella

Magnetic Resonance in Chemistry 2017 (DOI: 10.1002/mrc.4695)

Abstract

A 1H-1H TOCSY experiment incorporating 13C multiplicity information is proposed. In addition, broadband 1H homodecoupling in the indirect dimension can be implemented using a perfect BIRD module that affords exclusive 1H chemical shift evolution with full decoupling of all heteronuclear and homonuclear (including 2JHH) coupling constants. As a complement to the normal TOCSY and the recent PSYCHE-TOCSY experiments, this novel multiplicity-edited TOCSY experiment distinguishes between CH/CH3 (phased up) and CH2 (phased down) cross-peaks which facilitates resonance analysis and assignment.

SeRMN contribution to Symmetry 2017 Conference

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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 [1]. DNP allows boosting NMR sensitivity 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 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.

NMR could improve the detection of “date rape” drug GHB

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“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: https://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

NMR identification of monstrous mycobacterial lipids in cell wall of Mycobacterium abcessus

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” 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: https://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.


A New Chirally Organized Trifluoromethylanthrylmethanol Derivative and Its Application as Chiral Solvating Agent

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ChemistrySelect Journal“A New Chirally Organized Trifluoromethylanthrylmethanol Derivative and Its Application as Chiral Solvating Agent” By Eva Monteagudo, Pere de March, Ángel Álvarez‐Larena and Albert Virgili. ChemistrySelect, 2017, 2, pp. 7362-7367 DOI:10.1002/slct.201701429

The synthesis and structure of 1,1′‐(((10,10’‐(1,1′‐binaphthalene)‐2,2′‐diylbis(oxy))bis(methylene))bis(anthracene‐10,9‐diyl))bis(2,2,2‐trifluoroethanol), 4, is reported. This compound owns both axial and central chirality allowing its use as a chiral solvating agent (CSA) for the enantiomeric composition determination of several mixtures of chiral aromatic alcohols and amines using NMR. The study of the resulting diastereoisomeric complexes was carried out by determining its stoichiometry and association binding constants.

Removal of pharmaceuticals from hospital wastewater by Pleurotus ostreatus. Identification of pharmaceuticals metabolites by NMR

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“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: https://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.