Category Archives: Methods & Applications

Posts describing the use of the nmr spectroscopy (MRS) methodology, either from a practical point of view (how to perform certain experiment), or from a more theoretical perspective (description of techniques and their application).

MRI/MRS in treated Alzheimer mice

Differential effects of apoE and apoJ mimetic peptides on the action of an anti-Aβ scFv in 3xTg-AD mice” by L. Montoliu-Gaya, J. Güell-Bosch, G. Esquerda-Canals, A.R. Roda, G. Serra-Mir, S. Lope-Piedrafita, J.L. Sánchez-Quesada, S. Villegas. Biochem Pharmacol. 2018, 155:380-392. DOI: 10.1016/j.bcp.2018.07.012.

Anti-Aβ immunotherapy has emerged as a promising approach to treat Alzheimer’s disease (AD). The single-chain variable fragment scFv-h3D6 is an anti-Aβ antibody fragment that lacks the Fc region, which is associated with the induction of microglial reactivity by the full-length monoclonal antibody bapineuzumab. ScFv-h3D6 was previously shown to restore the levels of apolipoprotein E (apoE) and apolipoprotein J (apoJ) in a tripletransgenic- AD (3xTg-AD) mouse model. Since apoE and apoJ play an important role in the development of AD, we aimed to study the in vivo effect of the combined therapy of scFv-h3D6 with apoE and apoJ mimetic peptides (MPs).

Magnetic Resonance Imaging showed a general tendency of the different treatments to protect against the reduction in brain volume. This protection was further suggested by MRS, since all the treatments tended to recover the levels of Ala, which is involved in the alanine-glucose cycle, and NAA, which is a marker for cell viability. All the treatments in the present work recovered the IL-33 levels, and apoE-MP showed a potent anti-inflammatory effect in terms of glial activation that was decreased in the presence of scFv-h3D6, whereas the combination of apoJ-MP and scFvh3D6 was not detrimental. Moreover, the endogenous apoE and apoJ levels were decreased by scFvh3D6, but the MPs induced a simultaneous increase in both apolipoproteins, which reflects a coordinated expression between them.

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

“Exopolysaccharides from olive brines could reduce the adhesion of ETEC K88 to intestinal epithelial cells”  by Y. Zhu, G. Gonzalez-Ortiz, R. Jiménez-Díaz, M. Pérez-Trujillo, T. Parella, P. Lopez-Colom, SM Martín-Orúe. Food & function, 2018, 9, 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

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.

In order to do that it is studied the accurate and efficient measurement of isotropic (homo- and heteronuclear scalar coupling constants) for the 2D structre determination, as well as the anisotropic parameters (RDCs, RCSAs and RQCs) for the 3D structure analysis. These anisotropic parameters allows us the discrimination of the different conformers of a molecule and can be found in weakly alignment media. PMMA gel provides an easy, reusable, and practical way to obtain this weakly alignment media. In this work, It has been researched the best way to get these anisotropic parameters, developing and adapting new pulse sequences to this type of media.

To get the correct structure (2D and 3D) it is important to obtain a precise and accurate measurement. In this thesis the great accuracy of isotropic and anisotropic parameters has been achieved through the use of resolution improvement techniques such as Pure-Shift, Spectral Aliasing and Non Uniform Sampling.

In addition it has been applied protocols for the easy automatization and measurement of coupling constants in isotropic and anisotropic media.

Also it has been design improved pulse sequences to achieve the measurement of longer- range heteronuclear connectivities that will  increase the amount of information that we will have available to do the structural analysis

Each experiment has been discussed from a methodological point of view. An assessment on its application has been also performed.

Pulse Programs and Data Set Examples:

Publication 1:
Extending long-range heteronuclear NMR connectivities by HSQMBC-COSY and HSQMBC-TOCSY experiments Saurí, J.; Marcó, N.; Williamson, R. T.; Martin, G. E.; Parella, T. J. Magn. Reson. 2015, 258, 25–32.
DOI 10.1016/j.jmr.2015.06.004

Pulse Program Code for Bruker:

Data set Example:

 

Publication 2:
Ultra high-resolution HSQC: Application to the efficient and accurate measurement of heteronuclear coupling constants  Marcó, N.; Fredi, A.; Parella, T. Chem. Commun. 2015, 51 (15), 3262–3265.
DOI 10.1039/C4CC10279G

Pulse Program Code for Bruker:

Data set Example:

 

Publication 3:
Isotropic/Anisotropic NMR Editing by Resolution-Enhanced NMR Spectroscopy Marcó, N.; Gil, R. R.; Parella, T. ChemPhysChem 2018, 19, 9, 1024-1029.
DOI: 10.1002/cphc.201800094

Pulse Programs Code for Bruker:

 

Publication 4:
Structural discrimination from in situ measurement of 1DCH and 2DHH residual dipolar coupling constants Marcó, N.; Gil, R. R.; Parella, T. Magn. Reson. Chem. 2017, 55 (6), 540–545.
DOI 10.1002/mrc.4575

Pulse Programs Code for Bruker:

Data set Example:

 

Publication 5:
Perfect 1JCH-resolved HSQC: Efficient measurement of one-bond proton-carbon coupling constants along the indirect dimension Marcó, N.; Souza, A. A.; Nolis, P.; Gil, R. R.; Parella, T. J. Magn. Reson. 2017, 276, 37–42.
DOI: 10.1016/j.jmr.2017.01.002

Pulse Programs Code for Bruker:

Data set Example:

 

Publication 6:
1JCH NMR Profile: Identification of key structural features and functionalities by visual observation and direct measurement of one-bond proton-carbon coupling constants Marcó, N.; Souza, A. A.; Nolis, P.; Cobas, C.; Gil, R. R.; Parella, T. J. Org. Chem. 2017, 82 (4), 2040–2044.
DOI: 10.1021/acs.joc.6b02873

Pulse Programs Code for Bruker:

 

Publication 7:
2JHH-resolved HSQC: Exclusive determination of geminal proton-proton coupling constants Marcó, N.; Nolis, P.; Gil, R. R.; Parella, T. J. Magn. Reson. 2017, 282, 18–26.
DOI: 10.1016/j.jmr.2017.06.014

Pulse Programs Code for Bruker:

 

NEW BOOK RELEASE: “Preclinical MRI methods and protocols”

“Preclinical MRI: Methods and Protocols” by Maria Luisa Garcia Martin and Pilar Lopez Larrubia (Editors). Part of the Methods in Molecular Biology book series (MIMB, volume 1718). DOI: 10.1007/978-1-4939-7531-0.

This book was conceived with the idea of providing an update on a wide variety of preclinical MRI methods and protocols to help technicians and researchers interested in this technology. The basics of MRI physics are introduced, followed by chapters describing updated methodology and protocols for some standard and more advanced MRI techniques covering diffusion, perfusion, functional imaging, in-vivo spectroscopy (proton and heteronuclear), susceptibility contrast MRI… The book also contains some chapters where some applications of those methods are illustrated in animal models of several diseases including cancer, stroke and neurodegeneration. Protocols are described in a step-by-step approach, with interesting notes and tips at the end of each chapter, which -a priori- should allow the new worker to obtain successful results with the first attempt ;o) .

Precise characterization of mycobacterial cell wall lipid PTTM

Molecule confirmation and structure characterization of pentatriacontatrienyl mycolate in Mycobacterium smegmatisby 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.

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

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

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

” 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.


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

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

“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.

 

Generation of a new model of patellar tendinopathy in rats which mimics the human sports pathology: A pilot study

“Generation of a new model of patellar tendinopathy in rats which mimics the human sports pathology: A pilot study”  by David Domínguez, Paola Contreras-Muñoz, Silvia Lope, Gil Rodas, G. and Mario Marotta. Apunts. Medicina de l’Esport, 2017, 52:194, 53-59. DOI: 10.1016/j.apunts.2017.01.002

Introduction: Patellar tendon pathophysiology is not still fully understood. The collection of clinical samples from athletes that could permit the analysis of the tendinopathy progression, especially in the early stages, is difficult. For that reason, the purpose of this study is to develop a new experimental animal model of patellar tendinopathy in rats which mimics the human tendinopathy by in vivo intratendinous collagenase injection in the proximal portion of the patellar tendon. Material and methods: The experimental model used was 8-week-old male Wistar rats (N = 4). The administration of collagenase was performed by ultrasound-guided puncture at the level of the proximal and deep portion of the patellar tendon in anesthetized animals. The tendon lesion was evaluated 48 h after injury by magnetic resonance and then, the animals were euthanized and the patellar tendons were collected for histological evaluation. Results: The collagenase-induced lesion model demonstrated important similarities with the human patellar tendinopathy in the region of the proximal insertion. Conclusions: The experimental model of patellar tendinopathy in rat model induces a degeneration and distortion of the patellar tendon architecture in its proximal portion, which closely mimics to that seen in human patellar tendinopathy, and could represent an excellent preclinical model for the study of new therapies focused on treatment of tendinopathy.

Mouse model of patellar tendinopathy