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.

PhD Thesis: Implementation of high-resolution MRSI methods in a pre-clinical scanner, and optimization for brain longitudinal studies of therapy response in mice glioma model.

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Last 26th July 2023 I successfully defended my PhD Thesis entitled: “PhD Thesis: Implementation of high-resolution MRSI methods in a pre-clinical scanner, and optimization for brain longitudinal studies of therapy response in mice glioma model.”

Abstract

Magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRS/MRSI) are non-invasive diagnostic techniques that use a strong magnetic field and radio waves to examine the chemical composition of living tissue. Working on the same principles as Magnetic Resonance Imaging (MRI), instead of producing images, MRS generates a spectrum of signals that can be used to identify the type and amount of molecules present in a tissue. The utility of MRS and MRSI has already been established in many studies, providing useful information about the chemical makeup of different regions of the brain, and allowing diagnosis of conditions such as Alzheimer’s disease, multiple sclerosis, and brain Glioblastoma (GB) tumors.

Preclinical glioblastoma studies looking forward to improving therapeutic outcomes are necessary since clinical GB has no current cure. These studies can greatly benefit from improved spatial resolution and homogeneity of the acquired MRSI grids. Hence, we can work towards improved acquisition schemes enhancing the quality of acquired data using MRS and MRSI. There exists a methodological consensus among spectroscopy experts where the Localized Adiabatic Spin Echo Refocused (semiLASER) data acquisition strategy has been ranked as the most likely localization technique to improve (pre) clinical MRS. SemiLASER uses adiabatic pulses to selectively excite and refocus the signal from a localized volume of interest in the brain. This results in a higher signal-to-noise ratio (SNR) and better spatial resolution compared to conventional data acquisition sequences.

Partial volume effects can occur in MRSI when the voxel (a 3D volume of interest) being measured contains a mixture of different neighbouring tissue types or compartments, such as grey and white matter or cerebrospinal fluid. This can lead to inaccurate quantification of metabolites, as the signal from one tissue can mix with the signal from another and affect overall pattern recorded. SemiLASER is designed to minimize partial volume effects by using adiabatic pulses to selectively excite and refocus the signal from a small region of interest within the voxel. This allows for more accurate quantification of metabolites within the region of interest, while reducing the contamination of the signal by other tissue types. In addition, semiLASER also employs an outer-volume suppression (OVS) technique to further reduce contamination from outside the region of interest. This involves using additional adiabatic pulses to selectively saturate the signal from outside the volume of interest, so that it does not contribute to the MRSI signal. Overall, the combination of selective excitation and OVS in semiLASER can help improve the accuracy of MRSI measurements and reduce partial volume effects.

Although, the clinical utility of semiLASER has been acknowledged, the pre-clinical use and implementation of semiLASER still remains a less explored area. Our group has a long record of using MRSI in therapy response monitoring of a murine model glioblastoma (the GL261 cell line) using a commercially available MRSI acquisition sequence. In our efforts towards bridging the barriers between pre-clinical and clinical research, we have implemented the clinically verified semiLASER sequence on a pre-clinical 7T Bruker Biospec USR scanner running the ParaVision 5.1 software package, which provides a graphical user interface for sequence programming and data acquisition. The single and multi-voxel semiLASER sequences were implemented with the idea that the developments generated during this PhD project will be replicable by other interested users.

The implemented SV-semiLASER and MRSI-semiLASER sequences for preclinical acquisitions were optimised to perform high resolution MRSI of living mouse brain. For this, sequences were duly verified and tested first in phantoms and later in-vivo, in wild-type (wt) and tumor bearing (GL261) mice. To do so, the Bruker pulse sequence implementation was first studied in detail to become familiar with the Bruker programming environment and a test sequence PRESS_Slice to localize the slice dimension was developed by modifying the Bruker stock PRESS sequence for single voxel localization. After careful evaluation of test sequence results, the semiLASER single and multi-voxel sequences were also implemented using a similar strategy.

The implemented SV-semiLASER sequence provided a ca. 1.4-fold improvement in SNR in phantoms and ca. 1.3-fold improvement in SNR for in-vivo subjects, in comparison to the stock Bruker PRESS (single volume acquisition) sequence. The MRSI-semiLASER sequence resulted in a ca. 1.3-fold improvement of SNR in phantoms and in-vivo subjects compared to the stock Bruker CSI-PRESS sequence. Combined with phase encoding strategies and volume reduction methods, higher spatial resolution and SNR was achieved for the implemented MRSI-semiLASER. The quantification analysis of the results was done using MATLAB based post-processing tools specially designed to process Bruker datasets and solutions for a faster post processing pipeline were proposed. The single voxel MRSI-semiLASER sequences were further simulated using NMRSCOPE-B virtual simulator, a jMRUI plug-in which positively correlated with the experimental results. Preliminary nosological images obtained using MRSI-semiLASER datasets and the SpectraClassifier tool previously developed in our group, and trained with GL261 tumors using already available CSI-PRESS data, suggested those classifiers could be robust enough to recognize the tumor region acquired with the semi-LASER sequence. Still, classifiers may require retraining for the evaluation of response to therapy, which is an ongoing project within the group.

The thesis dissertation can be downloaded in PDF format using the link below or from the official TXD and Teseo repositories (currently in progress):

Zoona Javed-PhD Thesis-Univ Autonoma Barcelona-July 2023Download

Acknowledgments

I would like to thank the financial support by the European Comission Marie Curie Initial Training Networks (ITN, call H2020-MSCA-ITN-2018, grant 813120 to project INSPiRE-MED); by the Ministry of Science and Insdustry (MCIN/AEI/10.13039/501100011033) (APC); and by Centro de Investigación Biomédica en Red—Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN http://www.ciber-bbn.es/en, CB06/01/0010), an initiative of the Instituto de Salud Carlos III (Spain) co-funded by the European Regional Development Fund (ERDF). I was recipient of a Marie Skłodowska-Curie early-stage researcher fellowship of the INSPiRE-MED project (Grant agreement ID: 813120)

Using nuclear magnetic resonance urine metabolomics to develop a prediction model of early stages of renal disease in subjects with type 2 diabetes

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J Ricardo Lucio-Gutiérrez, Paula Cordero-Pérez, Iris C Farías-Navarro, Ramiro Tijerina-Marquez, Concepción Sánchez-Martínez, José Luis Ávila-Velázquez, Pedro A García-Hernández, Homero Náñez-Terreros, Jordi Coello-Bonilla, Míriam Pérez-Trujillo, Teodor Parella, Liliana Torres-González, Noemí H Waksman-Minsky, Alma L Saucedo

Journal of Pharmaceutical and Biomedical Analysis, 2022, 19, 114885

Abstract

Type 2 diabetes mellitus (DM2) is a multimorbidity, long-term condition, and one of the worldwide leading causes of chronic kidney disease (CKD) –a silent disease, usually detected when non-reversible renal damage have already occurred. New strategies and more effective laboratory methods are needed for more opportune diagnosis of DM2-CKD. This study comprises clinical parameters and nuclear magnetic resonance (NMR)-based urine metabolomics data from 60 individuals (20–65 years old, 67.7% females), sorted in 5 experimental groups (healthy subjects; diabetic patients without any clinical sign of CKD; and patients with mild, moderate, and severe DM2-CKD), according to KDIGO. DM2-CKD produces a continuous variation of the urine metabolome, characterized by an increase/decrement of a group of metabolites that can be used to monitor CKD progression (trigonelline, hippurate, phenylalanine, glycolate, dimethylamine, alanine, 2-hydroxybutyrate, lactate, and citrate). NMR profiles were used to obtain a statistical model, based on partial least squares analysis (PLS-DA) to discriminate among groups. The PLS-DA model yielded good validation parameters (sensitivity, specificity, and area under the curve (AUC) of the receiver operating characteristic curve (ROC) plot: 0.692, 0.778 and 0.912, respectively) and, thus, it can differentiate between subjects with DM2-CKD in early stages, from subjects with a mild or severe condition. This metabolic signature exhibits a molecular variation associated to DM2-CKD, and data suggests it can be used to predict risk of DM2-CKD in patients without clinical signs of renal disease, offering a new alternative to current diagnosis methods.

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

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Dolors Izquierdo, Montserrat Roura, Míriam Pérez-Trujillo, Sandra Soto-Heras, María-Teresa Paramio

International Journal of Molecular Sciences 2022, 23(8), 4141

Abstract

In goats, embryo oocyte competence is affected by follicle size regardless the age of the females. In previous studies we have found differences in blastocyst development between oocytes coming of small (<3 mm) and large follicles (>3 mm) in prepubertal (1–2 months-old) goats. Oocyte competence and Follicular Fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze 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. Not significant differences were found in follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production.

Representative 1H NMR spectra of follicular fluid samples of (a) adult and (b) prepubertal goats. Spectra were acquired at 298.0 K and at a magnetic field of 600 MHz, with suppression of the residual water signal.

(a) PCA scores plot (PC1-PC2) from 1H NMR spectral data of follicular fluid samples of prepubertal (n = 16; blue dots) and adult (n = 40; black dots) goats. (b) PCA heat map loadings plot (PC1-PC2) with some discriminant variables assigned.

PhD Thesis: Advances in NMR spectroscopic methodology and applications: time-efficient methods, ultra long-range heteronuclear correlation experiments and enantiospecific analysis of complex mixtures.

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Last 21st October 2021 I defended my PhD Thesis entitled: Advances in NMR spectroscopic methodology and applications: time-efficient methods, ultra long-range heteronuclear correlation experiments and enantiospecific analysis of complex mixtures.

The thesis is divided into the following topics:

  • the development of Nuclear Magnetic Resonance (NMR) experiments focused on efficiency in terms of time;
  • establishing new pulse sequences that facilitate the study of long-distance coupling constants fundamental for structural elucidation;
  • the development of a reliable method that allows the differentiated analysis of enantiomers (enantiospecific) directly from its original mixture (in situ) and from multiple molecules simultaneously (multicomponent).

The NMR experiments developed using the MFA (Multiple Fid Acquisition) approach based on the “afterglow magnetization”, allows a considerable reduction of the experimental time by acquiring several experiments at the same time, which are stored in different FIDs that can be visualized separately. In this thesis several works are presented that allow to make a structural elucidation of organic molecules in a fast, simple and unambiguous way, among them MFA-COSY / RELAY3, MFA-COSY / TOCSY, MFA-HMBC / HMBC-COSY, MFA-MBOB -COSY, MFA-TOCSY / TOCSY, MFA-HSQC / HSQC and the MFA-HSQC / Pure-shiftHSQC. Furthermore, with an adequate combination of MFA and “Spectral Aliasing” (SA), a new experiment is presented, which in addition to the experimental time improves spectral resolution and facilitates structural identification. The SA, despite being a powerful experiment to avoid signal overlapping has an important disadvantage related to the identification of each signal, to avoid this problem, for a few extra seconds, we acquired the two heteronuclear experiments in 2D, the HSQC with “Spectral Aliasing” and the standard HSQC to facilitate signal assignment.

Furthermore, in terms of improving the spectral resolution, this thesis presents two experiments following the Pure-shift methodology to eliminate the proton-proton coupling constant, using BIRD (BIlinear Rotation Decoupling) to perform heteronuclear decoupling, minimizing signal overlapping. The novelty of this work is based on the detection of multiple nuclei in the same 2D spectrum, nitrogen and carbon in the indirect dimension (F1) and proton in F2, is what is known as “Time-Shared NMR experiments”. In addition, using the same approach, a second experiment is presented that allows the calculation (via direct observation) of the heteronuclear proton-carbon and proton-nitrogen coupling constants simultaneously. The measurement of long-distance heteronuclear coupling constants remains a challenge in NMR spectroscopy, due to their tiny values and to the difficulty of their measurement. A modification in the LR-HSQMBC (changing some 180º pulses of the experiment by selective pulses irradiated in an area of the spectrum) allows the measurement of very small coupling constants (of up to 6 separation bonds). In this work, the advantages of the new LR-selHSQMBC NMR experiment are exposed and the advantages and disadvantages of both experiments are compared.

Finally, this thesis presents an innovative work related to the enantiospecific and simultaneous detection of multiple pairs of enantiomers in a mixture without prior separation or derivatization of the sample components and with minimal sample manipulation. This method is based on NMR spectroscopy and on the use of a chiral solvating agent (CSA) as chiral auxiliary. This work shows, as a proof of concept, the simultaneous enantiospecific detection of multiple enantiomeric pairs directly within the original mixture. This is demonstrated with an aqueous mixture of the essential amino acids in their D and L forms.

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

ACKNOWLEDGMENTS

I would like to thank the financial support for this research by Spanish MINECO projects “Diseño y Aplicación de Nuevas Metodologías en Resonancia Magnética Nuclear” (CTQ2015-64436-P) and “Metodologías Modernas en Resonancia Magnética Nuclear de Moleculas Pequeñas” (PGC2018-095808-B-I00) and for the grant BES-2016-078903 awarded by Agencia Estatal de Investigación.

SeRMN contribution to SMASH Small Molecule NMR Conference

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Kumar Motiram-Corral is presenting at SMASH 2021 Conference a talk entitled In situ Enantiospecific Detection of Multiple Metabolites in Mixtures using NMR Spectroscopy, related to some of our recent research work. The presentation will be 1st of September in the section “Unveiling the Unknown – New Methods in Structure Elucidation“.

L. T. Kuhn, K. Motiram-Corral, T. J. Athersuch, T. Parella, M. Pérez-Trujillo, Angew. Chem. Int. Ed. 59 (2020) 23615.

SeRMN contribution at EUROMAR 2021 Conference

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Some of our recent research work was presented at the European NMR meeting Euromar 2021 that was going to take place at Portoroz (Slovenia), but which was finally virtual from the 5th to the 8th of July 2021.

· Míriam Pérez-Trujillo presented the talk In situ Enantiospecific Detection of Multiple Metabolites in Mixtures using NMR Spectroscopy in the “Metabolomics” session. In this talk our last advances in enantiodifferentiation using NMR were shown and discussed.

To date, the enantiospecific analysis of mixtures necessarily requires prior separation of the individual components. The simultaneous enantiospecific detection of multiple chiral molecules in a mixture represents a major challenge, which would lead to a significantly better understanding of the underlying biological processes; e.g. via enantiospecifically analyzing metabolites in their native environment. Here, we report on the first in situ enantiospecific detection of a thirty-nine-component mixture. As a proof of concept, eighteen essential amino acids (AAs) at physiological concentrations were simultaneously enantiospecifically detected using NMR spectroscopy and a chiral solvating agent. This work represents a first step towards the simultaneous multicomponent enantiospecific analysis of complex mixtures, a capability that will have substantial impact on metabolism studies, metabolic phenotyping, chemical reaction monitoring, and many other fields where complex mixtures containing chiral molecules require efficient characterization.

L. T. Kuhn, K. Motiram-Corral, T. J. Athersuch, T. Parella, M. Pérez-Trujillo, Angew. Chem. Int. Ed. 59 (2020) 23615.

SeRMN contributions at 10th GERMN biennial /9th IberAmerican/7th Iberian NMR Meeting

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Some of the SeRMN staff has presented our recent research work at the biannual Spanish and IberAmerican NMR meeting, 10th GERMN biennial /9th IberAmerican/7th Iberian NMR Meeting. This year it was a virtual meeting taking place from 26 to 29 April 2021.

Pau Nolis presented an oral communication entitled “Reducing experimental time using Multiple Fid Acquisition“. P. Nolis, K. Motiram-Corral, M. Pérez-Trujillo, T. Parella.

Speeding-up NMR molecular analysis is an important research field which has been continuously advancing since NMR early days. The relevant benefits are clear and evident: i) reduce analysis time per sample => reduce analysis cost; ii) gain spectrometer time to analyze new samples => improve spectrometer efficiency. Multiple FID Acquisition (MFA) strategy consists in the design of NMR pulse sequence experiments accommodating N acquisition windows, each registering different relevant structural information. This strategy is faster
than perform a traditional sequential acquisition of N separated experiments. Several design strategies and practical experiments will be shown and discussed.

Míriam Pérez-Trujillo presented an oral communication entitled “Simultaneous Enantiospecific Detection of Multiple Metabolites in Mixtures using NMR Spectroscopy“. L. T. Kuhn, K. Motiram-Corral, T. J. Athersuch, T. Parella, M. Pérez-Trujillo.

Chirality plays a fundamental role in nature, but its detection and quantification still face many limitations. To date, the enantiospecific analysis of mixtures necessarily requires prior separation of the individual components. The simultaneous enantiospecific detection of multiple chiral molecules in a mixture represents a major challenge, which would lead to a
significantly better understanding of the underlying biological processes; e.g. via enantiospecifically analyzing metabolites in their native environment. Here, we report on the first in situ enantiospecific detection of a thirty-ninecomponent mixture. As a proof of concept, eighteen essential amino acids (AAs) at physiological concentrations were simultaneously enantiospecifically detected using NMR spectroscopy and a chiral solvating agent. This work
represents a first step towards the simultaneous multicomponent enantiospecific analysis of complex mixtures, a capability that will have substantial impact on metabolism studies, metabolic phenotyping, chemical reaction monitoring, and many other fields where complex mixtures containing chiral molecules require efficient characterization.

Special Issue: NMR-Based Metabolomics

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Special Issue: NMR-Based Metabolomics, by Míriam Pérez-Trujillo* and Toby J. Athersuch*

Molecules2021, 26(11), 3283; https://doi.org/10.3390/molecules26113283

This article belongs to the Special Issue NMR-Based Metabolomics

Nuclear magnetic resonance (NMR) spectroscopy remains one of the core analytical platforms for metabolomics, providing complementary chemical information to others, such as mass spectrometry, and offering particular advantages in some areas of research on account of its inherent robustness, reproducibility, and phenomenal dynamic range. While routine experimental protocols for profiling and related statistical analysis pipelines have been established, they often present considerable challenges to the analyst, including spectral overlap, accurate and precise quantification, and chemical shift variation. Consequently, there is still much activity across all areas of NMR spectroscopic analysis in relation to metabolomics. Furthermore, there remain many biological systems and sample types that have not been extensively explored using NMR spectroscopy-based metabolomics.In this Special Issue, several advances in methodology, and new applications in the field of NMR-based metabolomics, have been presented. In addition, the SI includes authoritative review articles focused on the state-of-the-art of quantitative NMR spectroscopy in biomedical metabolomics applications, and novel applications in the agri-food sector.

Simultaneous Enantiospecific Detection of Multiple Compounds in Mixtures using NMR Spectroscopy

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Simultaneous Enantiospecific Detection of Multiple Compounds in Mixtures using NMR Spectroscopy, by Lars T. Kuhn, Kumar Motiram-Corral, Toby J. Athersuch, Teodor Parella, Míriam Pérez-Trujillo*

Angew. Chem. Int. Ed., 2020 / doi:10.1002/anie.202011727

Chirality plays a fundamental role in nature, but its detection and quantification still face many limitations. To date, the enantiospecific analysis of mixtures necessarily requires prior separation of the individual components. The simultaneous enantiospecific detection of multiple chiral molecules in a mixture represents a major challenge, which would lead to a significantly better understanding of the underlying biological processes; e.g. via enantiospecifically analysing metabolites in their native environment. Here, we report on the first in situ enantiospecific detection of a thirty‐nine‐component mixture. As a proof of concept, eighteen essential amino acids at physiological concentrations were simultaneously enantiospecifically detected using NMR spectroscopy and a chiral solvating agent. This work represents a first step towards the simultaneous multicomponent enantiospecific analysis of complex mixtures, a capability that will have substantial impact on metabolism studies, metabolic phenotyping, chemical reaction monitoring, and many other fields where complex mixtures containing chiral molecules require efficient characterisation.

Simultaneous enantiospecific detection of a mixture of amino acids by NMR spectroscopy

This work has been selected to be presented as a talk at 2021 scientific conferences:

· 42nd FGMR (German Chemical Society, Magnetic Resonance Section) Annual Discussion Meeting – Virtual, Sep 27 to Oct 1.

· SMASH- Small Molecule NMR Conference 2021 – Virtual, Aug 30 to Sep 2.

· Euromar 2021 Conference – Virtual, 5 to 8 July.

· 10th GERMN (Spanish NMR group of the Real Sociedad Española de Química) biennial & 9th IberoAmerican NMR Meeting – Virtual, 26 to 19 April.

Electrochemical dehalogenation of dibromomethane and 1,2‐dibromoethane to non‐toxic products using a carbon fiber brush electrode

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by David Fernández‐Verdejo, Mira LK Sulonen, Míriam Pérez‐Trujillo, Ernest Marco‐Urrea, Albert Guisasola, Paqui Blánquez,  J. Chem. Technol. Biotechnol. 2020. https://doi.org/10.1002/jctb.6542

Dibromomethane (DBM) and 1,2‐dibromoethane (DBA) are two brominated volatile contaminants used in several industrial applications which are often detected in groundwater. The electrochemical degradation of DBM and DBA was studied at different cathode potentials (−0.8, −1 and −1.2 V versus Standard Hydrogen Electrode) in aqueous solution using an inexpensive graphite fiber brush electrode.

The degradation followed first‐order kinetics with respect to the nominal concentration of the brominated compounds, and the kinetic constant increased concomitantly with the decrease of the cathode potential. During the electrochemical dehalogenation 96.8% and 99.8% of the bromide in DBM and DBA was released as bromine ions, respectively. The main non‐brominated compounds detected during the degradation of DBM and DBA were methane and ethene, respectively. In addition, traces of formic acid for DBM and acetic acid for DBA degradation were detected by NMR spectroscopy. The non‐toxicity of the effluent was confirmed by a Microtox test. The efficient electrochemical degradation of DBM and DBA and the lack of toxic products open the door for a simple and non‐toxic electrochemical approach for removing aliphatic brominated compounds from aquifers and other water sources.