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.

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: 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.


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: 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.

 

Metronomic treatment in immunocompetent preclinical glioblastoma

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“Metronomic treatment in immunocompetent preclinical GL261 glioblastoma: effects of cyclophosphamide and temozolomide” by by L. Ferrer-Font,  N. Arias-Ramos, S. Lope-Piedrafita, M. Julià-Sapé , M. Pumarola, C. Arús  and A. P. Candiota. NMR Biomed. 2017. DOI: 10.1002/nbm.3748. 

Glioblastoma (GBM) causes poor survival in patients even when applying aggressive treatment. In preceding years, efforts have focused in new therapeutic regimens with conventional drugs to activate immune responses that may enhance tumor regression and prevent regrowth, as for example the “metronomic” approaches.

We have evaluated whether metronomic CPA or TMZ administration could increase survival in orthotopic GL261 in C57BL/6 mice, an immunocompetent model. Longitudinal in vivo studies with CPA (140 mg/Kg) or TMZ (range 140-240 mg/Kg) metronomic administration (every 6 days) were performed in tumor-bearing mice. Tumor evolution was monitored at 7T with T2-weighted MRI, Diffusion weighted imaging and MRSI-based nosological images of response to therapy. Obtained results demonstrated that both treatments resulted in increased survival (38.6+21.0 days, n=30) compared to control (19.4+2.4 days, n=18). Also, it was found a clear edema appearance during chemotherapeutic treatment suggesting inflammatory associated processes. The necropsy performed in mice cured from GBM after high TMZ cumulative dosage (980-1400 mg/Kg) revealed lymphoma incidence.

Long-term fertilization determines different metabolomic profiles and responses in saplings of three rainforest tree species with different adult canopy position

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“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.

Chiral Recognition by Dissolution DNP NMR Spectroscopy of 13C-Labeled DL-Methionine

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“Chiral Recognition by Dissolution DNP NMR Spectroscopy of 13C-Labeled DL-Methionine” By Eva Monteagudo, Albert Virgili, Teodor Parella and Míriam Pérez-Trujillo.. Anal. Chem., 2017, 89 (9), pp 4939–4944 DOI: 10.1021/acs.analchem.7b00156

A method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in a millimolar aqueous solution using a chiral solvating agent was performed. Hyperpolarized 13C-labeled DL-methionine enantiomers were differently observed with a single-scan 13C NMR experiment, while the chiral auxiliary at thermal equilibrium remained unobserved. The method developed entails a step forward in the chiral recognition of small molecules by NMR spectroscopy, opening new possibilities in situations where the sensitivity is limited, for example, when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like 13C, is required. The advantages and current limitations of the method, as well as future perspectives, are discussed.

Solving JHH measurement in overcrowded regions

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Accurate measurement of JHH in overlapped signals by a TOCSY-edited SERF Experiment

André Fredi, Pau Nolis* and Teodor Parella*

Magnetic Resonance in Chemistry: DOI: 10.1002/mrc.4572

Selective Refocusing (GSERF or the recent PSYCHEDELIC) experiments were originally designed to determine all proton-proton coupling constants (JHH) for a selected proton resonance. They work for isolated signals on which selective excitation can be successfully applied but, as happens in other selective experiments, fail for overlapped signals. To circumvent this limitation, a doubly-selective TOCSY-GSERF scheme is presented for the measurement of JHH in protons resonating in crowded regions. This new experiment takes advantage of the editing features of an initial TOCSY transfer to uncover hidden resonances that become accessible to perform the subsequent frequency-selective refocusing.

RDC measurements and application

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joceah_v081i019.inddApplication to the structural discrimination of small molecules containing multiple stereocenters. One-Shot determination of residual dipolar couplings.
Castañar, L.; Garcia, M.; Hellemann, E.; Nolis, P.; Gil, R.; Parella, T.

 

A novel approach for the fast and efficient structural discrimination of molecules containing multiple stereochemical centers is described. A robust Jresolved HSQC experiment affording highly resolved 1JCH/1TCHsplittings along the indirect dimension and homodecoupled 1H signals in the detected dimension is proposed. The experiment enables in-situ distinction of both isotropic and anisotropic components of molecules dissolved incompressed PMMA gels, allowing a rapid and direct one-shot determination of accurate residual dipolar coupling constants from a single NMR spectrum

 

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Mycobacteria clumping increase their capacity to damage macrophages

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frontiersmicrobiology

 

“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.

fig8

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.

SeRMN contribution to the 32nd AETE (European Embryo Transfer Association) Meeting

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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.

fig4

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.