Martial Piotto

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions.

SummaryA novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H2O at 5 °C is presented.

Toward improved grading of malignancy in oligodendrogliomas using metabolomics.

In spite of having been the object of considerable attention, the histopathological grading of oligodendrogliomas is still controversial. The determination of reliable biomarkers capable of improving the malignancy grading remains an essential step in working toward better therapeutic management of patients. Therefore the metabolome of 34 human brain biopsies, histopathologically classified as low‐grade (LGO, N = 10) and high‐grade (HGO, N = 24) oligodendrogliomas, was studied using high‐resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) and multivariate statistical analysis. The classification model obtained afforded a clear distinction between LGOs and HGOs and provided some useful insights into the different metabolic pathways that underlie malignancy grading. The analysis of the most discriminant metabolites in the model revealed the presence of tumoral hypoxia in HGOs. The statistical model was then used to study biopsy samples that were classified as intermediate oligodendrogliomas (N = 6) and glioblastomas (GBMs) (N = 30) by histopathology. The results revealed a gradient of tumoral hypoxia increasing in the following direction: LGOs, intermediate oligodendrogliomas, HGOs, and GBMs. Moreover upon analysis of the clinical evolution of the patients, the metabolic classification seems to provide a closer correlation with the actual patient evolution than the histopathological analysis. Magn Reson Med 59:959–965, 2008.

Solution State NMR Structure and Dynamics of KpOmpA, a 210 Residue Transmembrane Domain Possessing a High Potential for Immunological Applications

The three-dimensional structure of the outer membrane protein A from Klebsiella pneumoniae transmembrane domain was determined by NMR.This protein induces specific humoral and cytotoxic responses, and is a potent carrier protein. This is one of the largest integral membrane proteins(210 residues) for which nearly complete resonance assignment, including side chains, has been achieved so far. The methodology rested on the use of 900 MHz 3D and 4D TROSY experiments recorded on a uniformly 15N,13C,2H-labeled sample and on a perdeuterated methyl protonated sample. The structure was refined from 920 experimental constraints, giving an ensemble of 20 best structures with an r.m.s. deviation of 0.54 A for the main chain atoms in the core eight-stranded beta-barrel. The protein dynamics was assessed, in a residue-specific manner, by 1H-15N NOEs (pico- to nanosecond timescale), exchange broadening (millisecond to second) and 1H-2H chemical exchange (hour-weeks).

Solution structure of a truncated anti‐MUC1 DNA aptamer determined by mesoscale modeling and NMR

Mucin 1 is a well‐established target for the early diagnosis of epithelial cancers. The nucleotides of the S1.3/S2.2 DNA aptamer involved in binding to variable number tandem repeat mucin 1 peptides have been identified using footprinting experiments. The majority of these binding nucleotides are located in the 25‐nucleotide variable region of the total aptamer. Imino proton and 2D NMR spectra of truncated and total aptamers in supercooled water reveal common hydrogen‐bonding networks and point to a similar secondary structure for this 25‐mer sequence alone or embedded within the total aptamer. NMR titration experiments confirm that the TTT triloop structure is the primary binding site and show that the initial structure of the truncated aptamers is conserved upon interaction with variable number tandem repeat peptides. The thermal dependence of the NMR chemical shift data shows that the base‐paired nucleotides melt cooperatively at 47 ± 4 °C. The structure of the 25‐mer oligonucleotide was determined using a new combined mesoscale molecular modeling, molecular dynamics and NMR spectroscopy investigation. It contains three Watson–Crick pairs, three consecutive mispairs and four Watson–Crick pairs capped by a TTT triloop motif. The 3D model structures (PDB 2L5K) and biopolymer chain elasticity molecular models are consistent with both NMR and long unconstrained molecular dynamics (10 ns) in explicit water, respectively.

Metabolomic pattern of childhood neuroblastoma obtained by 1H-high-resolution magic angle spinning (HRMAS) NMR spectroscopy†

The aim of this preliminary study is to characterize by 1H high‐resolution magic angle spinning NMR spectroscopy (HRMAS) the metabolic content of intact biopsy samples obtained from 12 patients suffering from neuroblastoma (NB).

Slow-spinning low-sideband HR-MAS NMR spectroscopy: delicate analysis of biological samples.

High-Resolution Magic-Angle Spinning (HR-MAS) NMR spectroscopy has become an extremely versatile analytical tool to study heterogeneous systems endowed with liquid-like dynamics. Spinning frequencies of several kHz are however required to obtain NMR spectra, devoid of spinning sidebands, with a resolution approaching that of purely isotropic liquid samples. An important limitation of the method is the large centrifugal forces that can damage the structure of the sample. In this communication, we show that optimizing the sample preparation, particularly avoiding air bubbles, and the geometry of the sample chamber of the HR-MAS rotor leads to high-quality low-sideband NMR spectra even at very moderate spinning frequencies, thus allowing the use of well-established solution-state NMR procedures for the characterization of small and highly dynamic molecules in the most fragile samples, such as live cells and intact tissues.

HR-MAS NMR Spectroscopy of Reconstructed Human Epidermis: Potential for the in Situ Investigation of the Chemical Interactions between Skin Allergens and Nucleophilic Amino Acids

High-resolution magic angle spinning (HR-MAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques. In this article, we explore a new domain of application of HR-MAS, namely, reconstructed human epidermis (RHE) and the in situ observation of chemical interactions between skin sensitizers and nucleophilic amino acids. First, the preparation, storage, and analysis of RHE were optimized, and this work demonstrated that HR-MAS NMR was well adapted for investigating RHE with spectra of good quality allowing qualitative as well as quantitative studies of metabolites. Second, in order to study the response of RHE to chemical sensitizers, the ((13)C)methyldodecanesulfonate was chosen as an NMR probe, and we compared adducts formed on human serum albumin (HSA) in solution and adducts formed in RHE. Thus, while the modification of proteins or peptides in solution takes several days to lead to a significant amount of modification, in RHE the modifications of nucleophilic amino acids were observable already at 24 h. The chemioselectivity also appeared to be different with major modifications taking place on histidine, methionine, and cysteine residues in RHE, while on HSA, significant modifications were observed on lysine residues with the formation of methylated and dimethylated amino groups. We thus demonstrated that RHE could be used to investigate in situ chemical interactions taking place between skin sensitizers and nucleophilic amino acids. This opens perspectives for the molecular understanding of the skin immune system activation by sensitizing chemicals.

The assessment of the quality of the graft in an animal model for lung transplantation using the metabolomics 1H high-resolution magic angle spinning NMR spectroscopy

Standards are needed to control the quality of the lungs from nonheart‐beating donors as potential grafts. This was here assessed using the metabolomics 1H high‐resolution magic angle spinning NMR spectroscopy. Selective perfusion of the porcine bilung block was set up 30 min after cardiac arrest with cold Perfadex®. Lung alterations were analyzed at 3, 6, and 8 h of cold ischemia as compared to baseline and to nonperfused lung. Metabolomics analysis of lung biopsies allowed identification of 35 metabolites. Levels of the majority of the metabolites increased over time at 4°C without perfusion, indicating cellular degradation, whereas levels of glutathione decreased. When lung was perfused at 4°C, levels of the majority of the metabolites remained stable, including levels of glutathione. Levels of uracil by contrast showed a reverse profile, as its signal increased over time in the absence of perfusion while being totally absent in perfused samples. Our results showed glutathione and uracil as potential biomarkers for the quality of the lung. The metabolomics 1H high‐resolution magic angle spinning NMR spectroscopy can be efficiently applied for the assessment of the quality of the lung as an original technique characterized by a rapid assessment of intact biopsy samples without extraction and can be implemented in hospital environment. Magn Reson Med, 2012.

Metabolomic profile of the adrenal gland: from physiology to pathological conditions

In this study, we i) assessed the metabolic profile of the normal adrenal cortex and medulla of adult human subjects by means of (1)H-high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy; ii) compared the biochemical profile of adenoma (Ad), adrenal cortical carcinoma (ACC), and pheochromocytoma (PCC) samples with that of healthy adrenal tissue samples; and iii) investigated the metabolic differences between ACCs and Ads as well as between ACCs and PCCs. Sixty-six tissue samples (13 adrenal cortical tissue, eight medullary tissue, 13 Ad, 12 ACC, and 20 PCC samples) were analyzed. Adrenaline and noradrenaline were undetectable in cortical samples representing the metabolic signature of the tissue derived from neural crest. Similarity between the metabolic profile of Ads and that of the normal adrenal cortex was shown. Inversely, ACC samples clearly made up a detached group exhibiting the typical stigmata of neoplastic tissue such as choline-containing compounds, biochemical markers of anaerobic processes, and increased glycolysis. Significantly higher levels of lactate, acetate, and total choline-containing compounds played a major role in the differentiation of ACCs from Ads. Moreover, the high fatty acid content of ACCs contributed to the cluster identification of ACCs. Of the 14 sporadic PCC samples, 12 exhibited predominant or exclusive noradrenaline secretion. The noradrenaline:adrenaline ratio was inverted in the normal medullary tissue samples. Multiple endocrine neoplasia type 2- and NF1-related PCC samples exhibited both adrenaline and noradrenaline secretion. In the von Hippel-Lindau disease-related PCC samples, only noradrenaline secretion was detected by HRMAS NMR spectroscopy. This study is one of the first applications of metabolomics to adrenal pathophysiology and it is the largest study to report HRMAS NMR data related to the adrenal cortex and adrenal cortical tumors.

Ultra fast in vivo microwave irradiation for enhanced metabolic stability of brain biopsy samples during HRMAS NMR analysis

High resolution magic-angle spinning (HRMAS) NMR spectroscopy is a well established technique for ex vivo metabolite investigations but experimental factors such as ischemic delay or mechanical stress due to continuous spinning deserve further investigations. Cortical brain samples from rats that underwent ultrafast in vivo microwave irradiation (MWp group) were compared to similar samples that underwent standard nitrogen freezing with and without exposure to domestic microwaves (FN and FN+MWd groups). One dimensional (1)H HRMAS NMR spectra were acquired and 16 metabolites of interest were quantified. Within each group 3 samples underwent long lasting acquisition (up to 15 h). Statistically significant differences in metabolite concentrations were observed between groups for metabolites associated to post mortem biochemical changes and/or anaerobic glycolysis including several neurotransmitters. Spectral assessment over time showed a drastic reduction of biochemical variations in both MW groups. Only 2/16 metabolites exhibited significant signal variations after 15 h of continuous spinning and acquisition in the MWp group. This number increased to 10 in the FN group. We confirmed limited anaerobic metabolism and post mortem degradation after ultra fast in vivo MW irradiation. Furthermore, spectra obtained after MWp and MWd irradiation exhibited an extremely stable spectral pattern over extended periods of continuous acquisition.