Towards real-time metabolic profiling of a biopsy specimen during a surgical operation by 1H high resolution magic angle spinning nuclear magnetic resonance: a case report

<p>Abstract</p> <p>Introduction</p> <p>Providing information on cancerous tissue samples during a surgical operation can help surgeons delineate the limits of a tumoral invasion more reliably. Here, we describe the use of metabolic profiling of a colon biopsy specimen by high resolution magic angle spinning nuclear magnetic resonance spectroscopy to evaluate tumoral invasion during a simulated surgical operation.</p> <p>Case presentation</p> <p>Biopsy specimens (n = 9) originating from the excised right colon of a 66-year-old Caucasian women with an adenocarcinoma were automatically analyzed using a previously built statistical model.</p> <p>Conclusions</p> <p>Metabolic profiling results were in full agreement with those of a histopathological analysis. The time-response of the technique is sufficiently fast for it to be used effectively during a real operation (17 min/sample). Metabolic profiling has the potential to become a method to rapidly characterize cancerous biopsies in the operation theater.</p

Helical Stacking in DNA 3-Way Junctions Containing 2 Unpaired Pyrimidines - Proton NMR-Studies

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5\u27-TT- and 5\u27-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M. A., and D. J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines

NMR studies of biomolecules

The study of the structure and behavior of biomolecules is a key step to understanding their biological functions. The advent of 2D NMR, 20 years ago, allowed scientists to study small biomolecules in solution. Using this methodology we have studied the solution structure of a dithiophosphate oligonucleotide and of the signal peptide of the rat liver aldehyde dehydrogenase in a micellar environment. The dithiophosphate oligonucleotide exists preferentially as a hairpin in solution whereas the regular oligonucleotide exists as a duplex. The hairpin can be converted to a duplex form at higher salt concentrations. The signal peptide in a dodecylphosphocholine micelle exists as a helix-turn-helix structure, with one of the helices being much more stable than the other. It is possible to substantially increase the range of the biomolecules studied by NMR by adding a third dimension to the classical 2D experiment. We present the results of two 3D NMR experiments. A homonuclear 3D NOESY-TOCSY experiment applied to a oligonucleotide (a dodecamer) and a heteronuclear 3D NOESY-HMQC experiment applied to the protein bovine heart acid phosphatase (BHAP) which contains 157 amino acids and has been uniformly labelled with \sp{15}N

Fluorine NMR spectroscopy and computational calculations for assessing intramolecular hydrogen bond involving fluorine and for characterizing the dynamic of a fluorinated molecule

Intramolecular hydrogen bonds directly involving fluorine atoms are important for pre-organizing the molecule in a defined conformation and for improving the cell permeability of a molecule. Several NMR observable parameters can be used for detecting these intramolecular hydrogen bonds. The two fluorine atoms of the molecule 2-chloro-6-fluoro-N-(4-fluoro-1-methyl-1H-indazol-3yl)benzamide, which is a binder of the target telomeric repeat-containing RNA (TERRA), display through-space JFF coupling values in water and different aprotic solvents. A correlation between the dielectric constant of the solvent and the value of JFF coupling was established. The conformation of the molecule with large JFF is consistent with the presence of a transient intramolecular hydrogen bond involving the two fluorine atoms and the NH of the amide. However, the molecule is very flexible in solution and can adopt at least another conformer of lower population with a larger distance between the two fluorine atoms. The kinetics and thermodynamic parameters of this conformational equilibrium have been characterized using different 19F NMR experiments. Ab initio calculations performed in different solvents reveal the presence of several conformers which are separated by low energy barriers. The computed weighted distance between the two fluorine atoms is in agreement with the JFF values measured experimentally. The conformational flexibility of the molecule could be relevant for its binding to the telomeric repeat-containing RNA

Etudes structurales et dynamiques de la protéine membranaire kpOMPA par RMN en phase liquide et solide

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Etude par RMN HRMAS de molécules greffées sur support solide (Etude par RMN VASS de protéines dans des milieux orientés)

Ce travail comporte deux parties. La première partie décrit des études structurales secondaires de peptides greffés sur des résines par RMN haute résolution à l'angle magique (HRMAS). Nous avons notamment montré par RMN HRMAS que les peptides poly-Aib (acide -aminoisobutyrique) et les homo-peptides C -tétrasubstitués [L-( Me)Leu]n (n = 1-5) adoptent une structure régulière en hélice 310 même lorsqu'ils sont liés de manière covalente à une matrice polymérique. De tels systèmes s'avèrent en effet très utiles pour les études de reconnaissance moléculaire ou comme catalyseurs en synthèse asymétrique. La deuxième partie est consacrée à l'étude par RMN VASS (Variable Angle Sample Spinning) des milieux orientés. Des spectres RMN d'ubiquitine en présence de bicelles à une concentration de 25% poids/volume ont été enregistrés dans des conditions de rotation de l'échantillon à différents angles de rotation. Pour un axe de rotation égal à l'angle magique ( = 54,7), l'HSQC 1H/15N enregistrée sans découplage 1H dans la dimension indirecte correspond au spectre classique obtenu sur une protéine dans une solution isotrope et permet la mesure purement isotrope des constantes de couplage scalaire 1JNH. Pour un angle de rotation inférieur à l'angle magique ( 54,7) les bicelles s'orientent avec leur normale le long de l'axe de rotation. Cet alignement de bicelles crée des conditions anisotropes qui donnent lieu à l'observation de constantes dipolaires résiduelles sur l'ubiquitine. L'ampleur de ces constantes dipolaires résiduelles dépend directement de l'angle que le rotor fait avec le champ magnétique principal. En changeant cet angle de manière contrôlée, les constantes dipolaires résiduelles peuvent être aussi bien augmentées ou diminuées offrant ainsi la possibilité d'étudier simultanément une grande gamme de constantes dipolaires dans un même échantillon.This work is divided into two parts. The first part is dedicated to the secondary structural study of peptides grafted to a resin by High Resolution Magic Angle Spinning (HRMAS) NMR. We have shown by HRMAS NMR that poly-Aib ( -aminoisobutyric acid) peptides and C -tetrasubstituted homo-peptides [L-( Me)Leu]n (n = 1-5) adopt a regular 310-helical structure even when covalently bound to a polymeric matrix. Such systems are very useful in molecular recognition studies or as catalysts in asymmetric synthesis. The second part is dedicated to the study of oriented media by Variable Angle Sample Spinning (VASS) NMR. NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle ( = 54,7), the 1H/15N HSQC recorded without any 1H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of pure isotropic scalar J-couplings 1JNH. For an angle of rotation inferior to the magic angle ( 54,7) the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Ultrafast high-resolution magic-angle-spinning NMR spectroscopy

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