High-resolution Magic Angle Spinning NMR Spectroscopy Research Articles

Metabolite profiles of medulloblastoma for rapid and non-invasive detection of molecular disease groups

The malignant childhood brain tumour, medulloblastoma, is classified clinically into molecular groups which guide therapy. DNA-methylation profiling is the current classification 'gold-standard', typically delivered 3-4 weeks post-surgery. Pre-surgery non-invasive diagnostics thus offer significant potential to improve early diagnosis and clinical management. Here, we determine tumour metabolite profiles of the four medulloblastoma groups, assess their diagnostic utility using tumour tissue and potential for non-invasive diagnosis using invivo magnetic resonance spectroscopy (MRS). Metabolite profiles were acquired by high-resolution magic-angle spinning NMR spectroscopy (MAS) from 86 medulloblastomas (from 59 male and 27 female patients), previously classified by DNA-methylation array (WNT (n=9), SHH (n=22), Group3 (n=21), Group4 (n=34)); RNA-seq data was available for sixty. Unsupervised class-discovery was performed and a support vector machine (SVM) constructed to assess diagnostic performance. The SVM classifier was adapted to use only metabolites (n=10) routinely quantified from invivo MRS data, and re-tested. Glutamate was assessed as a predictor of overall survival. Group-specific metabolite profiles were identified; tumours clustered with good concordance to their reference molecular group (93%). GABA was only detected in WNT, taurine was low in SHH and lipids were high in Group3. The tissue-based metabolite SVM classifier had a cross-validated accuracy of 89% (100% for WNT) and, adapted to use metabolites routinely quantified invivo, gave a combined classification accuracy of 90% for SHH, Group3 and Group4. Glutamate predicted survival after incorporating known risk-factors (HR=3.39, 95% CI 1.4-8.1, p=0.025). Tissue metabolite profiles characterise medulloblastoma molecular groups. Their combination with machine learning can aid rapid diagnosis from tissue and potentially invivo. Specific metabolites provide important information; GABA identifying WNT and glutamate conferring poor prognosis. Children with Cancer UK, Cancer Research UK, Children's Cancer North and a Newcastle University PhD studentship.

Molecular Level Characterisation of the Surface of Carbohydrate-Functionalised Mesoporous silica Nanoparticles (MSN) as a Potential Targeted Drug Delivery System via High Resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy.

Atomistic level characterisation of external surface species of mesoporous silica nanoparticles (MSN) poses a significant analytical challenge due to the inherently low content of grafted ligands. This study proposes the use of HR-MAS NMR spectroscopy for a molecular level characterisation of the external surface of carbohydrate-functionalised nanoparticles. MSN differing in size (32 nm, 106 nm, 220 nm) were synthesised using the sol-gel method. The synthesised materials displayed narrow particle size distribution (based on DLS and TEM results) and a hexagonal arrangement of the pores with a diameter of ca. 3 nm as investigated with PXRD and N2 physisorption. The surface of the obtained nanoparticles was functionalised with galactose and lactose using reductive amination as confirmed by FTIR and NMR techniques. The functionalisation of the particles surface did not alter the pore architecture, structure or morphology of the materials as confirmed with TEM imaging. HR-MAS NMR spectroscopy was used for the first time to investigate the structure of the functionalised MSN suspended in D2O. Furthermore, lactose was successfully attached to the silica without breaking the glycosidic bond. The results demonstrate that HR-MAS NMR can provide detailed structural information on the organic functionalities attached at the external surface of MSN within short experimental times.

High-Resolution Magic-Angle Spinning NMR Spectroscopy for Evaluation of Cell Shielding by Virucidal Composites Based on Biogenic Silver Nanoparticles, Flexible Cellulose Nanofibers and Graphene Oxide.

Antiviral and non-toxic effects of silver nanoparticles onto in vitro cells infected with coronavirus were evaluated in this study using High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy. Silver nanoparticles were designed and synthesized using an orange flavonoid—hesperetin (HST)—for reduction of silver(I) and stabilization of as obtained nanoparticles. The bio-inspired process is a simple, clean, and sustainable way to synthesize biogenic silver nanoparticles (AgNP@HST) with diameters of ∼20 nm and low zeta potential (−40 mV), with great colloidal stability monitored for 2 years. The nanoparticles were used for the fabrication of two types of antiviral materials: colloids (AgNP@HST spray) and 3D flexible nanostructured composites. The composites, decorated with AgNP@HST (0.05 mmol L−1), were made using cellulose nanofibers (CNF) obtained from orange peel and graphene oxide (GO), being denominated CNF@GO@AgNP@HST. Both materials showed high virucidal activity against coronaviruses in cell infection in vitro models and successfully inhibited the viral activity in cells. HR-MAS 1H-NMR technique was used for determining nanomaterials’ effects on living cells and their influences on metabolic pathways, as well as to study viral effects on cells. It was proven that none of the manufactured materials showed toxicity towards the intact cells used. Furthermore, viral infection was reverted when cells, infected with the coronavirus, were treated using the as-fabricated nanomaterials. These significant results open possibilities for antiviral application of 3D flexible nanostructured composite such as packaging papers and filters for facial masks, while the colloidal AgNP@HST spray can be used for disinfecting surfaces, as well as a nasal, mouth, and eye spray.

Metabolomic Impact of Lidocaine on a Triple Negative Breast Cancer Cell Line.

Background: Metabolomics and onco-anesthesia are two emerging research fields in oncology. Metabolomics (metabolites analysis) is a new diagnostic and prognostic tool that can also be used for predicting the therapeutic or toxic responses to anticancer treatments. Onco-anesthesia studies assess the impact of anesthesia on disease-free and overall survival after cancer surgery. It has been shown that local anesthetics (LA), particularly lidocaine (LIDO), exert antitumor properties both in vitro and in vivo and may alter the biologic fingerprints of cancer cells. As LA are known to impair mitochondrial bioenergetics and byproducts, the aim of the present study was to assess the impact of LIDO on metabolomic profile of a breast cancer cell line. Methods: Breast cancer MDA-MB-231 cells were exposed for 4 h to 0.5 mM LIDO or vehicle (n = 4). The metabolomic fingerprint was characterized by high resolution magic angle spinning NMR spectroscopy (HRMAS). The multivariate technique using the Algorithm to Determine Expected Metabolite Level Alteration (ADEMA) (Cicek et al., PLoS Comput. Biol., 2013, 9, e1002859), based on mutual information to identify expected metabolite level changes with respect to a specific condition, was used to determine the metabolites variations caused by LIDO. Results: LIDO modulates cell metabolites levels. Several pathways, including glutaminolysis, choline, phosphocholine and total choline syntheses were significantly downregulated in the LIDO group. Discussion: This is the first study assessing the impact of LIDO on metabolomic fingerprint of breast cancer cells. Among pathways downregulated by LIDO, many metabolites are reported to be associated with adverse prognosis when present at a high titer in breast cancer patients. These results fit with the antitumor properties of LIDO and suggest its impact on metabolomics profile of cancer cells. These effects of LIDO are of clinical significance because it is widely used for local anesthesia with cutaneous infiltration during percutaneous tumor biopsy. Future in vitro and preclinical studies are necessary to assess whether metabolomics analysis requires modification of local anesthetic techniques during tumor biopsy.

NMR-based metabolomics revealed metabolic changes in energy production for viral replication and immunological response in rock bream (Oplegnathus fasciatus) tissues during rock bream iridovirus (RBIV) infection

Outbreaks of rock bream iridovirus (RBIV) infection in rock bream (Oplegnathus fasciatus) were continuously recorded, and these infections lead to vast economic losses in the aquaculture industry. However, metabolomic, proteomic, and transcriptomic studies in rock bream infected with RBIV are limited. In this study, metabolic responses in tissues of rock bream with different infection degrees of control (0C) and heavy-infected fish (0H) at 0 week, and fish with low viral titer at 3 week (3 L)-were observed by high-resolution magic-angle spinning (HR-MAS) NMR spectroscopy-based metabolomics, with multivariate, and pathway-based analyses being used to aid interpretation. In the liver, significant metabolic pathways related to energy metabolism such as glutamine and glutamate metabolism and the TCA cycle were observed only in 0H. Additionally, 0C and 3 L were clustered in the OPLS-DA (orthogonal partial least squares discriminant analysis) score plot, and 3 L showed no significant pathways compared to 0C. Thus, it is assumed that lightly infected fish (3 L) were survived from heavy-infection after 3 weeks. In the spleen, the metabolites relevant to energy production and immune response including pyruvate metabolism, the TCA cycle and branched-chain amino acid (BCAA) metabolism were changed in 0H. These results suggest that RBIV induced rapid and high energy production in the spleen by promoting both the Warburg effect, and the TCA cycle for their replication. In the kidney, elevation of the kynurenine pathway, which is related to the immunological response, and decreased osmolytes such as trimethylamine N-oxide and betaine were observed in 0H. We found that RBIV induced various disturbances in metabolism in host cells through viral replication and protective responses in the host. This study is the first metabolomics study of rock bream infected in RBIV using HR-MAS NMR spectroscopy, providing insights into how cellular metabolism is affected during RBIV infection. These results can be used as a resource to evaluate the mechanism of host-pathogen interactions.

MBRS-69. METABOLITE PROFILING OF SHH MEDULLOBLASTOMA IDENTIFIES A SUBSET OF CHILDHOOD TUMOURS ENRICHED FOR HIGH-RISK MOLECULAR BIOMARKERS AND CLINICAL FEATURES

SHH medulloblastoma patients have a variable prognosis. Infants (<3–5 years at diagnosis) are associated with a good prognosis, while disease-course in childhood is associated with specific prognostic biomarkers (MYCN amplification, TP53 mutation, LCA histology; all high-risk). There is an unmet need to identify prognostic subgroups of SHH tumours rapidly in the clinical setting, to aid in real-time risk stratification and disease management. Metabolite profiling is a powerful technique for characterising tumours. High resolution magic angle spinning NMR spectroscopy (HR-MAS) can be performed on frozen tissue samples and provides high quality metabolite information. We therefore assessed whether metabolite profiles could identify subsets of SHH tumours with prognostic potential. Metabolite concentrations of 22 SHH tumours were acquired by HR-MAS and analysed using unsupervised hierarchical clustering. Methylation profiling assigned the infant and childhood SHH subtypes, and clinical and molecular features were compared between clusters. Two clusters were observed. A significantly higher concentration of lipids was observed in Cluster 1 (t-test, p=0.012). Cluster 1 consisted entirely of childhood-SHH whilst Cluster 2 included both childhood-SHH and infant-SHH subtypes. Cluster 1 was enriched for high-risk markers - LCA histology (3/7 v. 0/5), MYCN amplification (2/7 v. 0/5), TP53 mutations (3/7 v. 1/5) and metastatic disease - whilst having a lower proportion of TERT mutations (0/7 v. 2/5) than Cluster 2. These pilot results suggest that (i) it is possible to identify childhood-SHH patients linked to high-risk clinical and molecular biomarkers using metabolite profiles and (ii) these may be detected non-invasively in vivo using magnetic-resonance spectroscopy.

Metabolomic Profiling of Wildtype and Transgenic Giardia lamblia Strains by 1H HR-MAS NMR Spectroscopy.

Giardia lamblia, a causative agent of persistent diarrhea in humans, domestic animals, and cattle, is usually treated with nitro compounds. Consequently, enzymes involved in anaerobic nitro reduction have been investigated in detail as potential targets. Their role within the normal metabolic context is, however, not understood. Using 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, we analyzed the metabolomes of G. lamblia trophozoites overexpressing three nitroreductases (NR1–NR3) and thioredoxin reductase (TrxR), most likely a scavenger of reactive oxygen species, as suggested by the results published in this study. We compared the patterns to convenient controls and to the situation in the nitro drug resistant strain C4 where NR1 is downregulated. We identified 27 metabolites in G. lamblia trophozoites. Excluding metabolites of high variability among different wildtype populations, only trophozoites overexpressing NR1 presented a distinct pattern of nine metabolites, in particular arginine catabolites, differing from the respective controls. This pattern matched a differential pattern between wildtype and strain C4. This suggests that NR1 interferes with arginine and thus energy metabolism. The exact metabolic function of NR1 (and the other nitroreductases) remains to be elucidated.

High-Resolution Magic Angle Spinning (HRMAS) NMR Methods in Metabolomics.

High-resolution magic angle spinning (HRMAS) NMR spectroscopy enables the evaluation of metabolite profiles of intact tissue with high spectral resolution. The ability to preserve the tissue after analysis permits subsequent histopathological examination and enables the analyses of correlations between tissue metabolites and pathologies, thus making HRMAS NMR spectroscopy a powerful tool in the metabolomics field. Improved methods for the elimination of spinning sidebands that appear at low spinning rates preserve the integrity of tissue structures better and allow measurement of delicate tissues, such as clinical biopsy core samples. In the metabolomics field, HRMAS NMR has been established as a valuable tool for both untargeted and targeted metabolite profiling. In this chapter, we present protocols to perform HRMAS NMR spectroscopy experiments, including sample preparation, acquisition procedures, measurement parameters, histopathological examination techniques, spectral processing, and metabolite quantification and statistical analyses.

Impact of chemotherapy on metabolic reprogramming: Characterization of the metabolic profile of breast cancer MDA-MB-231 cells using 1H HR-MAS NMR spectroscopy

Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, studies investigating the effect of chemotherapy on the metabolism of breast cancer cells are still limited. We used 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to study the metabolic profile of human breast cancer MDA-MB-231 cells either untreated (control) or treated with tamoxifen, cisplatin, and doxorubicin. 1H HR-MAS NMR single pulse spectra evidenced signals from all mobile cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites. NMR spectra showed that phosphocholine (i.e., a biomarker of breast cancer malignant transformation) signals were stronger in control than in treated cells, but significantly decreased upon treatment with tamoxifen/cisplatin. NMR spectra acquired with Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence were interpreted only qualitatively because signal areas were attenuated according to their transverse relaxation times (T2). The CPMG method was used to identify soluble metabolites such as organic acids, amino acids, choline and derivatives, taurine, guanidine acetate, tyrosine, and phenylalanine. The fatty acid variations observed by single pulse as well as the lactate, acetate, glycine, and phosphocholine variations observed through CPMG 1H HR-MAS NMR have potential to characterize both responder and non-responder tumors in a molecular level. Additionally, we emphasized that comparable tumors (i.e., with the same origin, in this case breast cancer) may respond totally differently to chemotherapy. Our observations reinforce the theory that alterations in cellular metabolism may contribute to the development of a malignant phenotype and cell resistance.

Metabolic Profiling of Cells in Response to Drug Treatment using 1H High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy.

High-resolution magic angle spinning (HR-MAS) is an NMR technique that provides access to well resolved liquid-like 1H NMR spectra of semi-solid samples. Therefore, 1H HR-MAS NMR spectroscopy has become an important tool for the direct analysis of biological samples such as tissues and cells in a mostly non-destructive way. Here, we focus on the application of HR-MAS NMR combined with multivariate statistical methods used for metabolic profiling of cells and in particular for the study of cellular metabolic responses to drug exposure. The principles of HR-MAS and the metabolomic approach are briefly described. As an example, a study on the metabolic response of different cell types towards treatment with a highly cytotoxic hexacationic ruthenium metallaprism as potential anti-cancer drug is presented. Specific metabolites and metabolic pathways are suggested to be associated with the cellular response. The study demonstrates the potential of HR-MAS metabolomics applied to cells for addressing the intracellular processes involved in the treatment with organometallic drugs.

Polyurea Structure Characterization by HR-MAS NMR Spectroscopy

Owing to the presence of abundant interchain interactions such as hydrogen bonds, polyureas (PU) are only swellable or soluble in a limited number of highly protonic solvents, and the viscosity of the solutions obtained is very high, making their chemical structure characterization hard or even impossible by standard NMR. Accurate structure analysis is also hard by solid-state NMR due to low spectral resolution. The presence of a side reaction in their synthesis generating biuret cross-links is often invoked to explain their insolubility. Here we demonstrate that High Resolution Magic Angle Spinning (HR-MAS) NMR is an efficient tool for the chemical structure analysis even for cross-linked PU. With 1H, 13C, and 1H–15N HSQC combined, a variety of linear and cross-linked PU is analyzed by HR-MAS NMR, and conclusive information on their chemical structure is obtained, which reveals for the first time that the biuret group is absent in all PU.

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study.

The chemical cross-linking of β-cyclodextrin (β-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels. The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of β-cyclodextrin (β-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels. The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

(1)H HR-MAS NMR Spectroscopy and the Metabolite Determination of Typical Foods in Mediterranean Diet.

NMR spectroscopy has become an experimental technique widely used in food science. The experimental procedures that allow precise and quantitative analysis on different foods are relatively simple. For a better sensitivity and resolution, NMR spectroscopy is usually applied to liquid sample by means of extraction procedures that can be addressed to the observation of particular compounds. For the study of semisolid systems such as intact tissues, High-Resolution Magic Angle Spinning (HR-MAS) has received great attention within the biomedical area and beyond. Metabolic profiling and metabolism changes can be investigated both in animal organs and in foods. In this work we present a proton HR-MAS NMR study on the typical vegetable foods of Mediterranean diet such as the Protected Geographical Indication (PGI) cherry tomato of Pachino, the PGI Interdonato lemon of Messina, several Protected Designation of Origin (PDO) extra virgin olive oils from Sicily, and the Traditional Italian Food Product (PAT) red garlic of Nubia. We were able to identify and quantify the main metabolites within the studied systems that can be used for their characterization and authentication.

Anomalous diffusion of Ibuprofen in cyclodextrin nanosponge hydrogels: an HRMAS NMR study.

Ibuprofen sodium salt (IP) was encapsulated in cyclodextrin nanosponges (CDNS) obtained by cross-linking of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn) in two different preparations: CDNSEDTA 1:4 and 1:8, where the 1:n notation indicates the CD to EDTAn molar ratio. The entrapment of IP was achieved by swelling the two polymers with a 0.27 M solution of IP in D2O, leading to colourless, homogeneous hydrogels loaded with IP. The molecular environment and the transport properties of IP in the hydrogels were studied by high resolution magic angle spinning (HRMAS) NMR spectroscopy. The mean square displacement (MSD) of IP in the gels was obtained by a pulsed field gradient spin echo (PGSE) NMR pulse sequence at different observation times td. The MSD is proportional to the observation time elevated to a scaling factor α. The α values define the normal Gaussian random motion (α = 1), or the anomalous diffusion (α < 1, subdiffusion, α > 1 superdiffusion). The experimental data here reported point out that IP undergoes subdiffusive regime in CDNSEDTA 1:4, while a slightly superdiffusive behaviour is observed in CDNSEDTA 1:8. The transition between the two dynamic regimes is triggered by the polymer structure. CDNSEDTA 1:4 is characterized by a nanoporous structure able to induce confinement effects on IP, thus causing subdiffusive random motion. CDNSEDTA 1:8 is characterized not only by nanopores, but also by dangling EDTA groups ending with ionized COO− groups. The negative potential provided by such groups to the polymer backbone is responsible for the acceleration effects on the IP anion thus leading to the superdiffusive behaviour observed. These results point out that HRMAS NMR spectroscopy is a powerful direct method for the assessment of the transport properties of a drug encapsulated in polymeric scaffolds. The diffusion properties of IP in CDNS can be modulated by suitable polymer synthesis; this finding opens the possibility to design suitable systems for drug delivery with predictable and desired drug release properties.

PH-sensitive superabsorbent polymers: a potential candidate material for self-healing concrete

Superabsorbent polymers (SAPs) have already found their way in many applications. These ‘smart’ polymers undergo major characteristic changes by small environmental variations. In the present work, copolymer networks composed of acrylic acid, acrylamide and N,N′-methylenebisacrylamide have been synthesized using free radical precipitation polymerization. The polymers obtained have been characterized for their chemical structure, moisture (de)sorption and swelling behaviour using, respectively, attenuated total reflectance-infrared spectroscopy, high-resolution magic-angle spinning NMR spectroscopy, dynamic vapour sorption and swelling studies. The results indicated a remarkable moisture uptake capacity at high relative humidities of more than 90 % the original polymer weight with a negligible hysteresis. The latter implies that the SAPs developed are very promising water reservoir candidates, which become useful in concrete-related applications. Furthermore, the swelling data revealed that polymers with a low cross-linking density result in materials with superabsorbent properties. In addition, these SAPs show a pH-dependent swelling behaviour up to 450 times their original weight at pH 12.

Rapid Assessment of Fish Freshness and Quality by 1H HR-MAS NMR Spectroscopy

A new analytical method that allows the rapid assessment of fish freshness and quality is presented. The method is based on 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy and allows the rapid determination of two well-established indicators of fish freshness and quality: the K value and the trimethylamine nitrogen (TMA-N) content. The method is demonstrated on four different species of fish (sea bream, sea bass, trout, and red mullet) stored on ice at 0 °C. The results obtained are in agreement with more cumbersome methods classically used to determine the K value and the TMA-N concentration. The main advantage of the 1H HR-MAS NMR approach is to allow a direct measurement of these two parameters directly on unprocessed fish sample without using any preliminary extraction. The total analysis time, including sample preparation, is of the order of 40 min per sample.

Metabolic profiling of cervical tubercular lymphadenitis tissues by proton HR-MAS NMR spectroscopy

Proton metabolic profiling of incisional biopsied cervical lymph node tissue specimens of 109 patients suffering from tubercular (CTBL) and non-specific (NSCLA) lymphadenitis were analyzed by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. In the present study, 40 endogenous metabolites namely, myo-inositol (m-Ins), branched chain amino acids (BCAA), glutamate, serine, taurine (Tau) aromatic amino acids, choline (Cho) containing compounds and glucose were characterized. To the best of our knowledge, this is the first report on metabolic profiling of cervical tubercular lymph node tissues using HR-MAS NMR spectroscopy. The principal component analysis revealed a clear discrimination between CTBL and NSCLA tissues. Increase in the concentration of mobile poly unsaturated fatty acids, BCAA, Cho, Tau, glycine and a decrease in the concentration of lactate, phosphocholine and m-Ins was observed in CTBL cases. The partial least square discriminant analysis (PLS-DA) with R 2 = 0.95 and Q 2 = 0.92 provided >98 % of correct classification between the two groups. A PLS-DA training set model of 75 % (CTBL = 54, NSCLA = 27) of the subjects when subjected for prediction of 25 % cases (CTBL = 18, NSCLA = 10) as an unknown dataset provided more than 98 % of diagnostic accuracy in their respective histological categories. The receiver operator characteristic curve was generated from PLS-DA factor-1 projected an area under the curve of 0.962. The metabolic profile obtained from HR-MAS NMR spectroscopy may be used as surrogate markers in vivo MRS for differentiating between CTBL and NSCLA cases non-invasively.

Impact of ionic liquids on the structure of peptides proved by HR-MAS NMR spectroscopy

The present work reports on the first systematic study on the impact of ionic liquids on the structure of peptides via high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. The investigation is based on the use of six tetrapeptides of the general structure Ala-Xaa-Pro-Phe (Xaa: Ala, Asp, Glu, Gly, Lys and Phe) dissolved in three aqueous N,N′-dialkylimidazolium-derived ionic liquids. Analyses of individual chemical shift differences based on highly resolved one- and two-dimensional NMR spectra revealed a broad spectrum of strong peptide/ionic liquid interactions depending on the nature of the peptide and, most importantly, the composition of the molten salt. Alignments of ionic liquid specific interaction pattern with the effect of the respective ionic liquid on the cis/trans equilibrium state of the peptides' Xaa-Pro bond allowed for initial conclusions on the importance of individual modes of peptide/ionic liquid interactions on the structure of peptides at a molecular level. The reliability of the results obtained could be proven by a clear concentration dependency of individual peptide/ionic liquid interactions and cis/trans ratios of Xaa-Pro bonds as well as initial 2D ROESY NMR experiments.

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.