Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry and NMR Analysis Using an External Chamber.

We describe here a method to study and manipulate photorespiration in intact illuminated leaves. When the CO2/O2 mole fraction ratio changes, instant sampling is critical, to quench leaf metabolism and thus trace rapid metabolic modification due to gaseous conditions. To do so, we combine 13CO2 labeling and gas exchange, using a large custom leaf chamber to facilitate fast sampling by direct liquid nitrogen spraying. Moreover, the use of a high chamber surface area (about 130 cm2) allows one to sample a large amount of leaf material to carry out 13C-nuclear magnetic resonance (NMR) analysis and complementary analyses, such as isotopic analyses by high-resolution mass spectrometry (by both GC and LC-MS). 13C-NMR gives access to absolute 13C amounts at the specific carbon atom position in the labeled molecules and thereby provides an estimate of 13C-flux of photorespiratory intermediates. Since NMR analysis is not very sensitive and can miss minor metabolites, GC or LC-MS analyses are useful to monitor metabolites at low concentrations. Furthermore, 13C-NMR and high-resolution LC-MS allow to estimate isotopologue distribution in response to 13CO2 labeling while modifying photorespiration activity.

First analytical confirmation of drug-induced crystal nephropathy in felines caused by GS-441524, the active metabolite of Remdesivir.

GS-441524 is an adenosine nucleoside antiviral demonstrating significant efficacy in the treatment of feline infectious peritonitis (FIP), an otherwise fatal illness, resulting from infection with feline coronavirus. However, following the emergence of COVID-19, veterinary development was halted, and Gilead pursued clinical development of a GS-441524 pro-drug, resulting in the approval of Remdesivir under an FDA emergency use authorization. Despite lack of regulatory approval, GS-441524 is available without a prescription through various unlicensed online distributors and is commonly purchased by pet owners for the treatment of FIP. Herein, we report data obtained from the analytical characterization of two feline renal calculi, demonstrating the propensity for GS-441524 to cause renal toxicity through drug-induced crystal nephropathy in vivo. As definitive diagnosis of drug-induced crystal nephropathy requires confirmation of the lithogenic material to accurately attribute a mechanism of toxicity, renal stone composition and crystalline matrix were characterized using ultra-performance liquid chromatography photodiode array detection (UPLC-PDA), ultra-performance liquid chromatography mass spectrometry (LCMS), nuclear magnetic resonance (NMR) spectroscopy, X-ray powder diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). This work serves to provide the first analytical confirmation of GS-441524-induced crystal nephropathy in an effort to support toxicologic identification of adverse renal effects caused by administration of GS-441524 or any pro-drug thereof.

Impact of the delay in cryopreservation timing during biobanking procedures on human liver tissue metabolomics.

The liver is a highly specialized organ involved in regulating systemic metabolism. Understanding metabolic reprogramming of liver disease is key in discovering clinical biomarkers, which relies on robust tissue biobanks. However, sample collection and storage procedures pose a threat to obtaining reliable results, as metabolic alterations may occur during sample handling. This study aimed to elucidate the impact of pre-analytical delay during liver resection surgery on liver tissue metabolomics. Patients were enrolled for liver resection during which normal tissue was collected and snap-frozen at three timepoints: before transection, after transection, and after analysis in Pathology. Metabolomics analyses were performed using 1H Nuclear Magnetic Resonance (NMR) and Liquid Chromatography-Mass Spectrometry (LC-MS). Time at cryopreservation was the principal variable contributing to differences between liver specimen metabolomes, which superseded even interindividual variability. NMR revealed global changes in the abundance of an array of metabolites, namely a decrease in most metabolites and an increase in ß-glucose and lactate. LC-MS revealed that succinate, alanine, glutamine, arginine, leucine, glycerol-3-phosphate, lactate, AMP, glutathione, and NADP were enhanced during cryopreservation delay (all p<0.05), whereas aspartate, iso(citrate), ADP, and ATP, decreased (all p<0.05). Cryopreservation delays occurring during liver tissue biobanking significantly alter an array of metabolites. Indeed, such alterations compromise the integrity of metabolomic data from liver specimens, underlining the importance of standardized protocols for tissue biobanking in hepatology.

NMR-based metabolomics identification of potential serum biomarkers of disease progression in patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic and progressive neurological disorder, characterized by neuroinflammation and demyelination within the central nervous system (CNS). The etiology and the pathogenesis of MS are still unknown. Till now, no satisfactory treatments, diagnostic and prognostic biomarkers are available for MS. Therefore, we aimed to investigate metabolic alterations in patients with MS compared to controls and across MS subtypes. Metabolic profiles of serum samples from patients with MS (n = 90) and healthy control (n = 30) were determined by Nuclear Magnetic Resonance (1H-NMR) Spectroscopy using cryogenic probe. This approach was also utilized to identify significant differences between the metabolite profiles of the MS groups (primary progressive, secondary progressive, and relapsing-remitting) and the healthy controls. Concentrations of nine serum metabolites (adenosine triphosphate (ATP), tryptophan, formate, succinate, glutathione, inosine, histidine, pantothenate, and nicotinamide adenine dinucleotide (NAD)) were significantly higher in patients with MS compared to control. SPMS serum exhibited increased pantothenate and tryptophan than in PPMS. In addition, lysine, myo-inositol, and glutamate exhibited the highest discriminatory power (0.93, 95% CI 0.869-0.981; 0.92, 95% CI 0.859-0.969; 0.91, 95% CI 0.843-0.968 respectively) between healthy control and MS. Using NMR- based metabolomics, we identified a set of metabolites capable of classifying MS patients and controls. These findings confirmed untargeted metabolomics as a useful approach for the discovery of possible novel biomarkers that could aid in the diagnosis of the disease.

Evaluation of DFT methods for predicting geometries and NMR spectra of Bi(III) dithiocarbamate complexes with antitumor properties.

CONTEXT: Bismuth complexes with dithiocarbamate ligands have attracted attention because of their biological applications, such as antimicrobial, antileishmanial, and anticancer properties. These complexes have high cytotoxic activity against cancer cells, being more active than the standard drugs cisplatin, doxorubicin, and tamoxifen. In the present study, we investigated the ability of some DFT methods to reproduce the geometries and NMR spectra of the Bi(III) dithiocarbamate complexes, selected based on their proven antitumor activity. Our investigation revealed that the M06-L/def2-TZVP/ECP/CPCM method presented good accuracy in predicting geometries, while the TPSSh/def2-SVP/ECP/CPCM method proved effective in analyzing the 13C NMR spectra of these molecules. In general, all examined methods exhibited comparable performance in predicting 1H NMR signals. METHODS: Calculations were performed with the Gaussian 09 program using the def2-SVP and def2-TZVP basis sets, employing relativistic effective core potential (ECP) for Bi and using the CPCM solvent model. The exchange-correlation functionals BP86, PBE, OLYP, M06-L, B3LYP, B3LYP-D3, M06-2X, TPSSh, CAM-B3LYP, and ωB97XD were used in the study. Geometry optimizations were started from crystallographic structures available at the Cambridge Structural Database. The theoretical results were compared with experimental data using the mean root-mean-square deviation (RMSD), mean absolute deviations (MAD), and linear correlation coefficient (R2).

Influence of Silver Nanoparticles on the Metabolites of Two Transgenic Soybean Varieties: An NMR-Based Metabolomics Approach.

This study investigated the effect of AgNPs and AgNO3, at concentrations equivalent, on the production of primary and secondary metabolites on transgenic soybean plants through an NMR-based metabolomics. The plants were cultivated in a germination chamber following three different treatments: T0 (addition of water), T1 (addition of AgNPs), and T2 (addition of AgNO3). Physiological characteristics, anatomical analyses through microscopic structures, and metabolic profile studies were carried out to establish the effect of abiotic stress on these parameters in soybean plants. Analysis of the 1H NMR spectra revealed the presence of amino acids, organic acids, sugars, and polyphenols. The metabolic profiles of plants with AgNP and AgNO3 were qualitatively similar to the metabolic profile of the control group, suggesting that the application of silver does not affect secondary metabolites. From the PCA, it was possible to differentiate the three treatments applied, mainly based on the content of fatty acids, pinitol, choline, and betaine.

The diagnostic challenge of lack of choline level elevation on 1H-MR spectroscopy in grade II-III gliomas.

The accurate diagnosis of brain tumour is very important in modern neuro-oncology medicine. Magnetic resonance spectroscopy (MRS) is supposed to be a promising tool for detecting cancerous lesions. However, the interpretation of MRS data is complicated by the fact that not all cancerous lesions exhibit elevated choline (Cho) levels. The main goal of our study was to investigate the lack of Cho lesion /Cho ref elevation in the population of grade II-III gliomas. 89 cases of gliomas grade II and III were used for the retrospective analysis - glioma (astrocytoma or oligodendroglioma) grade II (74 out of 89 cases [83%]) and III (15 out of 89 cases [17%]) underwent conventional MRI extended by MRS before treatment. Histopathological diagnosis was obtained either by biopsy or surgical resection. Gliomas were classified to the group of no-choline elevation when the ratio of choline measured within the tumour (Cho lesion ) to choline from NABT (Cho ref ) were equal to or lower than 1. Significant differences were observed between ratios of Cho lesion /Cr lesion calculated for no-choline elevation and glial tumour groups as well as in the NAA lesion /Cr lesion ratio between the no-choline elevation group and glial tumour group. With consistent data concerning choline level elevation and slightly lower NAA value, the Cho lesion /NAA lesion ratio is significantly higher in the WHO II glial tumour group compared to the no-choline elevation cases ( p < 0.000). In the current study the results demonstrated possibility of lack of choline elevation in patients with grade II-III gliomas, so it is important to remember that the lack of elevated choline levels does not exclude neoplastic lesion.

NMR-based metabolomics approach to study the effect and related molecular mechanisms of Saffron essential oil against depression.

Depression currently ranks as the fourth leading cause of disability globally, affecting approximately 20% of the world's population. we established a chronic restraint stress (CRS) induced depression model in mice and employed fluoxetine as a reference drug. We assessed the therapeutic potential of saffron essential oil (SEO) and elucidated its underlying mechanisms through behavioral indices and NMR-based metabolomic analysis. The findings indicate that SEO ameliorates behavioral symptoms of depression, such as the number of entries into the central area, fecal count, latency to immobility, and duration of immobility in both the Tail Suspension Test (TST) and the Forced Swim Test (FST), along with correcting the dysregulation of 5-serotonin. Metabolomic investigations identified sixteen potential biomarkers across the liver, spleen, and kidneys. SEO notably modulated nine of these biomarkers: dimethylglycine, glycerol, adenosine, ß-glucose, α-glucose, uridine, mannose, sarcosine, and aspartate, with glycerol emerging as a common biomarker in both the liver and spleen. Pathway analysis suggests that these biomarkers participate in glycolysis, glycine serine threonine metabolism, and energy metabolism, potentially implicating a role in neural regulation. In summary, SEO effectively mitigates depressive-like behaviors in CRS mice, predominantly via modulation of glycolysis, amino acid metabolism, and energy metabolism, and potentially exerts antidepressant effects through neural regulation. Our study offers insights into small molecule metabolite alterations in CRS mice through a metabolomics lens, providing evidence for the antidepressant potential of plant essential oils and contributing to our understanding of the mechanisms of traditional Chinese medicine in treating depression.

7 Tesla magnetic resonance spectroscopic imaging predicting IDH status and glioma grading.

INTRODUCTION: With the application of high-resolution 3D 7 Tesla Magnetic Resonance Spectroscopy Imaging (MRSI) in high-grade gliomas, we previously identified intratumoral metabolic heterogeneities. In this study, we evaluated the potential of 3D 7 T-MRSI for the preoperative noninvasive classification of glioma grade and isocitrate dehydrogenase (IDH) status. We demonstrated that IDH mutation and glioma grade are detectable by ultra-high field (UHF) MRI. This technique might potentially optimize the perioperative management of glioma patients. METHODS: We prospectively included 36 patients with WHO 2021 grade 2-4 gliomas (20 IDH mutated, 16 IDH wildtype). Our 7 T 3D MRSI sequence provided high-resolution metabolic maps (e.g., choline, creatine, glutamine, and glycine) of these patients' brains. We employed multivariate random forest and support vector machine models to voxels within a tumor segmentation, for classification of glioma grade and IDH mutation status. RESULTS: Random forest analysis yielded an area under the curve (AUC) of 0.86 for multivariate IDH classification based on metabolic ratios. We distinguished high- and low-grade tumors by total choline (tCho) / total N-acetyl-aspartate (tNAA) ratio difference, yielding an AUC of 0.99. Tumor categorization based on other measured metabolic ratios provided comparable accuracy. CONCLUSIONS: We successfully classified IDH mutation status and high- versus low-grade gliomas preoperatively based on 7 T MRSI and clinical tumor segmentation. With this approach, we demonstrated imaging based tumor marker predictions at least as accurate as comparable studies, highlighting the potential application of MRSI for pre-operative tumor classifications.

Plasma Metabolite Profiling in the Search for Early-Stage Biomarkers for Lung Cancer: Some Important Breakthroughs.

Lung cancer is the leading cause of cancer-related mortality worldwide. In order to improve its overall survival, early diagnosis is required. Since current screening methods still face some pitfalls, such as high false positive rates for low-dose computed tomography, researchers are still looking for early biomarkers to complement existing screening techniques in order to provide a safe, faster, and more accurate diagnosis. Biomarkers are biological molecules found in body fluids, such as plasma, that can be used to diagnose a condition or disease. Metabolomics has already been shown to be a powerful tool in the search for cancer biomarkers since cancer cells are characterized by impaired metabolism, resulting in an adapted plasma metabolite profile. The metabolite profile can be determined using nuclear magnetic resonance, or NMR. Although metabolomics and NMR metabolite profiling of blood plasma are still under investigation, there is already evidence for its potential for early-stage lung cancer diagnosis, therapy response, and follow-up monitoring. This review highlights some key breakthroughs in this research field, where the most significant biomarkers will be discussed in relation to their metabolic pathways and in light of the altered cancer metabolism.

A modular motion compensation pipeline for prospective respiratory motion correction of multi-nuclear MR spectroscopy.

Magnetic resonance (MR) acquisitions of the torso are frequently affected by respiratory motion with detrimental effects on signal quality. The motion of organs inside the body is typically decoupled from surface motion and is best captured using rapid MR imaging (MRI). We propose a pipeline for prospective motion correction of the target organ using MR image navigators providing absolute motion estimates in millimeters. Our method is designed to feature multi-nuclear interleaving for non-proton MR acquisitions and to tolerate local transmit coils with inhomogeneous field and sensitivity distributions. OpenCV object tracking was introduced for rapid estimation of in-plane displacements in 2D MR images. A full three-dimensional translation vector was derived by combining displacements from slices of multiple and arbitrary orientations. The pipeline was implemented on 3 T and 7 T MR scanners and tested in phantoms and volunteers. Fast motion handling was achieved with low-resolution 2D MR image navigators and direct implementation of OpenCV into the MR scanner's reconstruction pipeline. Motion-phantom measurements demonstrate high tracking precision and accuracy with minor processing latency. The feasibility of the pipeline for reliable in-vivo motion extraction was shown on heart and kidney data. Organ motion was manually assessed by independent operators to quantify tracking performance. Object tracking performed convincingly on 7774 navigator images from phantom scans and different organs in volunteers. In particular the kernelized correlation filter (KCF) achieved similar accuracy (74%) as scored from inter-operator comparison (82%) while processing at a rate of over 100 frames per second. We conclude that fast 2D MR navigator images and computer vision object tracking can be used for accurate and rapid prospective motion correction. This and the modular structure of the pipeline allows for the proposed method to be used in imaging of moving organs and in challenging applications like cardiac magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI) guided radiotherapy.

Advancements in metabolomics research in benign gallbladder diseases: A review.

The burgeoning field of metabolomics has piqued the interest of researchers in the context of benign gallbladder diseases, which include conditions such as gallbladder polyps, gallstones, and cholecystitis, which are common digestive system disorders. As metabolomics continues to advance, researchers have increasingly focused their attention on its applicability in the study of benign gallbladder diseases to provide new perspectives for diagnostic, therapeutic, and prognostic evaluation. This comprehensive review primarily describes the techniques of liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, and nuclear magnetic resonance and their respective applications in the study of benign gallbladder disease. Metabolomics has made remarkable progress in various aspects of these diseases, ranging from early diagnosis, etiological research, assessment of disease progression and prognosis, and optimization of therapeutic strategies. However, challenges remain in the field of metabolomics in the study of benign gallbladder diseases. These include issues related to data processing and analysis, biomarker discovery and validation, interdisciplinary research integration, and the advancement of personalized medicine. This article attempts to summarize research findings to date, highlight future research directions, and provide a reference point for metabolomics research in benign gallbladder disease.

NMR Metabolomics of Primary Ovarian Cancer Cells in Comparison to Established Cisplatin-Resistant and -Sensitive Cell Lines.

Cancer cell lines are frequently used in metabolomics, such as in vitro tumor models. In particular, A2780 cells are commonly used as a model for ovarian cancer to evaluate the effects of drug treatment. Here, we compare the NMR metabolomics profiles of A2780 and cisplatin-resistant A2780 cells with those of cells derived from 10 patients with high-grade serous ovarian carcinoma (collected during primary cytoreduction before any chemotherapeutic treatment). Our analysis reveals a substantial similarity among all primary cells but significant differences between them and both A2780 and cisplatin-resistant A2780 cells. Notably, the patient-derived cells are closer to the resistant A2780 cells when considering the exo-metabolome, whereas they are essentially equidistant from A2780 and A2780-resistant cells in terms of the endo-metabolome. This behavior results from dissimilarities in the levels of several metabolites attributable to the differential modulation of underlying biochemical pathways. The patient-derived cells are those with the most pronounced glycolytic phenotype, whereas A2780-resistant cells mainly diverge from the others due to alterations in a few specific metabolites already known as markers of resistance.

Metabolite signature of human malignant thyroid tissue: A systematic review and meta-analysis.

BACKGROUND: Thyroid cancer (TC) is the predominant malignancy within the endocrine system. However, the standard method for TC diagnosis lacks the capability to identify the pathological condition of all thyroid lesions. The metabolomics approach has the potential to manage this problem by identifying differential metabolites. AIMS: This study conducted a systematic review and meta-analysis of the NMR-based metabolomics studies in order to identify significant altered metabolites associated with TC. METHODS: A systematic search of published literature in any language in three databases including Embase, PubMed, and Scopus was conducted. Out of 353 primary articles, 12 studies met the criteria for inclusion in the systematic review. Among these, five reports belonging to three articles were eligible for meta-analysis. The correlation coefficient of the orthogonal partial least squares discriminant analysis, a popular model in the multivariate statistical analysis of metabolomic data, was chosen for meta-analysis. The altered metabolites were chosen based on the fact that they had been found in at least three studies. RESULTS: In total, 49 compounds were identified, 40 of which were metabolites. The increased metabolites in thyroid lesions compared normal samples included lactate, taurine, alanine, glutamic acid, glutamine, leucine, lysine, phenylalanine, serine, tyrosine, valine, choline, glycine, and isoleucine. Lipids were the decreased compounds in thyroid lesions. Lactate and alanine were increased in malignant versus benign thyroid lesions, while, myo-inositol, scyllo-inositol, citrate, choline, and phosphocholine were found to be decreased. The meta-analysis yielded significant results for three metabolites of lactate, alanine, and citrate in malignant versus benign specimens. DISCUSSION: In this study, we provided a concise summary of 12 included metabolomic studies, making it easier for future researchers to compare their results with the prior findings. CONCLUSION: It appears that the field of TC metabolomics will experience notable advancement, leading to the discovery of trustworthy diagnostic and prognostic biomarkers.

Whole-brain deuterium metabolic imaging via concentric ring trajectory readout enables assessment of regional variations in neuronal glucose metabolism.

Deuterium metabolic imaging (DMI) is an emerging magnetic resonance technique, for non-invasive mapping of human brain glucose metabolism following oral or intravenous administration of deuterium-labeled glucose. Regional differences in glucose metabolism can be observed in various brain pathologies, such as Alzheimer's disease, cancer, epilepsy or schizophrenia, but the achievable spatial resolution of conventional phase-encoded DMI methods is limited due to prolonged acquisition times rendering submilliliter isotropic spatial resolution for dynamic whole brain DMI not feasible. The purpose of this study was to implement non-Cartesian spatial-spectral sampling schemes for whole-brain 2H FID-MR Spectroscopic Imaging to assess time-resolved metabolic maps with sufficient spatial resolution to reliably detect metabolic differences between healthy gray and white matter regions. Results were compared with lower-resolution DMI maps, conventionally acquired within the same session. Six healthy volunteers (4 m/2 f) were scanned for ~90 min after administration of 0.8 g/kg oral [6,6']-2H glucose. Time-resolved whole brain 2H FID-DMI maps of glucose (Glc) and glutamate + glutamine (Glx) were acquired with 0.75 and 2 mL isotropic spatial resolution using density-weighted concentric ring trajectory (CRT) and conventional phase encoding (PE) readout, respectively, at 7 T. To minimize the effect of decreased signal-to-noise ratios associated with smaller voxels, low-rank denoising of the spatiotemporal data was performed during reconstruction. Sixty-three minutes after oral tracer uptake three-dimensional (3D) CRT-DMI maps featured 19% higher (p = .006) deuterium-labeled Glc concentrations in GM (1.98 ± 0.43 mM) compared with WM (1.66 ± 0.36 mM) dominated regions, across all volunteers. Similarly, 48% higher (p = .01) 2H-Glx concentrations were observed in GM (2.21 ± 0.44 mM) compared with WM (1.49 ± 0.20 mM). Low-resolution PE-DMI maps acquired 70 min after tracer uptake featured smaller regional differences between GM- and WM-dominated areas for 2H-Glc concentrations with 2.00 ± 0.35 mM and 1.71 ± 0.31 mM, respectively (+16%; p = .045), while no regional differences were observed for 2H-Glx concentrations. In this study, we successfully implemented 3D FID-MRSI with fast CRT encoding for dynamic whole-brain DMI at 7 T with 2.5-fold increased spatial resolution compared with conventional whole-brain phase encoded (PE) DMI to visualize regional metabolic differences. The faster metabolic activity represented by 48% higher Glx concentrations was observed in GM- compared with WM-dominated regions, which could not be reproduced using whole-brain DMI with the low spatial resolution protocol. Improved assessment of regional pathologic alterations using a fully non-invasive imaging method is of high clinical relevance and could push DMI one step toward clinical applications.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Imaging of adeno-associated viral capsids for purposes of gene editing using CEST NMR/MRI.

PURPOSE: Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. METHODS: NMR experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800-MHz vertical scanner. In vitro experiments were performed for testing of CEST-NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase-polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. RESULTS: CEST-NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. CONCLUSION: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

Deuterium Metabolic Imaging Differentiates Glioblastoma Metabolic Subtypes and Detects Early Response to Chemoradiotherapy.

Metabolic subtypes of glioblastoma (GBM) have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. In this study, we used 2H magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived GBM xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial tricarboxylic acid cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H magnetic resonance spectroscopy and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for tricarboxylic acid cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response. SIGNIFICANCE: Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment.

An Update on MR Spectroscopy in Cancer Management: Advances in Instrumentation, Acquisition, and Analysis.

MR spectroscopy (MRS) is a noninvasive imaging method enabling chemical and molecular profiling of tissues in a localized, multiplexed, and nonionizing manner. As metabolic reprogramming is a hallmark of cancer, MRS provides valuable metabolic and molecular information for cancer diagnosis, prognosis, treatment monitoring, and patient management. This review provides an update on the use of MRS for clinical cancer management. The first section includes an overview of the principles of MRS, current methods, and conventional metabolites of interest. The remainder of the review is focused on three key areas: advances in instrumentation, specifically ultrahigh-field-strength MRI scanners and hybrid systems; emerging methods for acquisition, including deuterium imaging, hyperpolarized carbon 13 MRI and MRS, chemical exchange saturation transfer, diffusion-weighted MRS, MR fingerprinting, and fast acquisition; and analysis aided by artificial intelligence. The review concludes with future recommendations to facilitate routine use of MRS in cancer management. Keywords: MR Spectroscopy, Spectroscopic Imaging, Molecular Imaging in Oncology, Metabolic Reprogramming, Clinical Cancer Management © RSNA, 2024.

Effect of sirolimus on muscle in inclusion body myositis observed with magnetic resonance imaging and spectroscopy.

BACKGROUND: Finding sensitive clinical outcome measures has become crucial in natural history studies and therapeutic trials of neuromuscular disorders. Here, we focus on 1-year longitudinal data from quantitative magnetic resonance imaging (MRI) and phosphorus magnetic resonance spectroscopy (31P MRS) in a placebo-controlled study of sirolimus for inclusion body myositis (IBM), also examining their links to functional, strength, and clinical parameters in lower limb muscles. METHODS: Quantitative MRI and 31P MRS data were collected at 3 T from a single site, involving 44 patients (22 on placebo, 22 on sirolimus) at baseline and year-1, and 21 healthy controls. Assessments included fat fraction (FF), contractile cross-sectional area (cCSA), and water T2 in global leg and thigh segments, muscle groups, individual muscles, as well as 31P MRS indices in quadriceps or triceps surae. Analyses covered patient-control comparisons, annual change assessments via standard t-tests and linear mixed models, calculation of standardized response means (SRM), and exploration of correlations between MRI, 31P MRS, functional, strength, and clinical parameters. RESULTS: The quadriceps and gastrocnemius medialis muscles had the highest FF values, displaying notable heterogeneity and asymmetry, particularly in the quadriceps. In the placebo group, the median 1-year FF increase in the quadriceps was 3.2% (P < 0.001), whereas in the sirolimus group, it was 0.7% (P = 0.033). Both groups experienced a significant decrease in cCSA in the quadriceps after 1 year (P < 0.001), with median changes of 12.6% for the placebo group and 5.5% for the sirolimus group. Differences in FF and cCSA changes between the two groups were significant (P < 0.001). SRM values for FF and cCSA were 1.3 and 1.4 in the placebo group and 0.5 and 0.8 in the sirolimus group, respectively. Water T2 values were highest in the quadriceps muscles of both groups, significantly exceeding control values in both groups (P < 0.001) and were higher in the placebo group than in the sirolimus group. After treatment, water T2 increased significantly only in the sirolimus group's quadriceps (P < 0.01). Multiple 31P MRS indices were abnormal in patients compared to controls and remained unchanged after treatment. Significant correlations were identified between baseline water T2 and FF at baseline and the change in FF (P < 0.001). Additionally, significant correlations were observed between FF, cCSA, water T2, and functional and strength outcome measures. CONCLUSIONS: This study has demonstrated that quantitative MRI/31P MRS can discern measurable differences between placebo and sirolimus-treated IBM patients, offering promise for future therapeutic trials in idiopathic inflammatory myopathies such as IBM.