High-resolution Magic Angle Spinning Research Articles

Nanostructure and Molecular-Level Characterization of Aminoalkyl Methacrylate Copolymer and the Impact on Drug Solubilization Ability.

The effects of pH changes and saccharin (SAC) addition on the nanostructure and mobility of the cationic aminoalkyl methacrylate copolymer Eudragit E PO (EUD-E) and its drug solubilization ability were investigated. Small-angle X-ray scattering performed using synchrotron radiation and atomic force microscopy showed that the EUD-E nanostructure, which has a size of approximately several nanometers, changed from a random coil structure at low pH (pH 4.0-5.0) to a partially folded structure at high pH (pH 5.5-6.5). The EUD-E also formed a partially folded structure in a wide pH range of 4.5-6.5 when SAC was present, and the coil-to-globule transition was moderate with pH increase, compared with that when SAC was absent. The equilibrium solubility of the neutral drug naringenin (NAR) was enhanced in the EUD-E solution and further increased as the pH increased. The enlargement of the hydrophobic region of EUD-E in association with the coil-to-globule transition led to efficient solubilization of NAR. The interaction with SAC enhanced the mobility of the EUD-E chains in the hydrophobic region of EUD-E, resulting in changes in the drug-solubilizing ability. 1H high-resolution magic-angle spinning NMR measurements revealed that the solubilized NAR in the partially folded structure of EUD-E showed higher molecular mobility in the presence of SAC than in the absence of SAC. This study highlighted that solution pH and the presence of SAC significantly changed the drug solubilization ability of EUD-E, followed by changes in the EUD-E nanostructure, including its hydrophobic region.

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.

Grading of endometrial cancer using 1H HR-MAS NMR-based metabolomics

The tissue metabolomic characteristics associated with endometrial cancer (EC) at different grades were studied using high resolution (400 MHz) magic angle spinning (HR-MAS) proton spectroscopy. The metabolic profiles were obtained from 64 patients (14 with grade 1 (G1), 33 with grade 2 (G2) and 17 with grade 3 (G3) tumors) and compared with the profile acquired from 10 patients with the benign disorders. OPLS-DA revealed increased valine, isoleucine, leucine, hypotaurine, serine, lysine, ethanolamine, choline and decreased creatine, creatinine, glutathione, ascorbate, glutamate, phosphoethanolamine and scyllo-inositol in all EC grades in reference to the non-transformed tissue. The increased levels of taurine was additionally detected in the G1 and G2 tumors in comparison to the control tissue, while the elevated glycine, N-acetyl compound and lactate—in the G1 and G3 tumors. The metabolic features typical for the G1 tumors are the increased dimethyl sulfone, phosphocholine, and decreased glycerophosphocholine and glutamine levels, while the decreased myo-inositol level is characteristic for the G2 and G3 tumors. The elevated 3-hydroxybutyrate, alanine and betaine levels were observed in the G3 tumors. The differences between the grade G1 and G3 malignances were mainly related to the perturbations of phosphoethanolamine and phosphocholine biosynthesis, inositol, betaine, serine and glycine metabolism. The statistical significance of the OPLS-DA modeling was also verified by an univariate analysis. HR-MAS NMR based metabolomics provides an useful insight into the metabolic reprogramming in endometrial cancer.

Trehalose-deficient Acinetobacter baumannii exhibits reduced virulence by losing capsular polysaccharide and altering membrane integrity.

Acinetobacter baumannii has become a leading cause of bacterial nosocomial infections, in part, due to its ability to resist desiccation, disinfection and antibiotics. Several factors contribute to the tenacity and virulence of this pathogen, including production of a broad range of surface glycoconjugates, secretory systems and efflux pumps. We became interested in examining the importance of trehalose in A. baumannii after comparing intact bacterial cells by high-resolution magic angle spinning nuclear magnetic resonance and by noting high levels of this disaccharide, obscuring all other resonances in the spectrum. Since this was observed under normal growth conditions, we speculated that trehalose must serve additional functions beyond osmolyte homeostasis. Using the virulent isolate A. baumannii AB5075 and mutants in the trehalose synthesis pathway, osmoregulatory trehalose synthesis proteins A and B (△otsA and △otsB), we found that the trehalose-deficient △otsA showed increased sensitivity to desiccation, colistin, serum complement and peripheral blood mononuclear cells, while trehalose-6-phosphate producing △otsB behaved similar to the wild-type. The △otsA mutant also demonstrated increased membrane permeability and loss of capsular polysaccharide. These findings demonstrate that trehalose deficiency leads to loss of virulence in A. baumannii AB5075.

An integrated systems-level model of the toxicity of brevetoxin based on high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) metabolic profiling of zebrafish embryos

Brevetoxins (PbTx) are a well-recognized group of neurotoxins associated with harmful algal blooms, and specifically recurrent “Florida Red Tides,” in marine waters that are linked to impacts on both human and ecosystem health including well-documented “fish kills” and marine mammal mortalities in affected coastal waters. Understanding mechanisms and pathways of PbTx toxicity enables identification of relevant biomarkers to better understand these environmental impacts, and improve monitoring efforts, in relation to this toxin. Toward a systems-level understanding of toxicity, and identification of potential biomarkers, high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was utilized for metabolic profiling of zebrafish (Danio rerio) embryos, as an established toxicological model, exposed to PbTx-2 (the most common congener in marine waters). Metabolomics studies were, furthermore, complemented by an assessment of the toxicity of PbTx-2 in embryonic stages of zebrafish and mahi-mahi (Coryphaena hippurus), the latter representing an ecologically and geographically relevant marine species of fish, which identified acute embryotoxicity at environmentally relevant (i.e., parts-per-billion) concentrations in both species. HRMAS NMR analysis of intact zebrafish embryos exposed to sub-lethal concentrations of PbTx-2 afforded well-resolved spectra, and in turn, identification of 38 metabolites of which 28 were found to be significantly altered, relative to controls. Metabolites altered by PbTx-2 exposure specifically included those associated with (1) neuronal excitotoxicity, as well as associated neural homeostasis, and (2) interrelated pathways of carbohydrate and energy metabolism. Metabolomics studies, thereby, enabled a systems-level model of PbTx toxicity which integrated multiple metabolic, molecular and cellular pathways, in relation to environmentally relevant concentrations of the toxin, providing insight to not only targets and mechanisms, but potential biomarkers pertinent to environmental risk assessment and monitoring strategies.

Dysregulated Alanine as a Potential Predictive Marker of Glioma-An Insight from Untargeted HRMAS-NMR and Machine Learning Data.

Metabolic alterations play a crucial role in glioma development and progression and can be detected even before the appearance of the fatal phenotype. We have compared the circulating metabolic fingerprints of glioma patients versus healthy controls, for the first time, in a quest to identify a panel of small, dysregulated metabolites with potential to serve as a predictive and/or diagnostic marker in the clinical settings. High-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS-NMR) was used for untargeted metabolomics and data acquisition followed by a machine learning (ML) approach for the analyses of large metabolic datasets. Cross-validation of ML predicted NMR spectral features was done by statistical methods (Wilcoxon-test) using JMP-pro16 software. Alanine was identified as the most critical metabolite with potential to detect glioma with precision of 1.0, recall of 0.96, and F1 measure of 0.98. The top 10 metabolites identified for glioma detection included alanine, glutamine, valine, methionine, N-acetylaspartate (NAA), γ-aminobutyric acid (GABA), serine, α-glucose, lactate, and arginine. We achieved 100% accuracy for the detection of glioma using ML algorithms, extra tree classifier, and random forest, and 98% accuracy with logistic regression. Classification of glioma in low and high grades was done with 86% accuracy using logistic regression model, and with 83% and 79% accuracy using extra tree classifier and random forest, respectively. The predictive accuracy of our ML model is superior to any of the previously reported algorithms, used in tissue- or liquid biopsy-based metabolic studies. The identified top metabolites can be targeted to develop early diagnostic methods as well as to plan personalized treatment strategies.

Complementary Nuclear Magnetic Resonance-Based Metabolomics Approaches for Glioma Biomarker Identification in a Drosophila melanogaster Model.

Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort. Drosophila melanogaster is a compelling genetic model to better understanding human neurological diseases owing to its high conservation in signaling pathways and cellular content of the brain. Here, glioma has been induced in Drosophila by co-activating the epidermal growth factor receptor and the phosphatidyl-inositol-3 kinase signaling pathways. Complementary nuclear magnetic resonance (NMR) techniques were used to obtain metabolic profiles in the third instar larvae brains. Fresh organs were directly studied by 1H high resolution-magic angle spinning (HR-MAS) NMR, and brain extracts were analyzed by solution-state 1H-NMR. Statistical analyses revealed differential metabolic signatures, impacted metabolic pathways, and glioma biomarkers. Each method was efficient to determine biomarkers. The highlighted metabolites including glucose, myo-inositol, sarcosine, glycine, alanine, and pyruvate for solution-state NMR and proline, myo-inositol, acetate, and glucose for HR-MAS show very good performances in discriminating samples according to their nature with data mining based on receiver operating characteristic curves. Combining results allows for a more complete view of induced disturbances and opens the possibility of deciphering the biochemical mechanisms of these tumors. The identified biomarkers provide a means to rebalance specific pathways through targeted metabolic therapy and to study the effects of pharmacological treatments using Drosophila as a model organism.

Abstract LB209: Blood serum MRS metabolomics screening of human lung cancer prior to disease diagnosis

Abstract Blood serum MRS metabolomics screening of human lung cancer prior to disease diagnosis Lung cancer (LuCa), the leading cause of cancer deaths, is often diagnosed late. Low-dose spiral CT can detect small and early stage LuCa lesions, but cannot be used widely as an annual LuCa screening tool. Metabolomics detects global metabolite variations under physiology and pathology. Metabolomic profiles measured from blood may reveal LuCa at early stages as a screening tool to triage suspicious patients to CT tests. Blood sera obtained from LuCa patients prior to their diagnosis were studied with MRS to establish LuCa screening metabolomic profiles.Samples. Sera from non-small cell LuCa (NSCLC) patients and their age, gender and smoking habit matched healthy controls were grouped according to the design of training-testing-validation cohorts in this study. The training cohort included 25 NSCLC sera from patients at the time of diagnosis (TOD) and controls (Ctrl); the testing cohort consisted 25 sera collected 0.5 to 5 yrs prior to diagnosis (PTD) from the 25 NSCLC patients in the training cohort; and the validation cohort recruited sera collected less than 2 yrs PTD from additional 54 NSCLC patients and controls. MR Spectroscopy. High resolution magic angle spinning (HRMAS) MRS analysis of serum samples are performed at 4°C by a 600MHz Bruker spectrometer at 3,600Hz spinning rate. Spectra are analyzed with a MatLab-based curve fitting program. Data Analysis. 57 spectral regions were selected based on the training and testing cohorts. Following selections of these regions, all data analytical procedures, including principal component and canonical analyses, were performed on the training cohort and followed by the testing and validation cohorts. Analyses of native sera HRMAS MRS of the training cohort selected 57 spectral regions for statistical analyses, and PCA and canonical analysis were conducted to differentiate TOD from Ctrl groups, with the testing and validation cohorts passively followed the calculations to produce scores within each case.Using the mean plus one standard error (M+SE) as the threshold, calculated from the canonical score differences between TOD and PTD for each case, i.e. the difference of the two scores for each patient, a Kaplan-Meier survival analysis indicates better survival rate from their time of NSCLC diagnoses for patients with score differences higher than the threshold (p=0.031). Furthermore, for Stage I and IIA patients, survivals can be predicted (p=0.027) from their PTD samples, if their score values are higher than the M+SE threshold. Since neither testing nor validation cohorts were involved in the determination of values of the canonical score, by collectively examining all Stage I and IIA in both cohorts, the resulting Kaplan-Meier survival predicting capability by the threshold (established by the testing cohort) was enhanced significantly (p=0.0044). For 5-year survival, our data showed Sen=0.63, Spe=1.00, PPV=1.00, NPV=0.21, ACC=0.67, and F1=0.78. Thus, serum metabolomics may be a screening candidate for early detections with the potential to further probe into all the related metabolic pathways for better understanding of disease. Citation Format: Tjada Schult, Mara Lauer, Yannick Berker, Marcella Cardoso, Lindsey Vandergrift, Piet Habbel, Johannes Nowak, Martin Aryee, Mari Mino-Kenudson, David Christiani, Leo Cheng. Blood serum MRS metabolomics screening of human lung cancer prior to disease diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB209.

Localizing the Seizure Onset Site Through Metabolic Imaging of GABA.

In Vivo Gamma-Aminobutyric Acid Increase as a Biomarker of the Epileptogenic Zone: An Unbiased Metabolomics Approach Hamelin S, Stupar V, Maziere L, et al. Epilepsia. 2021;62(1):163-175.Objective:Following surgery, focal seizures relapse in 20% to 50% of cases due to the difficulty of delimiting the epileptogenic zone (EZ) by current imaging or electrophysiological techniques. Here, we evaluate an unbiased metabolomics approach based on ex vivo and in vivo nuclear magnetic resonance spectroscopy (MRS) methods to discriminate the EZ in a mouse model of mesiotemporal lobe epilepsy (MTLE).Methods:Four weeks after unilateral injection of kainic acid (KA) into the dorsal hippocampus of mice (KA-MTLE model), we analyzed hippocampal and cortical samples with high-resolution magic angle spinning (HRMAS) MRS. Using advanced multivariate statistics, we identified the metabolites that best discriminate the injected dorsal hippocampus (EZ) and developed an in vivo MEGAPRESS MRS method to focus on the detection of these metabolites in the same mouse model.Results:Multivariate analysis of HRMAS data provided evidence that γ-aminobutyric acid (GABA) is largely increased in the EZ of KA-MTLE mice and is the metabolite that best discriminates the EZ when compared with sham and, more importantly, when compared with adjacent brain regions. These results were confirmed by capillary electrophoresis analysis and were not reversed by a chronic exposition to an antiepileptic drug (carbamazepine). Then, using in vivo noninvasive GABA-edited MRS, we confirmed that a high GABA increase is specific to the injected hippocampus of KA-MTLE mice.Significance:Our strategy using ex vivo MRS-based untargeted metabolomics to select the most discriminant metabolite(s), followed by in vivo MRS-based targeted metabolomics, is an unbiased approach to accurately define the EZ in a mouse model of focal epilepsy. Results suggest that GABA is a specific biomarker of the EZ in MTLE.

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

Metabolic Profiling of Suprachiasmatic Nucleus Reveals Multifaceted Effects in an Alzheimer's Disease Mouse Model.

Background:Circadian rhythm disturbance is commonly observed in Alzheimer’s disease (AD). In mammals, these rhythms are orchestrated by the superchiasmatic nucleus (SCN). Our previous study in the Tg2576 AD mouse model suggests that inflammatory responses, most likely manifested by low GABA production, may be one of the underlying perpetrators for the changes in circadian rhythmicity and sleep disturbance in AD. However, the mechanistic connections between SCN dysfunction, GABA modulation, and inflammation in AD is not fully understood.Objective:To reveal influences of amyloid pathology in Tg2576 mouse brain on metabolism in SCN and to identify key metabolic sensors that couple SCN dysfunction with GABA modulation and inflammation.Methods:High resolution magic angle spinning (HR-MAS) NMR in conjunction with multivariate analysis was applied for metabolic profiling in SCN of control and Tg2576 female mice. Immunohistochemical analysis was used to detect neurons, astrocytes, expression of GABA transporter 1 (GAT1) and Bmal1.Results:Metabolic profiling revealed significant metabolic deficits in SCN of Tg2576 mice. Reductions in glucose, glutamate, GABA, and glutamine provide hints toward an impaired GABAergic glucose oxidation and neurotransmitter cycling in SCN of AD mice. In addition, decreased redox co-factor NADPH and glutathione support a redox disbalance. Immunohistochemical examinations showed low expression of the core clock protein, Bmal1, especially in activated astrocytes. Moreover, decreased expression of GAT1 in astrocytes indicates low GABA recycling in this cell type.Conclusion:Our results suggest that redox disbalance and compromised GABA signaling are important denominators and connectors between neuroinflammation and clock dysfunction in AD.

Molecular Characterization of the Organic Fraction of Municipal Solid Waste and Compositional Evolution during Oxidative Processes Assessed by HR-MAS 13C NMR Spectroscopy

The use of high-resolution magic angle spinning (HR-MAS) 13C NMR spectroscopy is proposed here as an innovative and non-destructive approach to investigate the chemical composition of the organic fraction of municipal solid waste (OFMSW) and for monitoring the evolution of their composition during the oxidative iron-based Fenton treatment to which the initial matrix is subjected. The high quality and the good resolution of the 13C HR-MAS NMR spectra allowed an accurate assignment and quantification of the various types of carbon present in the analyzed organic matrix. Moreover, the HR-MAS has also shown its effectiveness in monitoring the different oxidative processes to which the same initially organic matrix has been subjected. The results obtained from the HR-MAS spectra on the collected samples during the different oxidative experiments, indicate that Fenton treatment is able to modify the percentage of the different types of carbons as a function of the concentrations of both Fenton reactants, H2O2 and Fe2+ salt, and of the oxidative process time.

Integrative analysis of physiological responses to high fat feeding with diffusion tensor images and neurochemical profiles of the mouse brain

BackgroundObesity proceeds with important physiological and microstructural alterations in the brain, but the precise relationships between the diet and feeding status, its physiological responses, and the observed neuroimaging repercussions, remain elusive. Here, we implemented a mouse model of high fat diet (HFD) feeding to explore specific associations between diet, feeding status, phenotypic and endocrine repercussions, and the resulting microstructural and metabolic alterations in the brain, as detected by diffusion tensor imaging (DTI) and neurochemical metabolic profiling.MethodsBrain DTI images were acquired from adult male C57BL6/J mice after 6 weeks of HFD, or standard diet (SD) administrations, both under the fed, and overnight fasted conditions. Metabolomic profiles of the cortex (Ctx), hippocampus (Hipc), and hypothalamus (Hyp) were determined by 1H high-resolution magic angle spinning (HRMAS) spectroscopy, in cerebral biopsies dissected after microwave fixation. Mean diffusivity (MD), fractional anisotropy (FA) maps, and HRMAS profiles were complemented with determinations of phenotypic alterations and plasma levels of appetite-related hormones, measured by indirect calorimetry and multiplex assays, respectively. We used Z-score and alternating least squares scaling (ALSCAL) analysis to investigate specific associations between diet and feeding status, physiological, and imaging parameters.ResultsHFD induced significant increases in body weight and the plasma levels of glucose and fatty acids in the fed and fasted conditions, as well as higher cerebral MD (Ctx, Hipc, Hyp), FA (Hipc), and mobile saturated fatty acids resonances (Ctx, Hipc, Hyp). Z-score and ASLCAL analysis identified the precise associations between physiological and imaging variables.ConclusionsThe present study reveals that diet and feeding conditions elicit prominent effects on specific imaging and spectroscopic parameters of the mouse brain that can be associated to the alterations in phenotypic and endocrine variables. Together, present results disclose a neuro-inflammatory response to HFD, characterized primarily by vasogenic edema and compensatory responses in osmolyte concentrations.

HR-MAS NMR Applications in Plant Metabolomics.

Metabolomics is used to reduce the complexity of plants and to understand the underlying pathways of the plant phenotype. The metabolic profile of plants can be obtained by mass spectrometry or liquid-state NMR. The extraction of metabolites from the sample is necessary for both techniques to obtain the metabolic profile. This extraction step can be eliminated by making use of high-resolution magic angle spinning (HR-MAS) NMR. In this review, an HR-MAS NMR-based workflow is described in more detail, including used pulse sequences in metabolomics. The pre-processing steps of one-dimensional HR-MAS NMR spectra are presented, including spectral alignment, baseline correction, bucketing, normalisation and scaling procedures. We also highlight some of the models which can be used to perform multivariate analysis on the HR-MAS NMR spectra. Finally, applications of HR-MAS NMR in plant metabolomics are described and show that HR-MAS NMR is a powerful tool for plant metabolomics studies.

Unique Metabolomic Profile of Skeletal Muscle in Chronic Limb Threatening Ischemia.

Chronic limb threatening ischemia (CLTI) is the most severe manifestation of peripheral atherosclerosis. Patients with CLTI have poor muscle quality and function and are at high risk for limb amputation and death. The objective of this study was to interrogate the metabolome of limb muscle from CLTI patients. To accomplish this, a prospective cohort of CLTI patients undergoing either a surgical intervention (CLTI Pre-surgery) or limb amputation (CLTI Amputation), as well as non-peripheral arterial disease (non-PAD) controls were enrolled. Gastrocnemius muscle biopsy specimens were obtained and processed for nuclear magnetic resonance (NMR)-based metabolomics analyses using solution state NMR on extracted aqueous and organic phases and 1H high-resolution magic angle spinning (HR-MAS) on intact muscle specimens. CLTI Amputation specimens displayed classical features of ischemic/hypoxic metabolism including accumulation of succinate, fumarate, lactate, alanine, and a significant decrease in the pyruvate/lactate ratio. CLTI Amputation muscle also featured aberrant amino acid metabolism marked by elevated branched chain amino acids. Finally, both Pre-surgery and Amputation CLTI muscles exhibited pronounced accumulation of lipids, suggesting the presence of myosteatosis, including cholesterol, triglycerides, and saturated fatty acids. Taken together, these metabolite differences add to a growing body of literature that have characterized profound metabolic disturbance’s in the failing ischemic limb of CLTI patients.

Mutations Linked to Dilated Cardiomyopathy Perturb the Topology and Regulatory Response of Phospholamban

Phospholamban (PLN), a small 52-residue membrane protein expressed in cardiac tissue, functions as a regulatory subunit to the 110 kDa sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) calcium pump. The SERCA-PLN complex is responsible for restoring high SR Ca2+ concentrations prior to each cardiomyocyte contraction, in which PLN maintains SERCA's activity within a tight physiological window. PLN binds SERCA via a transmembrane protein-protein interaction that suppresses the affinity of SERCA's Ca2+ sites, but this effect is relieved upon phosphorylation of PLN's cytoplasmic extension by protein kinase A (PKA) upon β-adrenergic stimulation. PLN mutations that alter its activity or interfere with phosphorylation are linked to hereditary forms of dilated cardiomyopathy (DCM), although the reasons for this remain unclear due to a lack of structural data for the SERCA-PLN complex. Using structural restraints from high-resolution magic-angle spinning (MAS) and oriented sample (OS) solid-state NMR (ssNMR) spectroscopy, we have constructed dynamic models of the SERCA-PLN complex that capture a topological allosteric mechanism for PLN-driven communication of cytoplasmic regulatory stimuli to SERCA's Ca2+ binding sites. Further OS-ssNMR measurements and activity assays of DCM mutants of PLN displayed an altered topology and a dampened response to phosphorylation, which was consistent with our dynamic models and suggest disease onset may occur from an impaired β-adrenergic pathway. Overall, our recent observations by OS-ssNMR and modelling demonstrate that structural topology (i.e., helical tilt, rotation and secondary structure) is a critical determinant of SERCA regulation by PLN as well as by other homologous regulatory partners expressed in non-cardiac tissues.

Ellagitannin-Lipid Interaction by HR-MAS NMR Spectroscopy.

Ellagitannins have antimicrobial activity, which might be related to their interactions with membrane lipids. We studied the interactions of 12 different ellagitannins and pentagalloylglucose with a lipid extract of Escherichia coli by high-resolution magic angle spinning NMR spectroscopy. The nuclear Overhauser effect was utilized to measure the cross relaxation rates between ellagitannin and lipid protons. The shifting of lipid signals in 1H NMR spectra of ellagitannin–lipid mixture due to ring current effect was also observed. The ellagitannins that showed interaction with lipids had clear structural similarities. All ellagitannins that had interactions with lipids had glucopyranose cores. In addition to the central polyol, the most important structural feature affecting the interaction seemed to be the structural flexibility of the ellagitannin. Even dimeric and trimeric ellagitannins could penetrate to the lipid bilayers if their structures were flexible with free galloyl and hexahydroxydiphenoyl groups.

Sampling Method Affects HR-MAS NMR Spectra of Healthy Caprine Brain Biopsies.

The metabolic profiling of tissue biopsies using high-resolution–magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy may be influenced by experimental factors such as the sampling method. Therefore, we compared the effects of two different sampling methods on the metabolome of brain tissue obtained from the brainstem and thalamus of healthy goats by 1H HR-MAS NMR spectroscopy—in vivo-harvested biopsy by a minimally invasive stereotactic approach compared with postmortem-harvested sample by dissection with a scalpel. Lactate and creatine were elevated, and choline-containing compounds were altered in the postmortem compared to the in vivo-harvested samples, demonstrating rapid changes most likely due to sample ischemia. In addition, in the brainstem samples acetate and inositols, and in the thalamus samples ƴ-aminobutyric acid, were relatively increased postmortem, demonstrating regional differences in tissue degradation. In conclusion, in vivo-harvested brain biopsies show different metabolic alterations compared to postmortem-harvested samples, reflecting less tissue degradation. Sampling method and brain region should be taken into account in the analysis of metabolic profiles. To be as close as possible to the actual situation in the living individual, it is desirable to use brain samples obtained by stereotactic biopsy whenever possible.

Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

Alcoholmeter as a Simple and Accessible Way for Ethanol Determination in Alcohol-Based Hand Sanitizers

Expensive and complex methodologies are available to determine the ethanol concentration in alcohol gel samples. The aim of this article was to demonstrate that alcoholmeter could be used as an alternative method to determine ethanol in gel formulations. Alcohol gel samples were produced using: hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC) and Carbopol 940© (CBP). A factorial design was performed to evaluate the interaction between the ethanol concentration, glycerin and polymer contents in the samples in the recovery data of the ethanol content. Rheological analyses were also performed to identify the limiting factors to ethanol quantification. All the results were compared to high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) as a reference methodology. The results demonstrated that the alcoholmeter could be used to determine alcohol concentration, moreover the level of polymer HEC or HPMC, glycerin and ethanol has no effect in the determination. Yield stress, and not viscosity or flow index, appeared as the limiting factor to the use of alcoholmeter in non-acidified CBP samples. Acidification appears to be mandatory to determination of ethanol concentration in CBP samples. It was possible to achieve an inexpensive, handy and fast methodology to quantify alcohol in gelled samples, in the range of concentrations used in this article.