1H detection and dynamic nuclear polarization-enhanced NMR of Aß1-42 fibrils.

Several publications describing high-resolution structures of amyloid-ß (Aß) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aß1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aß1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aß and other amyloid systems.

Ex-Vivo 13C NMR Spectroscopy of Rodent Brain: TNF Restricts Neuronal Utilization of Astrocyte-Derived Metabolites.

Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using 13C NMR spectroscopy on brain extracts following intravenous [1,2-13C]-glucose (to probe glia and neuron metabolism), [2-13C]-acetate (probing astrocyte-specific metabolites), or [3-13C]-lactate. An increase in [4,5-13C]-glutamine and [2,3-13C]-lactate coupled with a decrease in [4,5-13C]-glutamate was observed in the [1,2-13C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-13C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-13C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-13C]-lactate and decrease in [4,5-13C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-13C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.

A Very Deep Graph Convolutional Network for 13C NMR Chemical Shift Calculations with Density Functional Theory Level Performance for Structure Assignment.

Nuclear magnetic resonance (NMR) chemical shift calculations are powerful tools for structure elucidation and have been extensively employed in both natural product and synthetic chemistry. However, density functional theory (DFT) NMR chemical shift calculations are usually time-consuming, while fast data-driven methods often lack reliability, making it challenging to apply them to computationally intensive tasks with a high requirement on quality. Herein, we have constructed a 54-layer-deep graph convolutional network for 13C NMR chemical shift calculations, which achieved high accuracy with low time-cost and performed competitively with DFT NMR chemical shift calculations on structure assignment benchmarks. Our model utilizes a semiempirical method, GFN2-xTB, and is compatible with a broad variety of organic systems, including those composed of hundreds of atoms or elements ranging from H to Rn. We used this model to resolve the controversial J/K ring junction problem of maitotoxin, which is the largest whole molecule assigned by NMR calculations to date. This model has been developed into user-friendly software, providing a useful tool for routine rapid structure validation and assignation as well as a new approach to elucidate the large structures that were previously unsuitable for NMR calculations.

Looking for Actives in the Haystack: Merging HRMS2-Based Molecular Networking, Chemometrics, and 13C NMR-Based Dereplication Approaches.

The identification of bioactive natural products (NPs) in complex mixtures has become an important subject of contemporary NP research. In an attempt to address this challenge, the present work proposes an integrated strategy that combines tandem mass spectrometry (MS2)-based molecular networking (MN), a partial least-squares (PLS) chemometric model, as well as 13C NMR-based dereplication using MixONat software. In addition, an advanced glycation end product (AGEs) assay was used for activity evaluation. The approach was implemented on a Garcinia parvifolia bark extract that comprised a high content of prenylated xanthones and had previously shown a notable inhibitory effect on AGE formation. As a main result, the proposed strategy permitted the identification of potentially active metabolites within complex mixtures and their annotation with a higher level of confidence by NMR data. Overall, this comprehensive approach provides a powerful and efficient solution for the targeting and annotating of active compounds in complex NP mixtures.

Hyperpolarized 13C NMR Reveals Pathway Regulation in Lactococcus lactis and Metabolic Similarities and Differences Across the Tree of Life.

The control of metabolic networks is incompletely understood, even for glycolysis in highly studied model organisms. Direct real-time observations of metabolic pathways can be achieved in cellular systems with 13C NMR using dissolution Dynamic Nuclear Polarization (dDNP NMR). The method relies on a short-lived boost of NMR sensitivity using a redistribution of nuclear spin states to increase the alignment of the magnetic moments by more than four orders of magnitude. This temporary boost in sensitivity allows detection of metabolism with sub-second time resolution. Here, we hypothesized that dDNP NMR would be able to investigate molecular phenotypes that are not easily accessible with more conventional methods. The use of dDNP NMR allows real-time insight into carbohydrate metabolism in a Gram-positive bacterium (Lactoccocus lactis), and comparison to other bacterial, yeast and mammalian cells shows differences in the kinetic barriers of glycolysis across the kingdoms of life. Nevertheless, the accumulation of non-toxic precursors for biomass at kinetic barriers is found to be shared across the kingdoms of life. We further find that the visualization of glycolysis using dDNP NMR reveals kinetic characteristics in transgenic strains that are not evident when monitoring the overall glycolytic rate only. Finally, dDNP NMR reveals that resting Lactococcus lactis cells use the influx of carbohydrate substrate to produce acetoin rather than lactate during the start of glycolysis. This metabolic regime can be emulated using suitably designed substrate mixtures to enhance the formation of the C4 product acetoin more than 400-fold. Overall, we find that dDNP NMR provides analytical capabilities that may help to clarify the intertwined mechanistic determinants of metabolism and the optimal usage of biotechnologically important bacteria.

A simple method for quantifying de novo lipogenesis rate and substrate selection in cell cultures by 13 C NMR isotopomer analysis of the crude lipid fraction.

PURPOSE: De novo lipogenesis (DNL) is critical for cell growth and maintenance, and acetyl-CoA precursors can be derived from different substrates. We developed a 13 C NMR analysis of lipid extracts from cultured microglia cells administered with [U-13 C]glucose that informs overall lipogenic activity as well as the contribution of glucose to lipogenic acetyl-CoA. METHODS: BV-2 microglial cell line cultured with glucose and glutamine was provided with [U-13 C]glucose and unlabeled glutamine for 24 h and studied in either the presence or absence of lipopolysaccharide (LPS). Cells were then extracted for lipids and the crude lipid fraction was analyzed by 13 C NMR. 13 C-isotopomer signals in the fatty acid ω - 1 and ω - 2 signals representing consecutive or non-consecutive enrichment of the fatty acid chain by [1,2-13 C2 ]acetyl-CoA were quantified and applied to a probabilistic model of acetyl-CoA precursor and fatty acid enrichment. RESULTS: Glucose contributed 72 ± 2% of lipogenic acetyl-CoA while DNL from all sources accounted for 16 ± 2% of lipid turnover. With LPS, there was a significant decrease in glucose contribution (59 ± 4%, p < 0.05) while DNL was unchanged (11 ± 3%). CONCLUSIONS: A simple 13 C NMR analysis of the crude lipid fractions of BV-2 cells administered with [U-13 C]glucose informs DNL activity and the contribution of glucose to the acetyl-CoA precursors. While DNL was preserved in the presence of LPS, there was redirection of lipogenic acetyl-CoA sources from glucose to other substrates. Thus, in the present article, we describe a novel and simple 13 C NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures.

Solid-state NMR spectroscopy identifies three classes of lipids in Cryptococcus neoformans melanized cell walls and whole fungal cells.

A primary virulence-associated trait of the opportunistic fungal pathogen Cryptococcus neoformans is the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin "ghosts") revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in whole C. neoformans cells, grown under either melanizing or nonmelanizing conditions; triglycerides (TGs), sterol esters (SEs), and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and nonmelanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected to C. neoformans melanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in nonmelanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate that C. neoformans lipids are more varied compositionally and functionally than previously recognized.

A Methionine Chemical Shift Based Order Parameter Characterizing Global Protein Dynamics.

Coupling of side chain dynamics over long distances is an important component of allostery. Methionine side chains show the largest intrinsic flexibility among methyl-containing residues but the actual degree of conformational averaging depends on the proximity and mobility of neighboring residues. The 13 C NMR chemical shifts of the methyl groups of methionine residues located at long distances in the same protein show a similar scaling with respect to the values predicted from the static X-ray structure by quantum methods. This results in a good linear correlation between calculated and observed chemical shifts. The slope is protein dependent and ranges from zero for the highly flexible calmodulin to 0.7 for the much more rigid calcineurin catalytic domain. The linear correlation is indicative of a similar level of side-chain conformational averaging over long distances, and the slope of the correlation line can be interpreted as an order parameter of the global side-chain flexibility.

Isolation and characterization of flavonoid compounds from Stachytarpheta jamaicensis (L.) Vahl and its role as anti-gastro ulcerative agent in rats.

CONTEXT: Stomach ulcer is one of the most common gastrointestinal problems in the world. OBJECTIVE: This study aimed to isolate flavonoid compounds from methanol extract of the aerial parts of Stachytarpheta jamaicensis (L.) Vahl. and evaluate its protective and therapeutic effects against gastric ulcer. MATERIALS AND METHODS: Chromatographic techniques were used for the identification of the isolated compounds. To explore the effects of the plant extract, it was administrated by oral gavage for one week either before or post-ethanol ulcer induction. Ranitidine was also evaluated as a reference drug. Stomach pH, gastric juice volume, lesions number, glutathione, superoxide dismutase, malondialdehyde, succinate dehydrogenase, lactate dehydrogenase, acid phosphatase, Interleukin-10, intracellular adhesion molecule-1, prostaglandin E2, and total protein levels were estimated in gastric tissue. Stomach histopathological features were also monitored. RESULTS: Six flavonoid compounds were isolated, where five of them were isolated for the first time (vitexin, isovitexin, apigenin 7,4'-dimethyl ether, 5,7,2'-trimethoxyflavone, and scutellarein), while apigenin was previously reported. Treatment with plant extract recorded amelioration in all the biochemical parameters. CONCLUSION: The methanol extract of plant aerial parts had prophylactic and treatment effects against gastric ulcer in rats, where its treatment effect exceeded its protective role. The extract recorded anti-inflammatory, and antioxidant effects due to the presence of flavonoid compounds.

A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR.

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5 mg of cholesterol in 31 min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2 h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol. Graphical abstract.

Triterpenes induced by young apple fruits in response to herbivore attack.

Apples Malus domestica, known as a rich source of triterpene acids, induced more variety and quantity of triterpene acids in response to herbivory or mechanical damage. There were 3 major induced compounds: pomaceic acid and euscaphic acid, both of which are known apple triterpene acids, and 2α,19α-dihydroxy-3-oxours-12-en-28-oic acid (named eriobotoric acid), which was first identified in apples. In this study, the 3 compounds' induction curves after damage, varietal differences in induction amounts, and physiological roles against pest insects were further investigated. Eriobotoric acid showed clear antifeedant activity against lepidopteran insect Spodoptera litura but not against apple pests.

A method for increasing the solubility of industrial azo pigments for 13 C and 1 H NMR spectra and the assignments of their 13 C and 1 H NMR peaks.

A method for increasing the solubilities of industrial azo pigments in DMSO by adding DBU (1,8-diaza-7-bicyclo[5.4.0]undecene) has been developed. This facilitated the acquisition of solution 13 C NMR spectra of the pigments. This method was applied to four types of azo pigments: naphthol AS (3-hydroxy-2-naphthoic acid anilide) pigments, naphthol pigments, pyrazolone pigments and acetoacetanilide pigments. This represents the first solution 13 C NMR spectra for naphthol AS pigments. Altogether 18 industrial azo pigments were analysed using 1D and 2D NMR techniques. The proton and corresponding carbon NMR resonances of these pigments have all been assigned.

Trichilones A-E: New Limonoids from Trichilia adolfi.

In addition to the trichilianones A-D recently reported from Trichilia adolfi, a continuing investigation of the chemical constituents of the ethanol extract of the bark of this medicinal plant yielded the five new limonoids 1-5. They are characterized by having four fused rings and are new examples of prieurianin-type limonoids, having a ε-lactone which in 4 and 5 is α, ß- unsaturated. The structures of the isolated metabolites were determined by high field NMR spectroscopy and HR mass spectrometry. The new metabolites were shown to have the ε-lactone fused with a tetrahydrofuran ring which is connected to an oxidized hexane ring joined with a cyclo-pentanone having a 3-furanyl substituent. As the crude extract possesses antileishmanial activity, the compounds were assayed for cytotoxic and antiparasitic activities in vitro in murine macrophage cells (raw 264.7 cells) and in Leishmania amazoniensis as well as L. braziliensis promastigotes. Metabolites 1-3 and 5 showed moderate cytotoxicity (between 30-94 µg/mL) but are not responsible for the antileishmanial effect of the extract.

Lactate topography of the human brain using hyperpolarized 13C-MRI.

Lactate is now recognized as an important intermediate in brain metabolism, but its role is still under investigation. In this work we mapped the distribution of lactate and bicarbonate produced from intravenously injected 13C-pyruvate over the whole brain using a new imaging method, hyperpolarized 13C MRI (N = 14, ages 23 to 77). Segmenting the 13C-lactate images into brain atlas regions revealed a pattern of lactate that was preserved across individuals. Higher lactate signal was observed in cortical grey matter compared to white matter and was highest in the precuneus, cuneus and lingual gyrus. Bicarbonate signal, indicating flux of [1-13C]pyruvate into the TCA cycle, also displayed consistent spatial distribution. One-way ANOVA to test for significant differences in lactate among atlas regions gave F = 87.6 and p < 10-6. This report of a "lactate topography" in the human brain and its consistent pattern is evidence of region-specific lactate biology that is preserved across individuals.

Deleterious effects of carbon-carbon dipolar coupling on RNA NMR dynamics.

Many regulatory RNAs undergo dynamic exchanges that are crucial for their biological functions and NMR spectroscopy is a versatile tool for monitoring dynamic motions of biomolecules. Meaningful information on biomolecular dynamics requires an accurate measurement of relaxation parameters such as longitudinal (R1) rates, transverse (R2) rates and heteronuclear Overhauser effect (hNOE). However, earlier studies have shown that the large 13C-13C interactions complicate analysis of the carbon relaxation parameters. To investigate the effect of 13C-13C interactions on RNA dynamic studies, we performed relaxation measurements on various RNA samples with different labeling patterns and compared these measurements with the computational simulations. For uniformly labeled samples, contributions of the neighboring carbon to R1 measurements were observed. These contributions increased with increasing magnetic field and overall correlation time ([Formula: see text]) for R1 rates, necessitating more careful analysis for uniformly labeled large RNAs. In addition, the hNOE measurements were also affected by the adjacent carbon nuclei. Unlike R1 rates, R1ρ rates showed relatively good agreement between uniformly- and site-selectively labeled samples, suggesting no dramatic effect from their attached carbon, in agreement with previous observations. Overall, having more accurate rate measurements avoids complex analysis and will be a key for interpreting 13C relaxation rates for molecular motion that can provide valuable insights into cellular molecular recognition events.

The Effect of Gadolinium Doping in [13 C6 ,2 H7 ]Glucose Formulations on 13 C Dynamic Nuclear Polarization at 3.35†T.

The promise of hyperpolarized glucose as a non-radioactive imaging agent capable of reporting on multiple metabolic routes has led to recent advances in its dissolution-DNP (dDNP) driven polarization using UV-light induced radicals and trityl radicals at high field (6.7 T) and 1.1 K. However, most preclinical dDNP polarizers operate at the field of 3.35 T and 1.4-1.5 K. Minute amounts of Gd3+ complexes have shown large improvements in solid-state polarization, which can be translated to improved hyperpolarization in solution. However, this Gd3+ effect seems to depend on magnetic field strength, metal ion concentration, and sample formulation. The effect of varying Gd3+ concentrations at 3.35 T has been described for 13 C-labeled pyruvic acid and acetate. However, it has not been studied for other compounds at this field. The results presented here suggest that Gd3+ doping can lead to various concentration and temperature dependent effects on the polarization of [13 C6 ,2 H7 ]glucose, not necessarily similar to the effects observed in pyruvic acid or acetate in size or direction. The maximal polarization for [13 C6 ,2 H7 ]glucose appears to be at a Gd3+ concentration of 2 mM, when irradiating for more than 2 h at the negative maximum of the DNP intensity profile. Surprisingly, for shorter irradiation times, higher polarization levels were determined at 1.50 K compared to 1.45 K, at a [Gd3+ ]=1.3 mM. This was explained by the build-up time constant and maximum at these temperatures.

Noninvasive Immunometabolic Cardiac Inflammation Imaging Using Hyperpolarized Magnetic Resonance.

RATIONALE: Current cardiovascular clinical imaging techniques offer only limited assessment of innate immune cell-driven inflammation, which is a potential therapeutic target in myocardial infarction (MI) and other diseases. Hyperpolarized magnetic resonance (MR) is an emerging imaging technology that generates contrast agents with 10- to 20 000-fold improvements in MR signal, enabling cardiac metabolite mapping. OBJECTIVE: To determine whether hyperpolarized MR using [1-13C]pyruvate can assess the local cardiac inflammatory response after MI. METHODS AND RESULTS: We performed hyperpolarized [1-13C]pyruvate MR studies in small and large animal models of MI and in macrophage-like cell lines and measured the resulting [1-13C]lactate signals. MI caused intense [1-13C]lactate signal in healing myocardial segments at both day 3 and 7 after rodent MI, which was normalized at both time points after monocyte/macrophage depletion. A near-identical [1-13C]lactate signature was also seen at day 7 after experimental MI in pigs. Hyperpolarized [1-13C]pyruvate MR spectroscopy in macrophage-like cell suspensions demonstrated that macrophage activation and polarization with lipopolysaccharide almost doubled hyperpolarized lactate label flux rates in vitro; blockade of glycolysis with 2-deoxyglucose in activated cells normalized lactate label flux rates and markedly inhibited the production of key proinflammatory cytokines. Systemic administration of 2-deoxyglucose after rodent MI normalized the hyperpolarized [1-13C]lactate signal in healing myocardial segments at day 3 and also caused dose-dependent improvement in IL (interleukin)-1ß expression in infarct tissue without impairing the production of key reparative cytokines. Cine MRI demonstrated improvements in systolic function in 2-DG (2-deoxyglucose)-treated rats at 3 months. CONCLUSIONS: Hyperpolarized MR using [1-13C]pyruvate provides a novel method for the assessment of innate immune cell-driven inflammation in the heart after MI, with broad potential applicability across other cardiovascular disease states and suitability for early clinical translation.

Computationally Assisted Structural Revision of Flavoalkaloids with a Seven-Membered Ring: Aquiledine, Isoaquiledine, and Cheliensisine.

Aquiledine and cheliensisine are flavoalkaloids isolated from Aquilegia ecalcarata and Goniothalamus cheliensis, respectively. Different structures have been proposed for these flavoalkaloids; however, their 1H and 13C NMR spectroscopic data were virtually identical. In this study, the structures of aquiledine and cheliensisine were revised on the basis of the DFT calculation of NMR data including DP4+ and J-DP4 analysis, as well as specific rotations. Similarly, the structure of isoaquiledine, a regioisomer of aquiledine, was also revised. A biosynthetic pathway of these flavoalkaloids is proposed.

Denigrins and Dactylpyrroles, Arylpyrrole Alkaloids from a Dactylia sp. Marine Sponge.

Seven new arylpyrrole alkaloids (1-7), along with four known compounds, were isolated from an extract of a Dactylia sp. nov. marine sponge, and their structures were elucidated by interpretation of NMR and MS spectroscopic data. Denigrins D-G (1-4) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A-C (5-7) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from 1H-15N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (1). Dictyodendrin F (8), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC50 value of 13 µM.

Reisolation and NMR characterization of the satratoxins G and H.

The exquisitely cytotoxic macrolides, satratoxins G and H, have been reisolated from a solvent extract of a rice culture inoculated with Stachybotrys chartarum to be used as high-purity reference compounds for analytical analyses. Extensive chromatographic separation realized the compounds that were fully recharacterized in two solvents by 1D- and 2D-NMR spectroscopy, revealing some discrepancies in the nuclear magnetic resonance (NMR) data as compared with the previously reported values found in the literature. Detailed spectra are provided in order to aid future identification and dereplication.