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.

Simultaneous fMRI and metabolic MRS of hyperpolarized [1-13C]pyruvate during nicotine stimulus in rat.

Functional MRI (fMRI) and MRS (fMRS) can be used to noninvasively map cerebral activation and metabolism. Recently, hyperpolarized 13C spectroscopy and metabolic imaging have provided an alternative approach to assess metabolism. In this study, we combined 1H fMRI and hyperpolarized [1-13C]pyruvate MRS to compare cerebral blood oxygenation level-dependent (BOLD) response and real-time cerebral metabolism, as assessed with lactate and bicarbonate labelling, during nicotine stimulation. Simultaneous 1H fMRI (multislice gradient echo echo-planar imaging) and 13C spectroscopic (single slice pulse-acquire) data were collected in urethane-anaesthetized female Sprague-Dawley rats (n = 12) at 9.4 T. Animals received an intravenous (i.v.) injection of either nicotine (stimulus; 88 µg/kg, n = 7, or 300 µg/kg, n = 5) or 0.9% saline (matching volume), followed by hyperpolarized [1-13C]pyruvate injection 60 s later. Three hours later, a second injection was administered: the animals that had previously received saline were injected with nicotine and vice versa, both followed by another hyperpolarized [1-13C]pyruvate i.v. injection 60 s later. The low-dose (88 µg/kg) nicotine injection led to a 12% ± 4% (n = 7, t-test, p ~ 0.0006 (t-value -5.8, degrees of freedom 6), Wilcoxon p ~ 0.0078 (test statistic 0)) increase in BOLD signal. At the same time, an increase in 13C-bicarbonate signal was seen in four out of six animals. Bicarbonate-to-total carbon ratios were 0.010 ± 0.004 and 0.018 ± 0.010 (n = 6, t-test, p ~ 0.03 (t-value -2.3, degrees of freedom 5), Wilcoxon p ~ 0.08 (test statistic 3)) for saline and nicotine experiments, respectively. No increase in the lactate signal was seen; lactate-to-total carbon was 0.16 ± 0.02 after both injections. The high (300 µg/kg) nicotine dose (n = 5) caused highly variable BOLD and metabolic responses, possibly due to the apparent respiratory distress. Simultaneous detection of 1H fMRI and hyperpolarized 13C-MRS is feasible. A comparison of metabolic response between control and stimulated states showed differences in bicarbonate signal, implying that the hyperpolarization technique could offer complimentary information on brain activation.

Influence of DNP Polarizing Agents on Biochemical Processes: TEMPOL in Transient Ischemic Stroke.

Hyperpolarization of 13C by dissolution dynamic nuclear polarization (dDNP) boosts the sensitivity of magnetic resonance spectroscopy (MRS), making possible the monitoring in vivo and in real time of the biochemical reactions of exogenously infused 13C-labeled metabolic tracers. The preparation of a hyperpolarized substrate requires the use of free radicals as polarizing agents. Although added at very low doses, these radicals are not biologically inert. Here, we demonstrate that the presence of the nitroxyl radical TEMPOL influences significantly the cerebral metabolic readouts of a hyperpolarized [1-13C] lactate bolus injection in a mouse model of ischemic stroke with reperfusion. Thus, the choice of the polarizing agent in the design of dDNP hyperpolarized MRS experiments is of great importance and should be taken into account to prevent or to consider significant effects that could act as confounding factors.

Conversion of Similar Xenochemicals to Dissimilar Products: Exploiting Competing Reactions in Whole-Cell Catalysis.

Many enzymes have latent activities that can be used in the conversion of non-natural reactants for novel organic conversions. A classic example is the conversion of benzaldehyde to a phenylacetyl carbinol, a precursor for ephedrine manufacture. It is often tacitly assumed that purified enzymes are more promising catalysts than whole cells, despite the lower cost and easier maintenance of the latter. Competing substrates inside the cell have been known to elicit currently hard-to-predict selectivities that are not easily measured inside the living cell. We employ NMR spectroscopic assays to rationally combine isomers for selective reactions in commercial S. cerevisiae. This approach uses internal competition between alternative pathways of aldehyde clearance in yeast, leading to altered selectivities compared to catalysis with the purified enzyme. In this manner, 4-fluorobenzyl alcohol and 2-fluorophenylacetyl carbinol can be formed with selectivities in the order of 90%. Modification of the cellular redox state can be used to tune product composition further. Hyperpolarized NMR shows that the cellular reaction and pathway usage are affected by the xenochemical. Overall, we find that the rational construction of ternary or more complex substrate mixtures can be used for in-cell NMR spectroscopy to optimize the upgrading of similar xenochemicals to dissimilar products with cheap whole-cell catalysts.

Short-chain fatty acids reprogram metabolic profiles with the induction of reactive oxygen species production in human colorectal adenocarcinoma cells.

Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.

How to improve the efficiency of a traditional dissolution dynamic nuclear polarization (dDNP) apparatus: Design and performance of a fluid path compatible dDNP/LOD-ESR probe.

Dissolution Dynamic Nuclear Polarization (dDNP) was invented almost twenty years ago. Ever since, hardware advancement has observed 2 trends: the quest for DNP at higher field and, more recently, the development of cryogen free polarizers. Despite the DNP community is slowly migrating towards "dry" systems, many "wet" polarizers are still in use. Traditional DNP polarizers can use up to 100 L of liquid helium per week, but are less sensitive to air contamination and have higher cooling power. These two characteristics make them very versatile when it comes to new methods development. In this study we retrofitted a 5 T/1.15 K "wet" DNP polarizer with the aim of improving cryogenic and DNP performance. We designed, built, and tested a new DNP insert that is compatible with the fluid path (FP) technology and a LOgitudinal Detected Electron Spin Resonance (LOD-ESR) probe to investigate radical properties at real DNP conditions. The new hardware increased the maximum achievable polarization and the polarization rate constant of a [1-13C]pyruvic acid-trityl sample by a factor 1.5. Moreover, the increased liquid He holding time together with the possibility to constantly keep the sample space at low pressure upon sample loading and dissolution allowed us to save about 20 L of liquid He per week.

Practical dissolution dynamic nuclear polarization.

This review article intends to provide insightful advice for dissolution-dynamic nuclear polarization in the form of a practical handbook. The goal is to aid research groups to effectively perform such experiments in their own laboratories. Previous review articles on this subject have covered a large number of useful topics including instrumentation, experimentation, theory, etc. The topics to be addressed here will include tips for sample preparation and for checking sample health; a checklist to correctly diagnose system faults and perform general maintenance; the necessary mechanical requirements regarding sample dissolution; and aids for accurate, fast and reliable polarization quantification. Herein, the challenges and limitations of each stage of a typical dissolution-dynamic nuclear polarization experiment are presented, with the focus being on how to quickly and simply overcome some of the limitations often encountered in the laboratory.

Protonation tuned dipolar order mediated 1H→13C cross-polarization for dissolution-dynamic nuclear polarization experiments.

A strategy of dipolar order mediated nuclear spin polarization transfer has recently been combined with dissolution-dynamic nuclear polarization (dDNP) and improved by employing optimized shaped radiofrequency pulses and suitable molecular modifications. In the context of dDNP experiments, this offers a promising means of transferring polarization from high-gamma 1H spins to insensitive 13C spins with lower peak power and lower energy compared with state-of-the-art cross-polarization schemes. The role of local molecular groups and the glassing matrix protonation level are both postulated to play a key role in the polarization transfer pathway via an intermediary reservoir of dipolar spin order. To gain appreciation of the mechanisms involved in the dipolar order mediated polarization transfer under dDNP conditions, we investigate herein the influence of the pivotal characteristics of the sample makeup: (i) revising the protonation level for the constituents of the DNP glass; and (ii) utilizing deuterated molecular derivatives. Experimental demonstrations are presented for the case of [1-13C]sodium acetate. We find that the proton sample molarity has a large impact on both the optimal parameters and the performance of the dipolar order mediated cross-polarization sequence, with the 13C signal build-up time drastically shortened in the case of high solvent protonation levels. In the case of a deuterated molecular derivative, we observe that the nearby 2H substituted methyl group is deleterious to the 1H→13C transfer phenomenon (particularly at low levels of sample protonation). Overall, increased solvent protonation makes the dipolar order governed polarization transfer significantly faster and more efficient. This study sheds light on the influential sample formulation traits which govern the dipolar order-controlled transfer of polarization and indicates that the polarization transfer efficiencies of deuterated molecules can be boosted and reach high performances simply by adequate solvent protonation.

Application of low-rank approximation using truncated singular value decomposition for noise reduction in hyperpolarized 13 C NMR spectroscopy.

Dissolution dynamic nuclear polarization allows in vivo studies of metabolic flux using 13 C-hyperpolarized tracers by enhancing signal intensity by up to four orders of magnitude. The T1 for in vivo applications is typically in the range of 10-50 s for the different 13 C-enriched metabolic substrates; the exponential loss of polarization due to various relaxation mechanisms leads to a strong reduction of the signal-to-noise ratio (SNR). A common solution to the problem of low SNR is the accumulation/averaging of consecutive spectra. However, some limitations related to long delays between consecutive scans occur: in particular, following biochemical kinetics and estimate apparent enzymatic constants becomes time critical when measurement scans are repeated with the typical delay of about 3 T1 . Here we propose a method to dramatically reduce the noise, and therefore also the acquisition times, by computing, via truncated singular value decomposition, a low-rank approximation to the individual complex time-domain signals. Moreover, this approach has the additional advantage that the phase correction can be applied to the spectra already denoised, thus greatly reducing phase correction errors. We have tested the method on (1) simulated data; (2) performing dissolution of hyperpolarized 1-13 C-pyruvate in standard conditions and (3) in vivo data sets, using a porcine model injected with hyperpolarized Na-1-13 C-acetate. It was shown that the presented method reduces the noise level in all the experimental data sets, allowing the retrieval of signals from highly noisy data without any prior phase correction pre-processing. The effects of the proposed approach on the quantification of metabolic kinetics parameters have to be shown by full quantification studies.

Hyperpolarization via dissolution dynamic nuclear polarization: new technological and methodological advances.

Dissolution-DNP is a method to boost liquid-state NMR sensitivity by several orders of magnitude. The technique consists in hyperpolarizing samples by solid-state dynamic nuclear polarization at low temperature and moderate magnetic field, followed by an instantaneous melting and dilution of the sample happening inside the polarizer. Although the technique is well established and the outstanding signal enhancement paved the way towards many applications precluded to conventional NMR, the race to develop new methods allowing higher throughput, faster and higher polarization, and longer exploitation of the signal is still vivid. In this work, we review the most recent advances on dissolution-DNP methods trying to overcome the original technique's shortcomings. The review describes some of the new approaches in the field, first, in terms of sample formulation and properties, and second, in terms of instrumentation.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

Degradation of refractory organic compounds from dinitrodiazophenol containing industrial wastewater through UV/H2O2 and UV/PS processes.

In this study, refractory organic compounds from dinitrodiazophenol (DDNP) containing industrial wastewater were degraded through two ultraviolet (UV)-based advanced oxidation processes: UV/hydrogen peroxide (UV/H2O2) and UV/potassium persulfate (UV/PS) processes. In both processes, the synergistic effects, operational parameters (i.e., oxidant dosage and initial pH value), and pseudo first-order constant k were systematically studied. Moreover, the reactive oxygen species formed in the UV/H2O2 and UV/PS processes were identified, and the degradation of refractory organic compounds was characterized through UV-visible spectra analysis. The improvement in biodegradability of DDNP industrial wastewater after treatment by different processes was compared. Both the UV/H2O2 (synergistic coefficient F = 61.34) and UV/PS (synergistic coefficient F = 54.85) processes showed significant, highly synergistic effects. The increase in oxidant dosage was beneficial in organic compound removal in both the UV/H2O2 and UV/PS processes, but excessive H2O2 showed a stronger inhibition of the increase in organic compound removal than that in the UV/PS process. In addition, an acidic environment was more conducive to organic compound degradation in the UV/H2O2 process, whereas the initial pH value had less of an influence on the UV/PS process. Under optimal conditions for the UV/H2O2 and UV/PS processes, the CN and COD removal efficiencies were 99.71%, 66.35%, 99.69%, and 70.81%, respectively, and the k values for COD removal were 0.0804 and 0.0824 min-1. Tests to identify reactive oxygen species showed that the hydroxyl radical was the predominant oxidizing species in the UV/H2O2 process, whereas the hydroxyl and sulfate radicals were both identified in the UV/PS process, and the sulfate radical contributed the most to the degradation of organic compounds. In addition, spectrum analysis revealed that the complex structure (e.g., benzene ring, nitro group, and diazo group) of refractory organic compounds from DDNP industrial wastewater was effectively destroyed by the UV/H2O2 and UV/PS processes, and both processes improved the biodegradability (biochemical oxygen demand for 5 days/chemical oxygen demand (BOD5/COD)) of DDNP industrial wastewater from 0.052 to 0.665 and 0.717, respectively. Overall, both the UV/H2O2 and UV/PS processes effectively degraded the refractory organic compounds from DDNP industrial wastewater, and the UV/PS process exhibited a higher organic compound removal efficiency and better applicability.

A Low-Temperature Broadband NMR Probe for Multinuclear Cross-Polarization.

Dissolution dynamic nuclear polarization (D-DNP) probes are usually designed for one or at most two specific nuclei. Investigation of multiple nuclei usually requires manufacturing a number of costly probes. In addition, changing the probe is a time-consuming process since a system that works at low temperature (usually between 1.2 and 4.2 K) must be warmed up, thus increasing the risks of contamination. Here, an efficient apparatus is described for D-DNP designed not only for microwave-enhanced direct observation of a wide range of nuclei S such as 1 H, 13 C, 2 H, 23 Na, and 17 O, but also for cross-polarization (CP) from I=1 H to such S nuclei. Unlike most conventional designs, the tuning and matching circuits are partly immersed in superfluid helium at temperatures down to 1.2 K. Intense radio-frequency (RF) fields with amplitudes on the order of 50 kHz or better can be applied simultaneously to both nuclei I and S using RF amplifiers with powers on the order of 90 and 80 W, respectively, without significant losses of liquid helium. The system can operate at temperatures over a wide range between 1.2 and 300 K.

Dinitrodiazophenol industrial wastewater treatment by a sequential ozone Fenton process.

The ozonation process is efficient in degrading aromatic substances and substances with unsaturated bonds, but cannot effectively destroy small-molecule organic compounds, which accumulate. Likewise, the Fenton process is a classic wastewater treatment method, but requires strict pH control and produces secondary pollution when the concentration of organic substances is high. In this study, we applied a 1stO3-2ndFenton sequential process to treat diazodinitrophenol (DDNP) industrial wastewater and provide suitable reaction conditions for Fenton process. For the 1stOzone process, organics removal increased as O3 dosage increased. At optimized operation, the 1stO3 process provided an acidic effluent (pH = 3) and reduced the organics concentration to a level suitable for the 2ndFenton process. Benzene ring substances as well as nitro group and diazo group compounds were greatly degraded in the 1stO3 process and were further mineralized in the 2ndFenton process. Additionally, the biodegradability of DDNP industrial wastewater was greatly improved. This is the first reported time that ozonation and the Fenton process have been integrated sequentially to treat an explosive production wastewater. The study provides a feasible chemical oxidation method for treating DDNP industrial wastewater by simply combining two classic treatment processes.

Targeted Metabolomics with Quantitative Dissolution Dynamic Nuclear Polarization.

Metabolite profiles and their isotopomer distributions can be studied noninvasively in complex mixtures with NMR. The advent of hyperpolarized 13C-NMR using quantitative dissolution Dynamic Nuclear Polarization (qdDNP) and isotope enrichment add sensitivity to such metabolic studies, enabling mapping and quantification of metabolic pathways and networks. Here we describe a sample preparation method, including cell incubation, extraction, and signal enhancement, for reproducible and quantitative analysis of hyperpolarized 13C-NMR metabolite spectra. We further illustrate how qdDNP can be applied to gain metabolic insights into living cells.

Application and methodology of dissolution dynamic nuclear polarization in physical, chemical and biological contexts.

Dissolution dynamic nuclear polarization (d-DNP) is a versatile method to enhance nuclear magnetic resonance (NMR) spectroscopy. It boosts signal intensities by four to five orders of magnitude thereby providing the potential to improve and enable a plethora of applications ranging from the real-time monitoring of chemical or biological processes to metabolomics and in-cell investigations. This perspectives article highlights possible avenues for developments and applications of d-DNP in biochemical and physicochemical studies. It outlines how chemists, biologists and physicists with various fields of interest can transform and employ d-DNP as a powerful characterization method for their research.

Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP-NMR: the impact of overexpressed membrane transporters.

Pyruvate membrane crossing and its lactate dehydrogenase-mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization-enhanced NMR. Hyperpolarized 13 C-1-labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate-to-lactate conversion rate monitored by dissolution dynamic nuclear polarization-NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed. The enzymatic activity of lactate dehydrogenase was also assessed in buffer solutions, following the real-time pyruvate-to-lactate conversion speeds at different enzyme concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization.

Hyperpolarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (D-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices). This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpolarization in spectroscopy and MRI. We show that D-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2,2,6,6-tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels. The powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner. This approach allows high polarization without the need for glass-forming agents and is applicable to a broad range of substrates, including peptides and metabolites. During dissolution, HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds. The resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion.

Synthesis of superparamagnetic iron oxide nanoparticles coated with a DDNP-carboxyl derivative for in vitro magnetic resonance imaging of Alzheimer's disease.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been proposed for use in magnetic resonance imaging as versatile ultra-sensitive nanoprobes for Alzheimer's disease imaging. In this work, we synthetized an efficient contrast agent of Alzheimer's disease using 1,1-dicyano-2-[6-(dimethylamino)naphthalene-2-yl]propene (DDNP) carboxyl derivative to functionalize the surface of SPIONs. The DDNP-SPIONs are prepared by conjugating DDNP carboxyl derivative to oleic acid-treated SPIONs through ligand exchange. The structure, size distribution and magnetic property were identified by IR, TGA-DTA, XRD, TEM, Zetasizer Nano and VSM. TEM and Zetasizer Nano observations indicated that the DDNP-SPIONs are relatively mono-dispersed spherical distribution with an average size of 11.7nm. The DDNP-SPIONs were then further analyzed for their MRI relaxation properties using MR imaging and demonstrated high T2 relaxivity of 140.57s(-1)FemM(-1), and the vitro experiment that DDNP-SPIONs binding to ß-Amyloid aggregates were then investigated by fluorophotometry, the results showed that the combination had induced the fluorescence enhancement of the DDNP-SPIONs and displayed tremendous promise for use as a contrast agent of Alzheimer's disease in MRI.