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Water exchange is increasingly recognized as an important biological process that can affect the study of biological tissue using diffusion MR. Methods to measure exchange, however, remain immature as opposed to those used to characterize restriction, with no consensus on the optimal pulse sequence (s) or signal model (s). In general, the trend has been towards data-intensive fitting of highly parameterized models. We take the opposite approach and show that a judicious sub-sample of diffusion exchange spectroscopy (DEXSY) data can be used to robustly quantify exchange, as well as restriction, in a data-efficient manner. This sampling produces a ratio of two points per mixing time: (i) one point with equal diffusion weighting in both encoding periods, which gives maximal exchange contrast, and (ii) one point with the same total diffusion weighting in just the first encoding period, for normalization. We call this quotient the Diffusion EXchange Ratio (DEXR). Furthermore, we show that it can be used to probe time-dependent diffusion by estimating the velocity autocorrelation function (VACF) over intermediate to long times (∼2-500ms). We provide a comprehensive theoretical framework for the design of DEXR experiments in the case of static or constant gradients. Data from Monte Carlo simulations and experiments acquired in fixed and viable ex vivo neonatal mouse spinal cord using a permanent magnet system are presented to test and validate this approach. In viable spinal cord, we report the following apparent parameters from just 6 data points: τk=17±4ms, fNG=0.72±0.01, Reff=1.05±0.01µm, and κeff=0.19±0.04µm/ms, which correspond to the exchange time, restricted or non-Gaussian signal fraction, an effective spherical radius, and permeability, respectively. For the VACF, we report a long-time, power-law scaling with ≈t-2.4, which is approximately consistent with disordered domains in 3-D. Overall, the DEXR method is shown to be highly efficient, capable of providing valuable quantitative diffusion metrics using minimal MR data.

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Signals undergoing chemical or conformational exchange in one-dimensional NMR spectra are often identified by deuterium exchange. In order to obtain quantitative information about the dynamic processes involved, one frequently used method is EXchange SpectroscopY (EXSY). To detect all exchange processes, the EXSY experiment requires the acquisition of time-consuming two-dimensional spectra. Here we report a faster alternative, an experiment which uses spatial encoding to extract similar information in a 1D exchange-edited experiment. Thereby, all protons are observed at once, but in different slices of the detection volume. The experiment can be carried out in a single scan to identify exchanging sites in a 1D spectrum by changes in signal intensity indicating exchange processes. If the exchanging partner, for example water is in molar excess the exchange-editing method easily identifies mobile protons by negative signals in the 1D 1H NMR spectrum.

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Solute translocation by membrane transport proteins is a vital biological process that can be tracked, on the sub-second timescale, using nuclear magnetic resonance (NMR). Fluorinated substrate analogues facilitate such studies because of high sensitivity of 19 F NMR and absence of background signals. Accurate extraction of translocation rate constants requires precise quantification of NMR signal intensities. This becomes complicated in the presence of J-couplings, cross-correlations, and nuclear Overhauser effects (NOE) that alter signal integrals through mechanisms unrelated to translocation. Geminal difluorinated motifs introduce strong and hard-to-quantify contributions from non-exchange effects, the nuanced nature of which makes them hard to integrate into data analysis methodologies. With analytical expressions not being available, numerical least squares fitting of theoretical models to 2D spectra emerges as the preferred quantification approach. For large spin systems with simultaneous coherent evolution, cross-relaxation, cross-correlation, conformational exchange, and membrane translocation between compartments with different viscosities, the only available simulation framework is Spinach. In this study, we demonstrate GLUT-1 dependent membrane transport of two model sugars featuring CF2 and CF2 CF2 fluorination motifs, with precise determination of translocation rate constants enabled by numerical fitting of 2D EXSY spectra. For spin systems and kinetic networks of this complexity, this was not previously tractable.

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For its size, the brain is the most metabolically active organ in the body. Most of its energy demand is used to maintain stable homeostatic physiological conditions. Altered homeostasis and active states are hallmarks of many diseases and disorders. Yet there is currently no direct and reliable method to assess homeostasis and absolute basal activity of cells in the tissue noninvasively without exogenous tracers or contrast agents. We propose a novel low-field, high-gradient diffusion exchange nuclear magnetic resonance (NMR) method capable of directly measuring cellular metabolic activity via the rate constant for water exchange across cell membranes. Exchange rates are 140 ± 16 s - 1 under normal conditions in viable ex vivo neonatal mouse spinal cords. High repeatability across samples suggest that values are absolute and intrinsic to the tissue. Using temperature and drug (ouabain) perturbations, we find that the majority of water exchange is metabolically active and coupled to active transport by the sodium-potassium pump. We show that this water exchange rate is sensitive primarily to tissue homeostasis and provides distinct functional information. In contrast, the apparent diffusion coefficient (ADC) measured with submillisecond diffusion times is sensitive primarily to tissue microstructure but not activity. Water exchange appears independently regulated from microstructural and oxygenation changes reported by ADC and T 1 relaxation measurements in an oxygen-glucose deprivation model of stroke; exchange rates remain stable for 30-40 min before dropping to levels similar to the effect of ouabain and never completely recovering when oxygen and glucose are restored.

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Despite their high conductivity, factors such as being fragile enough to face processing issues and interfacial incompatibility with lithium electrodes are some of the main drawbacks hindering the commercialization of inorganic (mainly oxide-based) solid electrolytes for use in all-solid-state lithium batteries. To this end, strategies such as the addition of solid polymer electrolytes have been proposed to improve the electrode-electrolyte interface. Hybrid electrolytes, which are usually composed of ceramic particles dispersed in a polymer, generally have a better affinity with electrodes and higher ionic conductivity than pure inorganic electrolytes. However, a significant downside of this strategy is that differences in lithium transportability between electrolyte layers can result in the formation of a high interfacial energy barrier across the cell. One strategy to ensure sufficient "wetting" of ceramics is to incorporate a liquid electrolyte directly into the solid inorganic electrolyte resulting in the formation of a hybrid liquid-ceramic electrolyte. To this end, liquid-ceramic hybrid electrolytes were prepared by adding LiG4TFSI, a solvate ionic liquid (SIL), to garnet, NASICON, and perovskite-type ceramic electrolytes. Although SIL addition resulted in increased ionic conductivity, comparisons between the pure SIL and the hybrid systems revealed that improvements were due to the SIL alone. A thorough investigation of the hybrid systems by solid-state nuclear magnetic resonance (NMR) revealed little to no lithium exchange between the ceramic and the SIL. This confirms that lithium conductivity preferentially occurs through the SIL in these hybrid systems. The primary role of the ceramic is to provide mechanical strength.

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In an asymptomatic 19-year-old who regularly underwent cardiopulmonary fitness testing for national lifeguard-accreditation, 129Xe MRI unexpectedly revealed an abnormally augmented RBC signal and RBC-to-alveolar-capillary-tissue ratio with spatially homogeneous ventilation, tissue barrier, and RBC images. Pulmonary function was normal, but cardiopulmonary follow-up including transthoracic and transesophageal echocardiogram, heart catheterization, and contrast-enhanced cardiac CT imaging led to the diagnosis of a large (20 × 27 mm) secundum atrial septal defect (ASD) with a net right-to-left shunt (Qp:Qs = 0.5) and normal pulmonary pressures. This novel, unexpected case revealed that 129Xe RBC signal intensity likely reflected erythrocytosis, compensatory to the abnormal cardiovascular hemodynamics that resulted from a large congenital ASD. Unlike ASD cases that present with dyspnea and exercise limitation, this 129Xe MRI abnormality was detected in an asymptomatic teenager. This is the first report of asymptomatic adult congenital heart disease diagnosed subsequent to novel 129Xe MRI that led to early intervention, avoiding long-term complications of cyanosis, including ventricular fibrosis and thromboembolic and bleeding risks.

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PURPOSE: Water Exchange Spectroscopy (WEX) is a direct measurement of the exchange rate ksw of labile protons from a solute to water in which the exchange time is varied. However the useful information can be masked by the T1-decay of the solvent pool. We propose Saturation-WEX and Phase Sensitive WEX (PS-WEX) as an extension upon the WEX approach to reduce T1-masking. Additionally PS-WEX takes advantage of the phase information contained in the WEX signal to improve the dynamic range. METHODS: By introducing an additional RF-pulse and fixing the exchange time delay the T1-dependence of the signal is reduced. By exploiting the phase sensitivity of the WEX pathway the dynamic range can be increased. This approach is validated using simulations as well as phantom measurements. RESULTS: The improved dynamic range is demonstrated in measurements. The fixed exchange time reduces the influence of the T1-decay on the signal curve leading to improved fit quality. CONCLUSION: Sat-WEX and PS-WEX are an extension to the well established WEX approach with a less complex fit equation and in the case of PS-WEX improved dynamic range, allowing more accurate quantification.

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ß-N-methyl-amino-L-alanine (BMAA) in the presence of bicarbonate (HCO3-) undergoes structural modifications generating two carbamate species, α-carbamate and ß-carbamate forms of BMAA. The chemical structure of BMAA and BMAA-carbamate adducts strongly suggest they may interact with divalent metal ions. The ability of BMAA to cross the blood-brain barrier and possibly interact with divalent metal ions may augment the neurotoxicity of these molecules. To understand the effects of divalent metal ions (Mg2+, Zn2+, and Cu2+) on the overall dynamic equilibrium between BMAA and its carbamate adducts, a systematic study using nuclear magnetic resonance (NMR) is presented. The chemical equilibria between BMAA, its carbamate adducts, and each of the divalent ions were studied using two-dimensional chemical exchange spectroscopy (EXSY). The NMR results demonstrate that BMAA preferentially interacts with Zn2+ and Cu2+, causing an overall reduction in the production of carbamate species by altering the dynamic equilibria. The NMR-based spectral changes due to the BMAA interaction with Cu2+ is more drastic than with the Zn2+, under the same stoichiometric ratios of BMAA and the individual divalent ions. However, the presence of Mg2+ does not significantly alter the dynamic equilibria between BMAA and its carbamate adducts. The NMR-based results are further validated using circular dichroism (CD) spectroscopy, observing the n ➔ π interaction in the complex formation of BMAA and the divalent metal ions, with additional verification of the interaction with Cu2+ using UV-Vis spectroscopy. Our results demonstrate that BMAA differentially interacts with divalent metal ions (Mg2+ < Zn2+ < Cu2+), and thus alters the rate of formation of carbamate products. The equilibria between BMAA, the bicarbonate ions, and the divalent metal ions may alter the total population of a specific form of BMAA-ion complex at physiological conditions and, therefore, add a level of complexity of the mechanisms by which BMAA acts as a neurotoxin.

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Membrane interactions of proteins play a role in essential cellular processes in both physiological and disease states. The structural flexibility of intrinsically disordered proteins (IDPs) allows for interactions with multiple partners, including membranes. However, determining conformational states of IDPs when interacting with membranes can be challenging. Here we describe the use of nuclear magnetic resonance (NMR), including dark-state exchange saturation transfer (DEST), to probe IDP-membrane interactions in order to determine whether there is an interaction, which residues participate, and the extent/nature of the interaction between the protein and the membrane. Using α-synuclein and tau as typical examples, we provide protocols for how the membrane interactions of IDPs can be probed, including details of how the samples should be prepared and guidelines on how to interpret the results.

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Diffusion exchange spectroscopy (DEXSY) provides a means to isolate the signal attenuation associated with exchange from other sources of signal loss. With the total diffusion weighting b1+b2=bs held constant, DEXSY signals acquired with b1=0 or b2=0 have no exchange weighting, while a DEXSY signal acquired with b1=b2 has maximal exchange weighting. The exchange rate can be estimated by fitting a diffusion exchange model to signals acquired with variable mixing times. Conventionally, acquired signals are normalized by a signal with b1=0 and b2=0 to remove the decay due to spin-lattice relaxation. Instead, division by a signal with equal bs but b1=0 or b2=0 reduces spin-lattice relaxation weighting of the apparent exchange rate (AXR). Furthermore, apparent diffusion-weighted R1 relaxation rates can be estimated from non-exchange-weighted DEXSY signals. Estimated R1 values are utilized to remove signal decay due to spin-lattice relaxation from exchange-weighted signals, permitting a more precise estimate of AXR with less data. Data reduction methods are proposed and tested with regards to statistical accuracy and precision of AXR estimates on simulated and experimental data. Simulations show that the methods are capable of accurately measuring the ground-truth exchange rate. The methods remain accurate even when the assumption that DEXSY signals attenuate with b is violated, as occurs for restricted diffusion. Experimental data was collected from fixed neonatal mouse spinal cord samples at 25 and 7°C using the strong static magnetic field gradient produced by a single-sided permanent magnet (i.e., an NMR MOUSE). The most rapid method for exchange measurements requires only five data points (an 80 s experiment as implemented) and achieves a similar level of accuracy and precision to the baseline method using 44 data points. This represents a significant improvement in acquisition speed, overcoming a barrier which has limited the use of DEXSY on living specimen.

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The conformational state of distinct prolines can determine the folding of a protein but equally other biological processes when coupled to a conformation-sensitive secondary reaction. For the neuronal tau protein, the importance of proline conformation is underscored by its interaction with different prolyl cis/trans isomerases. The proline conformation would gain even further importance after phosphorylation of the preceding residue by various proline-directed kinases. A number of molecular diseases including Alzheimer's disease and traumatic brain injury were thereby recently qualified as "cistauosis", as they would imply a cis conformation for the pThr231-Pro232 prolyl bond. We here investigate by NMR spectroscopy the conformation of all prolines in a functional Tau fragment, Tau[208-324]. Although we can detect and identify some minor conformers in the cis form, we show that all prolines are for over 90% in the trans conformation. Phosphorylation by CDK2/CycA3, which notably leads to complete modification of the Thr231 residue, does not change this conclusion. Our data hence disagree with the notion that specific prolyl bonds in tau would adopt preferentially the cis conformation.

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Unlike proteins, a given RNA sequence can adopt more than a single conformation. The two (or more) conformations are long-lived and have similar stabilities, but interconvert only slowly. Such bi- or multistability is often linked to the biological functions of the RNA. This unit describes how nuclear magnetic resonance (NMR) spectroscopy can be used to characterize the conformational dynamics of bistable RNAs.

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PURPOSE: To investigate inter-compartmental water exchange in two model myelinated tissues ex vivo using relaxation exchange spectroscopy. METHODS: Building upon a previously developed theoretical framework, a three-compartment (myelin, intra-axonal, and extra-axonal water) model of the inversion-recovery prepared relaxation exchange spectroscopy signal was applied in excised rat optic nerve and frog sciatic nerve samples to estimate the water residence time constants in myelin (τmyelin ). RESULTS: In the rat optic nerve samples, τmyelin = 138 ± 15 ms (mean ± standard deviation) was estimated. In sciatic nerve, which possesses thicker myelin sheaths than optic nerve, a much longer τmyelin = 2046 ± 140 ms was observed. CONCLUSION: Consistent with previous studies in rat spinal cord, the extrapolation of exchange rates in optic nerve to in vivo conditions indicates that τmyelin < 100 ms. This suggests that there is a significant effect of inter-compartmental water exchange on the transverse relaxation of water protons in white matter. The much longer τmyelin values in sciatic nerve supports the postulate that the inter-compartmental water exchange rate is mediated by myelin thickness. Together, these findings point to the potential for MRI methods to probe variations in myelin thickness in white matter.

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