Temperature is a hallmark parameter influencing almost all magnetic resonance properties (e.g., T\textsubscript{1}, T\textsubscript{2}, proton density, diffusion and more). In the pre-clinical setting, temperature has a large influence on animal physiology (e.g., respiration rate, heart rate, metabolism, cellular stress, and more) and needs to be carefully regulated, especially when the animal is under anesthesia and thermoregulation is disrupted. We present an open-source heating and cooling system capable of stabilizing the temperature of the animal. The system was designed using Peltier modules capable of heating or cooling a circulating water bath with active temperature feedback. Feedback was obtained using a commercial thermistor, placed in the animal rectum, and a proportional{\text -}integral{\text -}derivative (PID) controller capable of locking the temperature. Operation was demonstrated in a phantom as well as mouse and rat animal models, where the standard deviation of the temperature of the animal upon convergence was less than a tenth of a degree. An application where brain temperature of a mouse was modulated was demonstrated using an invasive optical probe and non-invasive magnetic resonance spectroscopic thermometry measurements.

The creation of radicals via electrical discharge is a unique approach capable of facilitating dynamic nuclear polarization (DNP) in solids without the need to co-dissolve them with exogenous polarization agents. This method has previously been shown to generate high proton polarization in a variety of organic and inorganic solid compounds. In this work, we extend the scope of applicability for these so-called electrical discharge-induced radicals (EDIRs), by showing their use in DNP of 13C nuclei of glucose. We achieved significant NMR signal enhancement in low temperature solid-state DNP experiments. This enhancement was further improved by the use of a frequency modulated microwave pump instead of conventional DNP with single-frequency microwaves. We demonstrated successful dissolution DNP experiments with such hyperpolarized solid glucose, achieving 13C polarization levels of up to ∼0.6% in the resulting solution (∼0.95% at the exit of the polarizer). This is to be compared with the ∼6% polarization levels achieved with a trityl radical mixed with glucose in a frozen aqueous solution using the same DNP setup and experimental conditions. It is concluded that the use of EDIRs can serve as a suitable polarizing approach for metabolic MRI using glucose, avoiding the need to employ diluted solutions and eliminating the filtration stage of the polarizing agents.

Detecting and mapping metabolism in tissues represents a major step in detecting, characterizing, treating and understanding cancers. Recently introduced deuterium metabolic imaging techniques could offer a noninvasive route for the metabolic imaging of animals and humans, based on using 2 H magnetic resonance spectroscopic imaging (MRSI) to detect the uptake of deuterated glucose and the fate of its metabolic products. In this study, 2 H6,6' -glucose was administered to mice cohorts that had been orthotopically implanted with two different models of pancreatic ductal adenocarcinoma (PDAC), involving PAN-02 and KPC cell lines. As the tumors grew, 2 H6,6' -glucose was administered as bolii into the animals' tail veins, and 2 H MRSI images were recorded at 15.2 T. 2D phase-encoded chemical shift imaging experiments could detect a signal from this deuterated glucose immediately after the bolus injection for both the PDAC models, reaching a maximum in the animals' tumors ~ 20 min following administration, and nearly total decay after ~ 40 min. The main metabolic reporter of the cancers was the 2 H3,3' -lactate signal, which MRSI could detect and localize on the tumors when these were 5 mm or more in diameter. Lactate production time traces varied slightly with the animal and tumor model, but in general lactate peaked at times of 60 min or longer following injection, reaching concentrations that were ~ 10-fold lower than those of the initial glucose injection. This 2 H3,3' -lactate signal was only visible inside the tumors. 2 H-water could also be detected as deuterated glucose's metabolic product, increasing throughout the entire time course of the experiment from its ≈10 mM natural abundance background. This water resonance could be imaged throughout the entire abdomen of the animals, including an enhanced presence in the tumor, but also in other organs like the kidney and bladder. These results suggest that deuterium MRSI may serve as a robust, minimally invasive tool for the monitoring of metabolic activity in pancreatic tumors, capable of undergoing clinical translation and supporting decisions concerning treatment strategies. Comparisons with in vivo metabolic MRI experiments that have been carried out in other animal models are presented and their differences/similarities are discussed.

Recent magnetic resonance studies in healthy and cancerous organs have concluded that deuterated metabolites possess highly desirable properties for mapping non-invasively and, as they happen, characterizing glycolysis and other biochemical processes in animals and humans. A promising avenue of this deuterium metabolic imaging (DMI) approach involves looking at the fate of externally administered 2H6,6′-glucose, as it is taken up and metabolized into different products as a function of time. This study employs deuterium magnetic resonance to follow the metabolism of wildtype and preeclamptic pregnant mice models, focusing on maternal and fetoplacental organs over ≈2 h post-injection. 2H6,6′-glucose uptake was observed in the placenta and in specific downstream organs such as the fetal heart and liver. Main metabolic products included 2H3,3′-lactate and 2H-water, which were produced in individual fetoplacental organs with distinct time traces. Glucose uptake in the organs of most preeclamptic animals appeared more elevated than in the control mice (p = 0.02); also higher was the production of 2H-water arising from this glucose. However, the most notable differences arose in the 2H3,3′-lactate concentration, which was ca. two-fold more abundant in the placenta (p = 0.005) and in the fetal (p = 0.01) organs of preeclamptic-like animals, than in control mice. This is consistent with literature reports about hypoxic conditions arising in preeclamptic and growth-restricted pregnancies, which could lead to an enhancement in anaerobic glycolysis. Overall, the present measurements suggest that DMI, a minimally invasive approach, may offer new ways of studying and characterizing health and disease in mammalian pregnancies, including humans.

Purpose Deuterium metabolic imaging (DMI) maps the uptake of deuterated precursors and their conversion into lactate and other markers of tumor metabolism. Even after leveraging 2 H’s short T 1 s, DMI’s signal‐to‐noise ratio (SNR) is limited. We hypothesize that a multi‐echo balanced steady‐state free precession (ME‐bSSFP) approach would increase SNR compared to chemical shift imaging (CSI), while achieving spectral isolation of the metabolic precursors and products. Methods Suitably tuned 2 H ME‐bSSFP (five echo times [TEs], ΔTE = 2.2 ms, repetition time [TR]/flip‐angle = 12 ms/60°) was implemented at 15.2T and compared to CSI (TR/flip‐angle = 95 ms/90°) regarding SNR and spectral isolation, in simulations, in deuterated phantoms and for the in vivo diagnosis of a mouse tumor model of pancreatic adenocarcinoma (N = 10). Results Simulations predicted an SNR increase vs. CSI of 3‐5, and that the peaks of 2 H‐water, 2 H 6,6’ ‐glucose, and 2 H 3,3’ ‐lactate can be well isolated by ME‐bSSFP; phantoms confirmed this. In vivo, at equal spatial resolution (1.25 × 1.25 mm 2 ) and scan time (10 min), 2 H 6,6’ ‐glucose’s and 2 H 3,3’ ‐lactate’s SNR were indeed higher for bSSFP than for CSI, three‐fold for glucose (57 ± 30 vs. 19 ± 11, P < .001), doubled for water (13 ± 5 vs. 7 ± 3, P = .005). The time courses and overall localization of all metabolites agreed well, comparing CSI against ME‐bSSFP. However, a clearer localization of glucose in kidneys and bladder, the detection of glucose‐avid rims in certain tumors, and a heterogeneous pattern of intra‐tumor lactate production could only be observed using ME‐bSSFP’s higher resolution. Conclusions ME‐bSSFP provides greater SNR per unit time than CSI, providing for higher spatial resolution mapping of glucose uptake and lactate production in tumors.

This study explored the usefulness of multiple quantitative MRI approaches to detect pancreatic ductal adenocarcinomas in two murine models, PAN‐02 and KPC. Methods assayed included 1 H T 1 and T 2 measurements, quantitative diffusivity mapping, magnetization transfer (MT) 1 H MRI throughout the abdomen and hyperpolarized 13 C spectroscopic imaging. The progress of the disease was followed as a function of its development; studies were also conducted for wildtype control mice and for mice with induced mild acute pancreatitis. Customized methods developed for scanning the motion‐ and artifact‐prone mice abdomens allowed us to obtain quality 1 H images for these targeted regions. Contrasts between tumors and surrounding tissues, however, were significantly different. Anatomical images, T 2 maps and MT did not yield significant contrast unless tumors were large. By contrast, tumors showed statistically lower diffusivities than their surroundings (≈8.3 ± 0.4 x 10 −4 for PAN‐02 and ≈10.2 ± 0.6 x 10 −4 for KPC vs 13 ± 1 x 10 −3 mm 2 s −1 for surroundings), longer T 1 relaxation times (≈1.44 ± 0.05 for PAN‐02 and ≈1.45 ± 0.05 for KPC vs 0.95 ± 0.10 seconds for surroundings) and significantly higher lactate/pyruvate ratios by hyperpolarized 13 C MR (0.53 ± 0.2 for PAN‐02 and 0.78 ± 0.2 for KPC vs 0.11 ± 0.04 for control and 0.31 ± 0.04 for pancreatitis‐bearing mice). Although the latter could also distinguish early‐stage tumors from healthy animal controls, their response was similar to that in our pancreatitis model. Still, this ambiguity could be lifted using the 1 H‐based reporters. If confirmed for other kinds of pancreatic tumors this means that these approaches, combined, can provide a route to an early detection of pancreatic cancer.

Diffusion-weighted MRI on rodents could be valuable to evaluate pregnancy-related dysfunctions, particularly in knockout models whose biological nature is well understood. Echo Planar Imaging’s sensitivity to motions and to air/water/fat heterogeneities, complicates these studies in the challenging environs of mice abdomens. Recently developed MRI methodologies based on SPatiotemporal ENcoding (SPEN) can overcome these obstacles, and deliver diffusivity maps at ≈150 µm in-plane resolutions. The present study exploits these capabilities to compare the development in wildtype vs vascularly-altered mice. Attention focused on the various placental layers—deciduae, labyrinth, trophoblast, fetal vessels—that the diffusivity maps could resolve. Notable differences were then observed between the placental developments of wildtype vs diseased mice; these differences remained throughout the pregnancies, and were echoed by perfusion studies relying on gadolinium-based dynamic contrast-enhanced MRI. Longitudinal monitoring of diffusivity in the animals throughout the pregnancies also showed differences between the development of the fetal brains in the wildtype and vascularly-altered mice, even if these disparities became progressively smaller as the pregnancies progressed. These results are analyzed on the basis of the known physiology of normal and preeclamptic pregnancies, as well as in terms of the potential that they might open for the early detection of disorders in human pregnancies.

Purpose: To develop an approach for improving the resolution and sensitivity of hyperpolarized 13C MRSI based on a priori anatomical information derived from featured, water-based 1H images. Methods: A reconstruction algorithm exploiting 1H MRI for the redefinition of the 13C MRSI anatomies was developed, based on a modification of the Spectroscopy with Linear Algebraic Modeling (SLAM) principle. To enhance 13C spatial resolution and reduce spillover effects without compromising SNR, this model was extended by endowing it with a search allowing smooth variations in the 13C MR intensity within the targeted regions of interest. Results: Experiments were performed in vitro on enzymatic solutions and in vivo on rodents, based on the administration of 13C-enriched hyperpolarized pyruvate and urea. The spectral images reconstructed for these substrates and from metabolic products based on predefined 1H anatomical compartments using the new algorithm, compared favorably with those arising from conventional Fourier-based analyses of the same data. The new approach also delivered reliable kinetic 13C results, for the kind of processes and timescales usually targeted by hyperpolarized MRSI. Conclusions: A simple yet flexible strategy is introduced to boost the sensitivity and resolution provided by hyperpolarized 13C MRSI, based on readily available 1H MR information.

Significance The placenta mediates the transfer of metabolites and nutrients, and its dysfunction leads to clinical syndromes that jeopardize both the fetus and mother. Current methods for assessing placental transport and metabolism in vivo are limited; the goal of this study is to validate the potential of minimally invasive technologies based on high-sensitivity, hyperpolarized 13 C MRI. Distinct placental crossing features were observed when administering different hyperpolarized metabolites to near-term rodents. In particular, [1- 13 C]pyruvate was observed to clearly localize in the placenta and to efficiently metabolize into lactate in healthy animals. By contrast, though transport appeared normal, a marked decrease in placental metabolism was observed in preeclamptic models. This opens avenues to diagnose placental disorders, which could translate into humans.

Transparency in the eye lens is maintained via specific, functional interactions among the structural βγ- and chaperone α-crystallins. Here, we report the structure and α-crystallin binding interface of the G18V variant of human γS-crystallin (γS-G18V), which is linked to hereditary childhood-onset cortical cataract. Comparison of the solution nuclear magnetic resonance structures of wild-type and G18V γS-crystallin, both presented here, reveal that the increased aggregation propensity of γS-G18V results from neither global misfolding nor the solvent exposure of a hydrophobic residue but instead involves backbone rearrangement within the N-terminal domain. αB-crystallin binds more strongly to the variant, via a well-defined interaction surface observed via chemical shift differences. In the context of the αB-crystallin structure and the finding that it forms heterogeneous multimers, our structural studies suggest a potential mechanism for cataract formation via the depletion of the finite αB-crystallin population of the lens.

We report the complete solid-state MAS NMR resonance assignment of a medium-sized, trimeric membrane protein, YadA-M. The protein YadA (Yersinia adhesin A) is an important virulence factor of enteropathogenic Yersinia species (such as Yersinia enterocolitica and Yersinia pseudotuberculosis). YadA is localized on the bacterial cell surface and is involved in adhesion to host cells and tissues. It is anchored in the outer membrane by a transmembrane anchor domain (YadA-M). This domain hosts the so-called autotransporter function of YadA: it transports its own N-terminal domain through the outer membrane. The assignment is based on a dataset that consisted of several MAS NMR correlation spectra, recorded on a single, uniformly 13C, 15N- labelled microcrystalline preparation. Except for the single C-terminal residue and the mobile strep tag, we were able to completely assign YadA-M. From this, secondary structure elements were predicted, which, combined with several long-range interstrand restraints, yielded the architecture of the β-sheet.

Die strukturelle Charakterisierung von unlöslichen Proteinen stützt sich auf Festkörper-NMR-Spektroskopie. Versuche mit Perdeuterierung und partiellem Rückaustausch, wie sie schon für kristalline Modellproteine vorgeschlagen wurden, erwiesen sich nun auch als vorteilhaft zur Detektion von Protonen in heterogenen Systemen wie Fibrillen, die aus dem Alzheimer'schen β-Amyloidpeptid Aβ40 gebildet werden, das β-Fass-Membranprotein OmpG und das α-helikale Membranprotein Bakteriorhodopsin. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Die Festkörper-NMR-Spektroskopie ist ein interessantes Verfahren für die Strukturbiologie, mangelnde Empfindlichkeit und Auflösung stehen aber einer verbreiteten Anwendung im Weg. Protonen sind ideale Detektionsziele, weil sie über das größte gyromagnetische Verhältnis verfügen; allerdings neigen sie auch zu starker Linienverbreiterung. B. Reif und Mitarbeiter beschreiben in ihrer Zuschrift auf S. 4601 ff., wie das Problem starker Kopplungen durch Perdeuterierung und teilweisen Rücktausch austauschbarer Protonen umgangen werden konnte. Ihr Ansatz war für fibrilläre und Membranproteine erfolgreich.

Structural characterization of insoluble proteins often relies on solid-state NMR spectroscopy. Perdeuteration and partial back-substitution of exchangeable protons, as proposed for crystalline model proteins, is now shown to lead to beneficial proton spectra for heterogeneous systems, such as fibrils formed by the Alzheimer's disease β-amyloid peptide Aβ40, the lipid reconstituted β-barrel membrane protein OmpG, and the α-helical membrane protein bacteriorhodopsin.

Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [ also known as vesicle-associated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.

The structure of the small heat shock protein αB-crystallin, associated with multiple sclerosis and Alzheimer's disease, has eluded biologists for years. Small angle X-ray scattering and solid-state NMR reveal a curved dimer that modulates substrate interactions upon a change in pH. The small heat shock protein αB-crystallin (αB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric αB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of ∼121° between the planes of the β-sandwich formed by α-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with β4 and β8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with β3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit αB oligomer with tetrahedral symmetry.

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3- 13 C]- and [2- 13 C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [ 13 C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13 C- 13 C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).