MATRESHCA: Microtesla Apparatus for Transfer of Resonance Enhancement of Spin Hyperpolarization via Chemical Exchange and Addition.

We present an integrated, open-source device for parahydrogen-based hyperpolarization processes in the microtesla field regime with a cost of components of less than $7000. The device is designed to produce a batch of 13C and 15N hyperpolarized (HP) compounds via hydrogenative or non-hydrogenative parahydrogen-induced polarization methods that employ microtesla magnetic fields for efficient polarization transfer of parahydrogen-derived spin order to X-nuclei (e.g., 13C and 15N). The apparatus employs a layered structure (reminiscent of a Russian doll "Matryoshka") that includes a nonmagnetic variable-temperature sample chamber, a microtesla magnetic field coil (operating in the range of 0.02-75 microtesla), a three-layered mu-metal shield (to attenuate the ambient magnetic field), and a magnetic shield degaussing coil placed in the overall device enclosure. The gas-handling manifold allows for parahydrogen-gas flow and pressure control (up to 9.2 bar of total parahydrogen pressure). The sample temperature can be varied either using a water bath or a PID-controlled heat exchanger in the range from -12 to 80 °C. This benchtop device measures 62 cm (length) × 47 cm (width) × 47 cm (height), weighs 30 kg, and requires only connections to a high-pressure parahydrogen gas supply and a single 110/220 VAC power source. The utility of the device has been demonstrated using an example of parahydrogen pairwise addition to form HP ethyl [1-13C]acetate (P13C = 7%, [c] = 1 M). Moreover, the Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) technique was employed to demonstrate efficient hyperpolarization of 13C and 15N spins in a wide range of biologically relevant molecules, including [1-13C]pyruvate (P13C = 14%, [c] = 27 mM), [1-13C]-α-ketoglutarate (P13C = 17%), [1-13C]ketoisocaproate (P13C = 18%), [15N3]metronidazole (P15N = 13%, [c] = 20 mM), and others. While the vast majority of the utility studies have been performed in standard 5 mm NMR tubes, the sample chamber of the device can accommodate a wide range of sample container sizes and geometries of up to 1 L sample volume. The device establishes an integrated, simple, inexpensive, and versatile equipment gateway needed to facilitate parahydrogen-based hyperpolarization experiments ranging from basic science to preclinical applications; indeed, detailed technical drawings and a bill of materials are provided to support the ready translation of this design to other laboratories.

X-ray beam diagnostics at the MID instrument of the European X-ray Free-Electron Laser Facility.

The Materials Imaging and Dynamics (MID) instrument at the European X-ray Free-Electron Laser Facility (EuXFEL) is equipped with a multipurpose diagnostic end-station (DES) at the end of the instrument. The imager unit in DES is a key tool for aligning the beam to a standard trajectory and for adjusting optical elements such as focusing lenses or the split-and-delay line. Furthermore, the DES features a bent-diamond-crystal spectrometer to disperse the spectrum of the direct beam to a line detector. This enables pulse-resolved characterization of the EuXFEL spectrum to provide X-ray energy calibration, and the spectrometer is particularly useful in commissioning special modes of the accelerator. Together with diamond-based intensity monitors, the imager and spectrometer form the DES unit which also contains a heavy-duty beamstop at the end of the MID instrument. Here, we describe the setup in detail and provide exemplary beam diagnostic results.

Highly Sensitive Switchable Sensors for Hydroxide on Glass Surfaces Based on Isoquinolinium-Quinolinium-substituted Acetylenes.

Bi-substituted acetylenes with a quinolinium and an isoquinolinium substituent are described, which reversibly form intensely colored adducts with O-nucleophiles and thus enable the detection of >0,5 ppm hydroxide on the surfaces of various glasses. Acids reconstitute the colorless bi-substituted acetylenes. The quinolinium and isoquinolinium rings are bound via their 2-, 3-, 4- and 1-, 3-, 4-positions to the triple bond, respectively. The choice of substitution sites of the hetarenium rings enables the design of mixed conjugated/cross-conjugated π-electron systems. Depending on the combination of binding sites, the frontier orbital profile, the triple bond polarization, the fluorescence behaviour, and the sensitivity to hydroxide differs.

Overcoming the Challenges of Hyperpolarizing Substrates with Parahydrogen-Induced Polarization in an MRI System.

Hyperpolarization of 13C nuclei in biomolecules and their administration as imaging agents enables in-vivo monitoring of metabolism. This approach has demonstrated potential for deriving imaging biomarkers for cancer detection, differentiation, and therapy efficacy assessment. The in situ generation of polarized substrates using a permanent addition of parahydrogen to an unsaturated precursor inside the bore of an MRI system used for subsequent imaging circumvents the need for a dedicated external polarizer. This approach reduces polarization loss associated with sample transfer, minimizes hardware requirements and cost, and results in reduced spatial requirements. However, performing INEPT-like pulsed sequences for heteronuclear spin-order transfer in the bore of an MRI system is challenged by poor uniformity of static and excitation magnetic field and molecular convection during the polarization transfer. Therefore, here we characterize these effects, implement a robust modification to the pulse sequence, and measure experimentally the polarization improvement upon modification of the sequence. After rigorous optimization of the parameters, we obtained a 13C polarization of 44.5% for 50 mM of the 1-13C site of ethyl acetate-d6. Our parahydrogen-induced polarization approach enhances the accessibility to hyperpolarized MRI, circumventing the need for an external polarizer.

Anionic N-Heterocyclic Carbenes from Mesoionic Imidazolium-4-pyrrolides: The Influence of Substituents, Solvents, and Charge on their 77Se NMR Chemical Shifts.

Mesoionic compounds are the starting material for the synthesis of unique anionic N-heterocyclic carbenes. Herein, mesoionic imidazolium pyrrolides synthesized from pyrrole-2-carbaldehyde via various N-alkyl-4-pyrroyl-imidazoles are described. These were converted into nine new 4-(pyrrol-2-yl)-substituted imidazolium salts and transformed into the mesoionic title compounds using an anion exchange resin. The DFT-calculated (B3LYP/6-311++G**) CREF values indicate a great potential for the formation of anionic N-heterocyclic carbenes by deprotonation, which were generated and reacted with selenium to obtain selenoureas. The 77Se NMR shifts investigated under systematic variation of conditions are dependent on the substitution pattern (ΔδSe = 133 ppm) and the steric demand of the substituents. Solvent dependencies of the 77Se NMR shifts were investigated applying toluene-d8, THF-d8, CDCl3, CD2Cl2, pyridine-d5, acetone-d6, DMSO-d6, CD3CN, AcOD, and MeOD. The influences of the referencing method on the 77Se shifts using external or internal Me2Se or Ph2Se2 and solvent can add up to ΔδSe = ca. 80 ppm. In addition, we observed a temperature dependence of both the selenoureas and the reference reagent Ph2Se2 as well as a 77Se shift difference of the analyte caused by interaction with internally added Ph2Se2. The negative charge of deprotonated selenoureas shifts the values by an additional -20 ppm.

SIRT7-dependent deacetylation of NPM promotes p53 stabilization following UV-induced genotoxic stress.

Adaptation to different forms of environmental stress is crucial for maintaining essential cellular functions and survival. The nucleolus plays a decisive role as a signaling hub for coordinating cellular responses to various extrinsic and intrinsic cues. p53 levels are normally kept low in unstressed cells, mainly due to E3 ubiquitin ligase MDM2-mediated degradation. Under stress, nucleophosmin (NPM) relocates from the nucleolus to the nucleoplasm and binds MDM2, thereby preventing degradation of p53 and allowing cell-cycle arrest and DNA repair. Here, we demonstrate that the mammalian sirtuin SIRT7 is an essential component for the regulation of p53 stability during stress responses induced by ultraviolet (UV) irradiation. The catalytic activity of SIRT7 is substantially increased upon UV irradiation through ataxia telangiectasia mutated and Rad3 related (ATR)-mediated phosphorylation, which promotes efficient deacetylation of the SIRT7 target NPM. Deacetylation is required for stress-dependent relocation of NPM into the nucleoplasm and MDM2 binding, thereby preventing ubiquitination and degradation of p53. In the absence of SIRT7, stress-dependent stabilization of p53 is abrogated, both in vitro and in vivo, impairing cellular stress responses. The study uncovers an essential SIRT7-dependent mechanism for stabilization of the tumor suppressor p53 in response to genotoxic stress.

Affecting Charges and Structures of {Bi6} Architectures by 12-Electron Transition Metal Fragments.

Molecules based on polyatomic bismuth substructures are currently attracting a lot of attention owing to this heavy and essentially non-toxic element's uncommon chemical and physical properties, which include unprecedented bonding properties. Hexaatomic {Bi6} substructures that underly more complex cluster structures were recently reported to adopt different structures or exhibit different structural details as a consequence of the charge of the {Bi6} unit. This leads to either crown-shaped cycles for a nominal Bi66- or differently distorted trigonal prisms for compositions close to Bi62-. It was predicted by quantum chemistry that Bi64- should adopt a distinctly distorted boat-like shape, yet a corresponding compound has remained elusive. Here, we report a proof of this assumption by the synthesis and isolation of [K(crypt-222)]2[Bi6{Zn(hmds)}2]∙1.5THF (1), comprising a bimetallic [Bi6{Zn(hmds)}2]2- cluster that fulfils the prediction for the geometric and electronic structure of the missing link (crypt-222 = 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8.8.8]hexa-cosane, hmds = hexamethyldisilazanid). A detailed quantum chemical study shows how the nature of Lewis-acidic transition metal complexes - in particular, 12-electron fragments - control and fine-tune the resulting {Bi6} architectures in accordance with the degree of electron-withdrawal from the polybismuthide core.

Toward Ultra-High-Quality-Factor Wireless Masing Magnetic Resonance Sensing.

It has recently been shown that a bolus of hyperpolarized nuclear spins can yield stimulated emission signals similar in nature to maser signals, potentially enabling new ways of sensing hyperpolarized contrast media, including most notably [1-13C]pyruvate that is under evaluation in over 50 clinical trials for metabolic imaging of cancer. The stimulated NMR signal emissions lasting for minutes do not require radio-frequency excitation, offering unprecedented advantages compared to conventional MR sensing. However, creating nuclear spin maser emission is challenging in practice due to stringent fundamental requirements, making practical in vivo applications hardly possible using conventional passive MR detectors. Here, we demonstrate the utility of a wireless NMR maser detector, the quality factor of which was enhanced 22-fold (to 1,670) via parametric pumping. This active-feedback technique breaks the intrinsic fundamental limit of NMR detector circuit quality factor. We show the use of parametric pumping to reduce the threshold requirement for inducing nuclear spin masing at 300 MHz resonance frequency in a preclinical MRI scanner. Indeed, stimulated emission from hyperpolarized protons was obtained under highly unfavorable conditions of low magnetic field homogeneity (T2* of 3 ms). Greater gains of the quality factor of the MR detector (up to 1 million) were also demonstrated.

Improving SWATH-MS analysis by deep-learning.

Data-independent acquisition (DIA) of tandem mass spectrometry spectra has emerged as a promising technology to improve coverage and quantification of proteins in complex mixtures. The success of DIA experiments is dependent on the quality of spectral libraries used for data base searching. Frequently, these libraries need to be generated by labor and time intensive data dependent acquisition (DDA) experiments. Recently, several algorithms have been published that allow the generation of theoretical libraries by an efficient prediction of retention time and intensity of the fragment ions. Sequential windowed acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) is a DIA method that can be applied at an unprecedented speed, but the fragmentation spectra suffer from a lower quality than data acquired on Orbitrap instruments. To reliably generate theoretical libraries that can be used in SWATH experiments, we developed deep-learning for SWATH analysis (dpSWATH), to improve the sensitivity and specificity of data generated by Q-TOF mass spectrometers. The theoretical library built by dpSWATH allowed us to increase the identification rate of proteins compared to traditional or library-free methods. Based on our analysis we conclude that dpSWATH is a superior prediction framework for SWATH-MS measurements than other algorithms based on Orbitrap data.

13C Radiofrequency Amplification by Stimulated Emission of Radiation Threshold Sensing of Chemical Reactions.

Conventional nuclear magnetic resonance (NMR) enables detection of chemicals and their transformations by exciting nuclear spin ensembles with a radio-frequency pulse followed by detection of the precessing spins at their characteristic frequencies. The detected frequencies report on chemical reactions in real time and the signal amplitudes scale with concentrations of products and reactants. Here, we employ Radiofrequency Amplification by Stimulated Emission of Radiation (RASER), a quantum phenomenon producing coherent emission of 13C signals, to detect chemical transformations. The 13C signals are emitted by the negatively hyperpolarized biomolecules without external radio frequency pulses and without any background signal from other, nonhyperpolarized spins in the ensemble. Here, we studied the hydrolysis of hyperpolarized ethyl-[1-13C]acetate to hyperpolarized [1-13C]acetate, which was analyzed as a model system by conventional NMR and 13C RASER. The chemical transformation of 13C RASER-active species leads to complete and abrupt disappearance of reactant signals and delayed, abrupt reappearance of a frequency-shifted RASER signal without destroying 13C polarization. The experimentally observed "quantum" RASER threshold is supported by simulations.

Quantification of Prostate Cancer Metabolism Using 3D Multiecho bSSFP and Hyperpolarized [1-13 C] Pyruvate: Metabolism Differs Between Tumors of the Same Gleason Grade.

BACKGROUND: Three-dimensional (3D) multiecho balanced steady-state free precession (ME-bSSFP) has previously been demonstrated in preclinical hyperpolarized (HP) 13 C-MRI in vivo experiments, and it may be suitable for clinical metabolic imaging of prostate cancer (PCa). PURPOSE: To validate a signal simulation framework for the use of sequence parameter optimization. To demonstrate the feasibility of ME-bSSFP for HP 13 C-MRI in patients. To evaluate the metabolism in PCa measured by ME-bSSFP. STUDY TYPE: Retrospective single-center cohort study. PHANTOMS/POPULATION: Phantoms containing aqueous solutions of [1-13 C] lactate (2.3 M) and [13 C] urea (8 M). Eight patients (mean age 67 ± 6 years) with biopsy-confirmed Gleason 3 + 4 (n = 7) and 4 + 3 (n = 1) PCa. FIELD STRENGTH/SEQUENCES: 1 H MRI at 3 T with T2 -weighted turbo spin-echo sequence used for spatial localization and spoiled dual gradient-echo sequence used for B0 -field measurement. ME-bSSFP sequence for 13 C MR spectroscopic imaging with retrospective multipoint IDEAL metabolite separation. ASSESSMENT: The primary endpoint was the analysis of pyruvate-to-lactate conversion in PCa and healthy prostate regions of interest (ROIs) using model-free area under the curve (AUC) ratios and a one-directional kinetic model (kP ). The secondary objectives were to investigate the correlation between simulated and experimental ME-bSSFP metabolite signals for HP 13 C-MRI parameter optimization. STATISTICAL TESTS: Pearson correlation coefficients with 95% confidence intervals and paired t-tests. The level of statistical significance was set at P < 0.05. RESULTS: Strong correlations between simulated and empirical ME-bSSFP signals were found (r > 0.96). Therefore, the simulation framework was used for sequence optimization. Whole prostate metabolic HP 13 C-MRI, observing the conversion of pyruvate into lactate, with a temporal resolution of 6 seconds was demonstrated using ME-bSSFP. Both assessed metrics resulted in significant differences between PCa (mean ± SD) (AUC = 0.33 ± 012, kP  = 0.038 ± 0.014) and healthy (AUC = 0.15 ± 0.10, kP  = 0.011 ± 0.007) ROIs. DATA CONCLUSION: Metabolic HP 13 C-MRI in the prostate using ME-bSSFP allows for differentiation between aggressive PCa and healthy tissue. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Metabolic Tumor Imaging with Rapidly Signal-Enhanced 1-13 C-Pyruvate-d3.

The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1-13 C-pyruvate-d3 , which is rapidly enhanced via para-hydrogen, and thus, the signal is amplified by several orders of magnitudes in less than a minute. Using as a model, human melanoma xenografts injected with signal-enhanced 1-13 C-pyruvate-d3, we show that the conversion of pyruvate into lactate can be monitored along with its kinetics, which could pave the way for rapidly detecting and monitoring changes in tumor metabolism.

Genetic and functional analysis of chymotrypsin-like protease (CTRL) in chronic pancreatitis.

BACKGROUND: Genetic predisposition is crucial in the pathogenesis of early-onset chronic pancreatitis (CP). So far, several genetic alterations have been identified as risk factors, predominantly in genes encoding digestive enzymes. However, many early-onset CP cases have no identified underlying cause. Chymotrypsins are a family of serine proteases that can cleave trypsinogen and lead to its degradation. Because genetic alterations in the chymotrypsins CTRC, CTRB1, and CTRB2 are associated with CP, we genetically and functionally investigated chymotrypsin-like protease (CTRL) as a potential risk factor. METHODS: We screened 1005 non-alcoholic CP patients and 1594 controls for CTRL variants by exome sequencing. We performed Western blots and activity assays to analyse secretion and proteolytic activity. We measured BiP mRNA expression to investigate the potential impact of identified alterations on endoplasmic reticulum (ER) stress. RESULTS: We identified 13 heterozygous non-synonymous CTRL variants: five exclusively in patients and three only in controls. Functionality was unchanged in 6/13 variants. Four alterations showed normal secretion but reduced (p.G20S, p.G56S, p.G61S) or abolished (p.S208F) activity. Another three variants (p.C201Y, p.G215R and p.C220G) were not secreted and already showed reduced or no activity intracellularly. However, intracellular retention did not lead to ER stress. CONCLUSION: We identified several CTRL variants, some showing potent effects on protease function and secretion. We observed these effects in variants found in patients and controls, and CTRL loss-of-function variants were not significantly more common in patients than controls. Therefore, CTRL is unlikely to play a relevant role in the development of CP.

Solid pseudopapillary neoplasms of the pancreas in childhood and adolescence-an analysis of the German Registry for Rare Pediatric Tumors (STEP).

Solid pseudopapillary neoplasms (SPNs) are the most common entity among pediatric pancreatic tumors. Still, these are rare tumors with an annual incidence of 0.1-0.2/1,000,000, and little is known about their optimal treatment. This analysis aimed to increase knowledge about the occurrence and treatment strategies of SPN in childhood. Data regarding diagnostics, treatment, and outcome of children aged 0-18 years with SPN recorded in the German Registry for Rare Pediatric Tumors (STEP) were analyzed. Thirty-eight patients were identified with a median age of 14.5 years at diagnosis (range: 8-18) and a female preponderance (81.6%). The most frequent location of the tumor was the pancreatic tail. In histopathological and immunohistochemical examination, pseudopapillary, solid, and cystic lesions as well as expression of beta-catenin, progesterone receptors, and cyclin D1 were the most common findings. All patients underwent surgical resection. Most patients underwent open resection, predominantly tail resection for tumors in the tail region and pylorus-preserving pancreaticoduodenectomy for tumors in the head region. The main postoperative sequela was exogenous pancreatic insufficiency (23.7%), especially with SPN in the pancreatic head. No recurrence occurred during follow-up, although two patients underwent resection with microscopic residue. CONCLUSION: SPN of the pancreas in childhood are low-grade malignancies with usually favorable treatment outcomes. However, therapy can lead to relevant long-term sequelae. To prevent recurrence, complete surgical resection is recommended, sparing as much healthy pancreatic tissue as possible. Interdisciplinary collaboration between specialists is essential to optimize treatment. Molecular genetic analysis of these tumors could improve understanding of their genesis. WHAT IS KNOWN: • Solid pseudopapillary neoplasms (SPNs) of the pancreas are very rare tumors in childhood. • Little is known about tumorigenesis, and there are no specific guidelines for treatment and follow-up in pediatric patients. WHAT IS NEW: • Characteristics, treatment, and outcome were comprehensively assessed in a large cohort of pediatric patients with SPN. • We propose recommendations for diagnosis, treatment, and follow-up of children with SPN, based on our analysis and considering published experience.

Evaluation of functional and metabolic tumor volume using voxel-wise analysis in childhood rhabdomyosarcoma.

BACKGROUND: Cross-sectional imaging-based morphological characteristics of pediatric rhabdomyosarcoma have failed to predict outcomes. OBJECTIVE: To evaluate the feasibility and possible value of generating tumor sub-volumes using voxel-wise analysis of metabolic and functional data from positron emission tomography/magnetic resonance imaging (PET/MR) or PET/computed tomography (CT) and MRI in rhabdomyosarcoma. MATERIALS AND METHODS: Thirty-four examinations in 17 patients who received PET/MRI or PET/CT plus MRI were analyzed. The volume of interest included total tumor volume before and after therapy. Apparent diffusion coefficients (ADC) and standard uptake values (SUV) were determined voxel-wise. Voxels were assigned to three different groups based on ADC and SUV: "viable tumor tissue," "intermediate tissue" or "possible necrosis." In a second approach, data were grouped into three clusters using the Gaussian mixture model. The ratio of these clusters to total tumor volume and changes due to chemotherapy were correlated with clinical and histopathological data. RESULTS: After chemotherapy, the proportion of voxels in the different groups changed significantly. A significant reduction of the proportion of voxels assigned to cluster 1 was found, from a mean of 36.4% to 2.5% (P < 0.001). There was a significant increase in the proportion of voxels in cluster 3 following chemotherapy from 24.8% to 81.6% (P = 0.02). The proportion of voxels in cluster 2 differed depending on the presence or absence of tumor recurrence, falling from 48% to 10% post-chemotherapy in the group with no tumor recurrence (P < 0.05) and from 29% to 23% (P > 0.05) in the group with tumor recurrence. CONCLUSION: Voxel-wise evaluation of multimodal data in rhabdomyosarcoma is feasible. Our initial results suggest that the different distribution of sub-volumes before and after therapy may have prognostic significance.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Two distinct modes for propagation of histone PTMs across the cell cycle.

Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment of these marks extends over several cell generations. Together, our results reveal how histone marks propagate and demonstrate that chromatin states oscillate within the cell cycle.

Parahydrogen-Induced Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation.

The feasibility of Carbon-13 Radiofrequency (RF) Amplification by Stimulated Emission of Radiation (C-13 RASER) is demonstrated on a bolus of liquid hyperpolarized ethyl [1-13 C]acetate. Hyperpolarized ethyl [1-13 C]acetate was prepared via pairwise addition of parahydrogen to vinyl [1-13 C]acetate and polarization transfer from nascent parahydrogen-derived protons to the carbon-13 nucleus via magnetic field cycling yielding C-13 nuclear spin polarization of approximately 6 %. RASER signals were detected from samples with concentration ranging from 0.12 to 1 M concentration using a non-cryogenic 1.4T NMR spectrometer equipped with a radio-frequency detection coil with a quality factor (Q) of 32 without any modifications. C-13 RASER signals were observed for several minutes on a single bolus of hyperpolarized substrate to achieve 21 mHz NMR linewidths. The feasibility of creating long-lasting C-13 RASER on biomolecular carriers opens a wide range of new opportunities for the rapidly expanding field of C-13 magnetic resonance hyperpolarization.

In Vivo Metabolic Imaging of [1-13 C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra-fast and low-cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1-13 C]pyruvate-d3 for injection in 6 minutes. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13 C]pyruvate-d3 polarized to ≈11 % (residual 250 mM methanol and 20 µM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles.

Biological liquid-liquid phase separation has gained considerable attention in recent years as a driving force for the assembly of subcellular compartments termed membraneless organelles. The field has made great strides in elucidating the molecular basis of biomolecular phase separation in various disease, stress response, and developmental contexts. Many important biological consequences of such "condensation" are now emerging from in vivo studies. Here we review recent work from our group and others showing that many proteins undergo rapid, reversible condensation in the cellular response to ubiquitous environmental fluctuations such as osmotic changes. We discuss molecular crowding as an important driver of condensation in these responses and suggest that a significant fraction of the proteome is poised to undergo phase separation under physiological conditions. In addition, we review methods currently emerging to visualize, quantify, and modulate the dynamics of intracellular condensates in live cells. Finally, we propose a metaphor for rapid phase separation based on cloud formation, reasoning that our familiar experiences with the readily reversible condensation of water droplets help understand the principle of phase separation. Overall, we provide an account of how biological phase separation supports the highly intertwined relationship between the composition and dynamic internal organization of cells, thus facilitating extremely rapid reorganization in response to internal and external fluctuations.