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Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppression for in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.

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Enhanced angiography based on magnetic resonance imaging (MRI) has emerged as a noninvasive, robust, and high-resolution imaging technique for the clinical evaluation of vascular diseases. However, the effects of clinical Gd-chelating contrast agents are unsatisfactory for MRI contrast enhancement owing to their short blood half-life caused by rapid vascular extravasation, especially in microvessels. To address these issues, nanoprobes based on red blood cell membrane-coated ultrasmall NaGdF4 nanoparticles that exhibit much higher longitudinal molar relaxivity (r1) than the clinically used contrast agent gadolinium diethylenetriaminepentaacetic acid have been developed. Furthermore, the appropriate hydrodynamic diameter and stealth nature aid the nanoprobes to reside longer within the blood vessels without extravasation, thereby increasing the contrast between the blood vessels and surrounding tissues. Through probe-enhanced three-dimensional (3D) dynamic contrast-enhanced MR angiography, the main arteries and veins of the mouse were readily discernible, and even tiny vessels with sub-millimeter diameters could be clearly depicted. With this level of outstanding MR angiography performance, the embolization and recanalization processes of the carotid artery can be serially monitored with high imaging resolution using only a single injection. Additionally, the results of clearance studies and the toxicity tests further highlight the safety features of the nanoprobe. To summarize, the nanoprobes used in this study exhibit less extravascular leakage and a longer blood half-life, thus successfully overcoming the defects of the conventional low-molecular-weight Gd-based contrast agents and demonstrating their potential usefulness in enhanced MR angiography.

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BACKGROUND: The prediction of alcohol consumption in youths and particularly biomarkers of resilience, is critical for early intervention to reduce the risk of subsequent harmful alcohol use. METHODS: At baseline, the longitudinal relaxation rate (R1), indexing grey matter myelination (i.e. myeloarchitecture), was assessed in 86 adolescents/young adults (mean age = 21.76, range: 15.75-26.67 years). The Alcohol Use Disorder Identification Test (AUDIT) was assessed at baseline, 1- and 2-year follow-ups (12- and 24-months post-baseline). We used a whole brain data-driven approach controlled for age, gender, impulsivity and other substance and behavioural addiction measures, such as problematic cannabis use, drug use-related problems, internet gaming, pornography use, binge eating, and levels of externalization, to predict the change in AUDIT scores from R1. RESULTS: Greater baseline bilateral anterior insular and subcallosal cingulate R1 (cluster-corrected family-wise error p < 0.05) predict a lower risk for harmful alcohol use (measured as a reduction in AUDIT scores) at 2-year follow-up. Control analyses show that other grey matter measures (local volume or fractional anisotropy) did not reveal such an association. An atlas-based machine learning approach further confirms the findings. CONCLUSIONS: The insula is critically involved in predictive coding of autonomic function relevant to subjective alcohol cue/craving states and risky decision-making processes. The subcallosal cingulate is an essential node underlying emotion regulation and involved in negative emotionality addiction theories. Our findings highlight insular and cingulate myeloarchitecture as a potential protective biomarker that predicts resilience to alcohol misuse in youths, providing novel identifiers for early intervention.

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We present 19F longitudinal and transverse relaxation studies for four differently fluorosubstituted L-tryptophans, which carry single F atoms in the indole ring, both in the context of the free amino acid and when located in the cyclophilin A protein. For the free 4F-, 5F-, 6F-, 7F-L-Trp, satisfactory agreement between experimentally measured and calculated relaxation rates was obtained, suggesting that the parameters used for calculating the rates for the indole frame are sufficiently accurate. We also measured and calculated relaxation rates for four differently 19F-tryptophan labeled cyclophilin A proteins, transferring the parameters from the free amino acid to the protein-bound moiety. Our results suggest that 19F relaxation data of the large and rigid indole ring in Trp are only moderately affected by protein motions and provide critical reference points for evaluating fluorine NMR relaxation in the future, especially in fluorotryptophan labeled proteins.

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BACKGROUND: There is limited evidence of parametric MR mapping to characterize carotid plaques associated with cerebral ischemic events. PURPOSE: To explore the apparent diffusion coefficients (ADCs) and longitudinal relaxation rates (R1 ) of carotid plaques, including areas of hemorrhage, lipid-rich/necrotic core (LR/NC) without hemorrhage, and fibrous tissue (Fbr) STUDY TYPE: Prospective. SUBJECTS: Twelve patients who underwent carotid endarterectomy. FIELD STRENGTH/SEQUENCE: R1 was measured using double angle Look-Locker acquisition on 3T systems. Single-shot spin-echo echo-planar imaging with fat suppression and outer-volume suppression (OVS-DWEPI) with b values of 10 and 500 s/mm2 was used for diffusion-weighted imaging. ASSESSMENT: A phantom study using diluted gadolinium solutions and polyvinyl alcohol solutions was used to validate the two protocols. Regions of interest (ROIs) were manually outlined on MR images for areas of LR/NC, hemorrhage, and Fbr based on histological cross-sections. Pixel-based R1 and ADC values in the ROIs were plotted for each component. The probability density function of the plots determined the optimum contours to separate the three components in the ADC-R1 plane. The LR/NC, hemorrhage, and Fbr regions were mapped on MR images based on the above results and compared to histological results. STATISTICAL TESTS: The R1 values of the phantom measurements were tested using Bland-Altman analysis. The accuracies of the MRI classification were calculated. RESULTS: R1 values <8 s-1 calculated using our method agreed with those calculated using an inversion-recovery fast-spin-echo sequence (error, ≤0.1 s-1 ). ADC values obtained using OVS-DWEPI were 4.1% higher than those obtained using standard echo-planar imaging. LR/NC (R1 , 0.4-1.2 s-1 ; ADC, 0-1.5 µm2 /ms), hemorrhage (R1 ≥ 1.5 s-1 ; ADC, 0.5-1.5 µm2 /ms), and Fbr (R1 , 0.2-0.8 s-1 ; ADC, 1.5-2.9 µm2 /ms) were separated on the plots. The accuracies of MRI classification were LR/NC, 0.86; hemorrhage, 0.79; and Fbr, 0.77. CONCLUSION: The combination of ADC and R1 values measured using our method enabled differentiation among LR/NC, hemorrhage, and Fbr. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;48:1657-1667.

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Objective: To reveal the immediate extent of trauma-induced neurodegenerative changes rostral to the level of lesion and determine the predictive clinical value of quantitative MRI (qMRI) following acute spinal cord injury (SCI). Methods: Twenty-four acute SCI patients and 23 healthy controls underwent a high-resolution T1-weighted protocol. Eighteen of those patients and 20 of controls additionally underwent a multi-parameter mapping (MPM) MRI protocol sensitive to the content of tissue structure, including myelin and iron. Patients were examined clinically at baseline, 2, 6, 12, and 24 months post-SCI. We assessed volume and microstructural changes in the spinal cord and brain using T1-weighted MRI, magnetization transfer (MT), longitudinal relaxation rate (R1), and effective transverse relaxation rate (R2*) maps. Regression analysis determined associations between acute qMRI parameters and recovery. Results: At baseline, cord area and its anterior-posterior width were decreased in patients, whereas MT, R1, and R2* parameters remained unchanged in the cord. Within the cerebellum, volume decrease was paralleled by increases of MT and R2* parameters. Early grey matter changes were observed within the primary motor cortex and limbic system. Importantly, early volume and microstructural changes of the cord and cerebellum predicted functional recovery following injury. Conclusions: Neurodegenerative changes rostral to the level of lesion occur early in SCI, with varying temporal and spatial dynamics. Early qMRI markers of spinal cord and cerebellum are predictive of functional recovery. These neuroimaging biomarkers may supplement clinical assessments and provide insights into the potential of therapeutic interventions to enhance neural plasticity.

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BACKGROUND AND PURPOSE: The longitudinal relaxation time of blood (T 1b) is influenced by haematocrit (Hct) which is known to vary in neonates. The purpose of this study was threefold: to obtain T 1b values in neonates, to investigate how the T 1b influences quantitative arterial spin labelling (ASL), and to evaluate if known relationships between T 1b and haematocrit (Hct) hold true when Hct is measured by means of a point-of-care device. MATERIALS AND METHODS: One hundred and four neonates with 120 MR scan sessions (3 T) were included. The T 1b was obtained from a T 1 inversion recovery sequence. T 1b-induced changes in ASL cerebral blood flow estimates were evaluated. The Hct was obtained by means of a point-of-care device. Linear regression analysis was used to investigate the relation between Hct and MRI-derived R1 of blood (the inverse of the T 1b). RESULTS: Mean T 1b was 1.85 s (sd 0.2 s). The mean T 1b in preterm neonates was 1.77 s, 1.89 s in preterm neonates scanned at term-equivalent age (TEA) and 1.81 s in diseased neonates. The T 1b in the TEA was significantly different from the T 1b in the preterm (p < 0.05). The change in perfusion induced by the T 1b was -11% (sd 9.1%, p < 0.001). The relation between arterial-drawn Hct and R1b was R1b = 0.80 × Hct + 0.22, which falls within the confidence interval of the previously established relationships, whereas capillary-drawn Hct did not correlate with R1b. CONCLUSION: We demonstrated a wide variability of the T 1b in neonates and the implications it could have in methods relying on the actual T 1b as for instance ASL. It was concluded that arterial-drawn Hct values obtained from a point-of-care device can be used to infer the T 1b whereas our data did not support the use of capillary-drawn Hct for T 1b correction.

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OBJECTIVE: Magnetic resonance imaging allows the noninvasive observation of PO2 changes between air breathing and oxygen breathing through quantification of the magnetic longitudinal relaxation time T1. Changes in PO2 are proportional to changes in the longitudinal relaxation rate ΔR1 (where ΔR1=1/T1oxygen-1/T1air). Knowledge of this response could inform clinical interventions using maternal oxygen administration antenatally to treat fetal growth restriction. We present in vivo measurements of the response of the fetal-placental unit to maternal hyperoxia. DESIGN: Prospective cohort. SETTING: Large tertiary maternity hospital. SAMPLE: Nine women undergoing low-risk pregnancy (21-33 weeks of gestation) and five nonpregnant adults. METHODS: During imaging the air supply to mothers was changed from medical air (21% oxygen) to medical oxygen (100% oxygen) and T1 was monitored over time in both the placenta and fetal brain using a periodically repeated magnetic resonance imaging sequence. To demonstrate that the method could detect a brain response, brain responses from five normal adult volunteers were measured using a similar imaging protocol. MAIN OUTCOME MEASURE: Changes in T1 following oxygen challenge. RESULTS: No significant ΔR1 (P=0.42, paired t-test) was observed in fetal brains. A significant placental ΔR1 (P=0.0002, paired t-test) of 0.02±0.01/s (mean±SD) was simultaneously observed in the same participants. In the brains of the nonpregnant adults, a significant ΔR1 (P=0.01, paired t-test) of 0.005±0.002/s was observed. CONCLUSION: Short-term maternal oxygen administration does not improve fetal brain oxygenation, in contrast to the response observed in the adult brain.

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PURPOSE: To test the hypothesis that a noninvasive dynamic contrast enhanced MRI (DCE-MRI) derived interstitial volume fraction (ve ) and/or distribution volume (VD ) were correlated with tumor cellularity in cerebral tumor. METHODS: T1 -weighted DCE-MRI studies were performed in 18 athymic rats implanted with U251 xenografts. After DCE-MRI, sectioned brain tissues were stained with Hematoxylin and Eosin for cell counting. Using a Standard Model analysis and Logan graphical plot, DCE-MRI image sets during and after the injection of a gadolinium contrast agent were used to estimate the parameters plasma volume (vp ), forward transfer constant (K(trans) ), ve , and VD . RESULTS: Parameter values in regions where the standard model was selected as the best model were: (mean ± S.D.): vp = (0.81 ± 0.40)%, K(trans) = (2.09 ± 0.65) × 10(-2) min(-1) , ve = (6.65 ± 1.86)%, and VD = (7.21 ± 1.98)%. The Logan-estimated VD was strongly correlated with the standard model's vp + ve (r = 0.91, P < 0.001). The parameters, ve and/or VD , were significantly correlated with tumor cellularity (r ≥ -0.75, P < 0.001 for both). CONCLUSION: These data suggest that tumor cellularity can be estimated noninvasively by DCE-MRI, thus supporting its utility in assessing tumor pathophysiology.

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Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two antiparallel ß sheets, two α helices and two 310 helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the µs-ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands.

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PURPOSE: Pregnancy complications such as preeclampsia and fetal growth restriction are sometimes thought to be caused by placental abnormalities associated with reduced oxygenation. Oxygen-enhanced MRI (R1 contrast) and BOLD MRI (R2 * contrast) have the potential to noninvasively investigate this oxygen environment at a range of gestational ages. METHODS: Scanning was carried out at 1.5 T under maternal air and oxygen breathing in a single placental slice in 14 healthy pregnant subjects of gestational ages 21-37 weeks. We report R1 changes using a respiratory-triggered inversion recovery-turbo spin-echo sequence, which is sensitive to changes in PO2 , and R2 * changes using a breathhold multiple gradient-recalled echo sequence sensitive to changes in oxygen saturation. RESULTS: Significant R1 increases (P < 0.005, paired t-test) and R2 * decreases (P < 0.0001, paired t-test) between air and oxygen breathing were demonstrated. ΔR1 decreased with gestational age (P < 0.0005, r = -0.835, Pearson correlation test). No significant effect of gestational age on R2 * change was observed. CONCLUSION: The results demonstrate the feasibility of non-invasive investigation of placental oxygenation using MRI and the sensitivity of R1 oxygen-enhanced MRI to gestational age. The techniques have the potential to provide unique noninvasive biomarkers in compromised pregnancies.

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