Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice.

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.

Tumor suppression by p53: fall of the triumvirate?

p53 is a key tumor suppressor protein that has numerous functions. Its primary mode of action has generally been ascribed to the induction of cell-cycle arrest, apoptosis, or senescence upon stress. Li et al. challenge this dogma with evidence that all three of these programs are dispensable for p53's tumor suppressive role.

Neurometabolic profile of the amygdala in smokers assessed with 1H-magnetic resonance spectroscopy.

Tobacco smoking is one of the main causes of premature death worldwide and quitting success remains low, highlighting the need to understand the neurobiological mechanisms underlying relapse. Preclinical models have shown that the amygdala and glutamate play an important role in nicotine addiction. The aims of this study were to compare glutamate and other metabolites in the amygdala between smokers and controls, and between different smoking states. Furthermore, associations between amygdalar metabolite levels and smoking characteristics were explored. A novel non-water-suppressed proton magnetic resonance spectroscopy protocol was applied to quantify neurometabolites in 28 male smokers (≥15 cigarettes/day) and 21 non-smoking controls, matched in age, education, verbal IQ, and weekly alcohol consumption. Controls were measured once (baseline) and smokers were measured in a baseline state (1-3 h abstinence), during withdrawal (24 h abstinence) and in a satiation state (directly after smoking). Baseline spectroscopy data were compared between groups by independent t-tests or Mann-Whitney-U tests. Smoking state differences were investigated by repeated-measures analyses of variance (ANOVAs). Associations between spectroscopy data and smoking characteristics were explored using Spearman correlations. Good spectral quality, high anatomical specificity (98% mean gray matter) and reliable quantification of most metabolites of interest were achieved in the amygdala. Metabolite levels did not differ between groups, but smokers showed significantly higher glutamine levels at baseline than satiation. Glx levels were negatively associated with pack-years and smoking duration. In summary, this study provides first insights into the neurometabolic profile of the amygdala in smokers with high anatomical specificity. By applying proton magnetic resonance spectroscopy, neurometabolites in smokers during different smoking states and non-smoking controls were quantified reliably. A significant shift in glutamine levels between smoking states was detected, with lower concentrations in satiation than baseline. The negative association between Glx levels and smoking quantity and duration may imply altered glutamate homeostasis with more severe nicotine addiction.

Nocturnal sodium oxybate increases the anterior cingulate cortex magnetic resonance glutamate signal upon awakening.

Clinical guidelines recommend sodium oxybate (SXB; the sodium salt of γ-hydroxybutyrate) for the treatment of disturbed sleep and excessive daytime sleepiness in narcolepsy, yet the underlying mode of action is elusive. In a randomised controlled trial in 20 healthy volunteers, we aimed at establishing neurochemical changes in the anterior cingulate cortex (ACC) following SXB-enhanced sleep. The ACC is a core neural hub regulating vigilance in humans. At 2:30 a.m., we administered in a double-blind cross-over manner an oral dose of 50 mg/kg SXB or placebo, to enhance electroencephalography-defined sleep intensity in the second half of nocturnal sleep (11:00 p.m. to 7:00 a.m.). Upon scheduled awakening, we assessed subjective sleepiness, tiredness and mood and measured two-dimensional, J-resolved, point-resolved magnetic resonance spectroscopy (PRESS) localisation at 3-Tesla field strength. Following brain scanning, we used validated tools to quantify psychomotor vigilance test (PVT) performance and executive functioning. We analysed the data with independent t tests, false discovery rate (FDR) corrected for multiple comparisons. The morning glutamate signal (at 8:30 a.m.) in the ACC was specifically increased after SXB-enhanced sleep in all participants in whom good-quality spectroscopy data were available (n = 16; pFDR < 0.002). Further, global vigilance (10th-90th inter-percentile range on the PVT) was improved (pFDR < 0.04) and median PVT response time was shorter (pFDR < 0.04) compared to placebo. The data indicate that elevated glutamate in the ACC could provide a neurochemical mechanism underlying SXB's pro-vigilant efficacy in disorders of hypersomnolence.

Impaired glutamate homeostasis in the nucleus accumbens in human cocaine addiction.

Cocaine addiction is characterized by overwhelming craving for the substance, which drives its escalating use despite adverse consequences. Animal models suggest a disrupted glutamate homeostasis in the nucleus accumbens to underlie addiction-like behavior. After chronic administration of cocaine, rodents show decreased levels of accumbal glutamate, whereas drug-seeking reinstatement is associated with enhanced glutamatergic transmission. However, due to technical obstacles, the role of disturbed glutamate homeostasis for cocaine addiction in humans remains only partially understood, and accordingly, no approved pharmacotherapy exists. Here, we applied a tailored proton magnetic resonance spectroscopy protocol that allows glutamate quantification within the human nucleus accumbens. We found significantly reduced basal glutamate concentrations in the nucleus accumbens in cocaine-addicted (N = 26) compared with healthy individuals (N = 30), and increased glutamate levels during cue-induced craving in cocaine-addicted individuals compared with baseline. These glutamatergic alterations, however, could not be significantly modulated by a short-term challenge of N-acetylcysteine (2400 mg/day on 2 days). Taken together, our findings reveal a disturbed accumbal glutamate homeostasis as a key neurometabolic feature of cocaine addiction also in humans. Therefore, we suggest the glutamatergic system as a promising target for the development of novel pharmacotherapies, and in addition, as a potential biomarker for a personalized medicine approach in addiction.

Improved prospective frequency correction for macromolecule-suppressed GABA editing with metabolite cycling at 3T.

PURPOSE: To combine metabolite cycling with J-difference editing (MC MEGA) to allow for prospective frequency correction at each transient without additional acquisitions and compare it to water-suppressed MEGA-PRESS (WS MEGA) editing with intermittent prospective frequency correction. METHODS: Macromolecule-suppressed gamma aminobutyric acid (GABA)-edited experiments were performed in a phantom and in the occipital lobe (OCC) (n = 12) and medial prefrontal cortex (mPFC) (n = 8) of the human brain. Water frequency consistency and average offset over acquisition time were compared. GABA multiplet patterns, signal intensities, and choline subtraction artifacts were evaluated. In vivo GABA concentrations were compared and related to frequency offset in the OCC. RESULTS: MC MEGA was more stable with 21% and 32% smaller water frequency SDs in the OCC and mPFC, respectively. MC MEGA also had 39% and 40% smaller average frequency offsets in the OCC and mPFC, respectively. Phantom GABA multiplet patterns and signal intensities were similar. In vivo GABA concentrations were smaller in MC MEGA than in WS MEGA, with median (interquartile range) of 2.52 (0.27) and 2.29 (0.19) institutional units (i.u.), respectively in the OCC scans without prior DTI, and 0.99 (0.3) and 1.72 (0.5), respectively in the mPFC. OCC WS MEGA GABA concentrations, but not MC MEGA GABA concentrations were moderately correlated with frequency offset. mPFC WS MEGA spectra contained significantly more subtraction artifacts than MC MEGA spectra. CONCLUSION: MC MEGA is feasible and allows for prospective frequency correction at every transient. MC MEGA GABA concentrations were not biased by frequency offsets and contained less subtraction artifacts compared to WS MEGA.

Neurometabolic alterations in the nucleus accumbens of smokers assessed with 1 H magnetic resonance spectroscopy: The role of glutamate and neuroinflammation.

Tobacco use is one of the leading causes of premature death and morbidity worldwide. For smokers trying to quit, relapse rates are high, even after prolonged periods of abstinence. Recent findings in animal models highlight the role of alterations in glutamatergic projections from the prefrontal cortex onto the nucleus accumbens (NAc) in relapse vulnerability. Moreover, inflammatory responses in the NAc have been reported during withdrawal. A novel proton magnetic resonance spectroscopy (1 H-MRS) protocol was applied in humans to measure molar concentrations for glutamate, its sum with glutamine (Glx), and myoinositol plus glycine (mI + Gly) in the NAc (19 smokers, 20 matched controls). Smokers were measured at baseline and during withdrawal and satiation. No difference between groups or smoking states was found for glutamate or Glx, but, in smokers, stronger craving and more severe nicotine dependence were associated with lower baseline glutamate and Glx levels, respectively. Interestingly, mI + Gly concentrations were higher during withdrawal than baseline and correlated negatively with nicotine dependence severity and pack years of smoking. The lack of glutamatergic changes between groups and smoking states may imply that glutamate homeostasis is not significantly altered in smokers or that changes are too small for detection by 1 H-MRS. Moreover, the observed increase in mI + Gly may imply that neuroinflammatory processes occur in the NAc during nicotine withdrawal. These findings shed light on neurobiological relapse mechanisms in smokers and may provide the opportunity to develop more effective treatment options targeting the glutamate and neuroinflammation system.

Highly accelerated time-of-flight magnetic resonance angiography using spiral imaging improves conspicuity of intracranial arterial branches while reducing scan time.

OBJECTIVE: To systematically compare time-of-flight magnetic resonance angiography (TOF-MRA) acquired with Compressed SENSE (TOF-CS) to spiral imaging (TOF-Spiral) for imaging of brain-feeding arteries. METHODS: Seventy-one patients (60.2 ± 19.5 years, 43.7% females, 28.2% with pathology) who underwent TOF-MRA after implementation of a new scanner software program enabling spiral imaging were analyzed retrospectively. TOF-CS (standard sequence; duration ~ 4 min) and the new TOF-Spiral (duration ~ 3 min) were acquired. Image evaluation (vessel image quality and detectability, diagnostic confidence (1 (diagnosis very uncertain) to 5 (diagnosis very certain)), quantitative measurement of aneurysm diameter or degree of stenosis according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria) was performed by two readers. Quantitative assessments of pathology were compared to computed tomography angiography (CTA) or digital subtraction angiography (DSA). RESULTS: TOF-CS showed higher image quality for intraosseous and intradural segments of the internal carotid artery while TOF-Spiral better depicted small intracranial vessels like the anterior choroidal artery. All vessel pathologies were correctly identified by both readers for TOF-CS and TOF-Spiral with high confidence (TOF-CS (4.4 ± 0.6 and 4.3 ± 0.8), TOF-Spiral (4.3 ± 0.7 and 4.3 ± 0.8)) and good inter-reader agreement (Cohen's kappa > 0.8). Quantitative assessments of aneurysm size or stenosis did not significantly differ between TOF-CS or TOF-Spiral and CTA or DSA (p > 0.05). CONCLUSIONS: TOF-Spiral for imaging of brain-feeding arteries enables reductions in scan time without drawbacks in diagnostic confidence. A combination of spiral imaging and CS may help to overcome shortcomings of both sequences alone and could further reduce acquisition times in the future. KEY POINTS: • TOF-MRA with Compressed SENSE is superior in depicting arteries at the skull base while spiral TOF-MRA is able to better depict small intracranial vessels. • Both TOF-MRA with Compressed SENSE and TOF-MRA with spiral imaging provide high diagnostic confidence for detection of pathologies of brain-feeding arteries. • Spiral TOF-MRA is faster (by 25% for the sequence used in this study) than TOF-MRA with Compressed SENSE, thus enabling clear reductions in scan time for the clinical setting.

In situ postmortem ethanol quantification in the cerebrospinal fluid by non-water-suppressed proton MRS.

Determination of the ethanol concentration in corpses with MRS would allow a reproducible forensic assessment by which evidence is collected in a noninvasive manner. However, although MRS has been successfully used to detect ethanol in vivo, it has not been applied to postmortem ethanol quantification in situ. The present study examined the feasibility of the noninvasive measurement of the ethanol concentration in human corpses with MRS. A total of 15 corpses with suspected alcohol consumption before demise underwent examination in a 3 T whole body scanner. To address the partial overlap of the ethanol and lactate signal in the postmortem spectrum, non-water-suppressed single voxel spectra were recorded in the cerebrospinal fluid (CSF) of the left lateral ventricle via the metabolite cycling technique. The ethanol signals were quantified using the internal water as reference standard, as well as based on a reference signal acquired in a phantom. The measured values were compared with biochemically determined concentrations in the blood (BAC) and CSF (CSFAC). In 8 of the 15 corpses a BAC above zero was determined (range 0.03-1.68 g/kg). In all of these 8 corpses, ethanol was measured in CSF with the proposed MRS protocol. The two applied MRS calibration strategies resulted in similar concentrations. However, the MRS measurements generally overestimated the ethanol concentration by 0.09 g/kg (4%) to 0.72 g/kg (45%) as compared with the CSFAC value. The presented MRS protocol allows the measurement of ethanol in the CSF in human corpses and provides an estimation of the ethanol concentration prior to autopsy. Observed deviations from biochemically determined concentrations are mainly explained by the approximate correction of the relaxation attenuation of the ethanol signal.

Effects of serotonin 2A/1A receptor stimulation on social exclusion processing.

Social ties are crucial for physical and mental health. However, psychiatric patients frequently encounter social rejection. Moreover, an increased reactivity to social exclusion influences the development, progression, and treatment of various psychiatric disorders. Nevertheless, the neuromodulatory substrates of rejection experiences are largely unknown. The preferential serotonin (5-HT) 2A/1A receptor agonist, psilocybin (Psi), reduces the processing of negative stimuli, but whether 5-HT2A/1A receptor stimulation modulates the processing of negative social interactions remains unclear. Therefore, this double-blind, randomized, counterbalanced, cross-over study assessed the neural response to social exclusion after the acute administration of Psi (0.215 mg/kg) or placebo (Pla) in 21 healthy volunteers by using functional magnetic resonance imaging (fMRI) and resting-state magnetic resonance spectroscopy (MRS). Participants reported a reduced feeling of social exclusion after Psi vs. Pla administration, and the neural response to social exclusion was decreased in the dorsal anterior cingulate cortex (dACC) and the middle frontal gyrus, key regions for social pain processing. The reduced neural response in the dACC was significantly correlated with Psi-induced changes in self-processing and decreased aspartate (Asp) content. In conclusion, 5-HT2A/1A receptor stimulation with psilocybin seems to reduce social pain processing in association with changes in self-experience. These findings may be relevant to the normalization of negative social interaction processing in psychiatric disorders characterized by increased rejection sensitivity. The current results also emphasize the importance of 5-HT2A/1A receptor subtypes and the Asp system in the control of social functioning, and as prospective targets in the treatment of sociocognitive impairments in psychiatric illnesses.

In vivo estimation of transverse relaxation time constant (T2 ) of 17 human brain metabolites at 3T.

PURPOSE: The transverse relaxation times T2 of 17 metabolites in vivo at 3T is reported and region specific differences are addressed. METHODS: An echo-time series protocol was applied to one, two, or three volumes of interest with different fraction of white and gray matter including a total number of 106 healthy volunteers and acquiring a total number of 128 spectra. The data were fitted with the 2D fitting tool ProFit2, which included individual line shape modeling for all metabolites and allowed the T2 calculation of 28 moieties of 17 metabolites. RESULTS: The T2 of 10 metabolites and their moieties have been reported for the first time. Region specific T2 differences in white and gray matter enriched tissue occur in 16 of 17 metabolites examined including single resonance lines and coupled spin systems. CONCLUSION: The relaxation time T2 is regions specific and has to be considered when applying tissue composition correction for internal water referencing. Magn Reson Med 80:452-461, 2018. © 2018 International Society for Magnetic Resonance in Medicine.

Isotropic resolution diffusion tensor imaging of lumbosacral and sciatic nerves using a phase-corrected diffusion-prepared 3D turbo spin echo.

PURPOSE: To perform in vivo isotropic-resolution diffusion tensor imaging (DTI) of lumbosacral and sciatic nerves with a phase-navigated diffusion-prepared (DP) 3D turbo spin echo (TSE) acquisition and modified reconstruction incorporating intershot phase-error correction and to investigate the improvement on image quality and diffusion quantification with the proposed phase correction. METHODS: Phase-navigated DP 3D TSE included magnitude stabilizers to minimize motion and eddy-current effects on the signal magnitude. Phase navigation of motion-induced phase errors was introduced before readout in 3D TSE. DTI of lower back nerves was performed in vivo using 3D TSE and single-shot echo planar imaging (ss-EPI) in 13 subjects. Diffusion data were phase-corrected per kz plane with respect to T2 -weighted data. The effects of motion-induced phase errors on DTI quantification was assessed for 3D TSE and compared with ss-EPI. RESULTS: Non-phase-corrected 3D TSE resulted in artifacts in diffusion-weighted images and overestimated DTI parameters in the sciatic nerve (mean diffusivity [MD] = 2.06 ± 0.45). Phase correction of 3D TSE DTI data resulted in reductions in all DTI parameters (MD = 1.73 ± 0.26) of statistical significance (P ≤ 0.001) and in closer agreement with ss-EPI DTI parameters (MD = 1.62 ± 0.21). CONCLUSION: DP 3D TSE with phase correction allows distortion-free isotropic diffusion imaging of lower back nerves with robustness to motion-induced artifacts and DTI quantification errors. Magn Reson Med 80:609-618, 2018. © 2018 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Quantitative magnetic resonance spectroscopy at 3T based on the principle of reciprocity.

Quantification of magnetic resonance spectroscopy signals using the phantom replacement method requires an adequate correction of differences between the acquisition of the reference signal in the phantom and the measurement in vivo. Applying the principle of reciprocity, sensitivity differences can be corrected at low field strength by measuring the RF transmitter gain needed to obtain a certain flip angle in the measured volume. However, at higher field strength the transmit sensitivity may vary from the reception sensitivity, which leads to wrongly estimated concentrations. To address this issue, a quantification approach based on the principle of reciprocity for use at 3T is proposed and validated thoroughly. In this approach, the RF transmitter gain is determined automatically using a volume-selective power optimization and complemented with information from relative reception sensitivity maps derived from contrast-minimized images to correct differences in transmission and reception sensitivity. In this way, a reliable measure of the local sensitivity was obtained. The proposed method is used to derive in vivo concentrations of brain metabolites and tissue water in two studies with different coil sets in a total of 40 healthy volunteers. Resulting molar concentrations are compared with results using internal water referencing (IWR) and Electric REference To access In vivo Concentrations (ERETIC). With the proposed method, changes in coil loading and regional sensitivity due to B1 inhomogeneities are successfully corrected, as demonstrated in phantom and in vivo measurements. For the tissue water content, coefficients of variation between 2% and 3.5% were obtained (0.6-1.4% in a single subject). The coefficients of variation of the three major metabolites ranged from 3.4-14.5%. In general, the derived concentrations agree well with values estimated with IWR. Hence, the presented method is a valuable alternative for IWR, without the need for additional hardware such as ERETIC and with potential advantages in diseased tissue.

Neurochemical dynamics of acute orofacial pain in the human trigeminal brainstem nuclear complex.

The trigeminal brainstem sensory nuclear complex is the first central relay structure mediating orofacial somatosensory and nociceptive perception. Animal studies suggest a substantial involvement of neurochemical alterations at such basal CNS levels in acute and chronic pain processing. Translating this animal based knowledge to humans is challenging. Human related examining of brainstem functions are challenged by MR related peculiarities as well as applicability aspects of experimentally standardized paradigms. Based on our experience with an MR compatible human orofacial pain model, the aims of the present study were twofold: 1) from a technical perspective, the evaluation of proton magnetic resonance spectroscopy at 3 T regarding measurement accuracy of neurochemical profiles in this small brainstem nuclear complex and 2) the examination of possible neurochemical alterations induced by an experimental orofacial pain model. Data from 13 healthy volunteers aged 19-46 years were analyzed and revealed high quality spectra with significant reductions in total N-acetylaspartate (N-acetylaspartate + N-acetylaspartylglutamate) (-3.7%, p = 0.009) and GABA (-10.88%, p = 0.041) during the pain condition. These results might reflect contributions of N-acetylaspartate and N-acetylaspartylglutamate in neuronal activity-dependent physiologic processes and/or excitatory neurotransmission, whereas changes in GABA might indicate towards a reduction in tonic GABAergic functioning during nociceptive signaling. Summarized, the present study indicates the applicability of 1H-MRS to obtain neurochemical dynamics within the human trigeminal brainstem sensory nuclear complex. Further developments are needed to pave the way towards bridging important animal based knowledge with human research to understand the neurochemistry of orofacial nociception and pain.

Accurate determination of brain metabolite concentrations using ERETIC as external reference.

Magnetic Resonance Spectroscopy (MRS) can provide in vivo metabolite concentrations in standard concentration units if a reliable reference signal is available. For 1 H MRS in the human brain, typically the signal from the tissue water is used as the (internal) reference signal. However, a concentration determination based on the tissue water signal most often requires a reliable estimate of the water concentration present in the investigated tissue. Especially in clinically interesting cases, this estimation might be difficult. To avoid assumptions about the water in the investigated tissue, the Electric REference To access In vivo Concentrations (ERETIC) method has been proposed. In this approach, the metabolite signal is compared with a reference signal acquired in a phantom and potential coil-loading differences are corrected using a synthetic reference signal. The aim of this study, conducted with a transceiver quadrature head coil, was to increase the accuracy of the ERETIC method by correcting the influence of spatial B1 inhomogeneities and to simplify the quantification with ERETIC by incorporating an automatic phase correction for the ERETIC signal. Transmit field ( B1+) differences are minimized with a volume-selective power optimization, whereas reception sensitivity changes are corrected using contrast-minimized images of the brain and by adapting the voxel location in the phantom measurement closely to the position measured in vivo. By applying the proposed B1 correction scheme, the mean metabolite concentrations determined with ERETIC in 21 healthy subjects at three different positions agree with concentrations derived with the tissue water signal as reference. In addition, brain water concentrations determined with ERETIC were in agreement with estimations derived using tissue segmentation and literature values for relative water densities. Based on the results, the ERETIC method presented here is a valid tool to derive in vivo metabolite concentration, with potential advantages compared with internal water referencing in diseased tissue.

Reactivity of NHC Alane Adducts towards N-Heterocyclic Carbenes and Cyclic (Alkyl)(amino)carbenes: Ring Expansion, Ring Opening, and Al-H Bond Activation.

The synthesis of mono-NHC alane adducts of the type (NHC)⋅AlH3 (NHC=Me2 Im (1), Me2 ImMe (2), iPr2 Im (3 and [D3 ]-3), iPr2 ImMe (4), Dipp2 Im (10); Im=imidazolin-2-ylidene, Dipp=2,6-diisopropylphenyl) and (NHC)⋅AliBu2 H (NHC=iPr2 Im (11), Dipp2 Im (12)) as well as their reactivity towards different types of carbenes is presented. Although the mono-NHC adducts remained stable at elevated temperatures, ring expansion occurred when (iPr2 Im)⋅AlH3 (3) was treated with a second equivalent of the carbene iPr2 Im to give (iPr2 Im)⋅AlH(RER-iPr2 ImH2 ) (6). In 6, {(iPr2 Im}AlH} is inserted into the NHC ring. In contrast, ring opening was observed with the sterically more demanding Dipp2 Im with the formation of (iPr2 Im)⋅AlH2 (ROR-Dipp2 ImH2 )H2 Al⋅(iPr2 Im) (9). In 9, two {(iPr2 Im)⋅AlH2 } moieties stabilize the ring-opened Dipp2 Im. If two hydridic sites are blocked, the adducts are stable with respect to further ring expansion or ring opening, as exemplified by the adducts (iPr2 Im)⋅AliBu2 H (11) and (Dipp2 Im)⋅AliBu2 H (12). The adducts (NHC)⋅AlH3 and (iPr2 Im)⋅AliBu2 H reacted with cAACMe by insertion of the carbene carbon atom into the Al-H bond to give (NHC)⋅AlH2 /iBu2 (cAACMe H) (13-18) instead of ligand substitution, ring-expansion, or ring-opened products.

Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation.

BACKGROUND: Casitas B-lineage lymphoma (Cbl or c-Cbl) is a RING ubiquitin ligase that negatively regulates protein tyrosine kinase (PTK) signalling. Phosphorylation of a conserved residue (Tyr371) on the linker helix region (LHR) between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available, they do not reveal how Tyr371 mutations affect Cbl's conformation. Here, we investigate how Tyr371 mutations affect Cbl's conformation in solution and how this relates to Cbl's ability to potentiate transformation in cells. RESULTS: To explore how Tyr371 mutations affect Cbl's properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2-Ub), small angle X-ray scattering studies to investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation potential in cells. Cbl Tyr371 mutants enhance E2-Ub binding and cause Cbl to adopt extended conformations in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are those that perturb the LHR-substrate-binding domain interaction. CONCLUSIONS: c-Cbl's LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl.

Motion correction and frequency stabilization for MRS of the human spinal cord.

Subject motion is challenging for MRS, because it can falsify results. For spinal cord MRS in particular, subject movement is critical, since even a small movement > 1 mm) can lead to a voxel shift out of the desired measurement region. Therefore, the identification of motion corrupted MRS scans is essential. In this investigation, MR navigators acquired simultaneously with the MRS data are used to identify a displacement of the spinal cord due to subject motion. It is shown that navigators are able to recognize substantial subject motion (>1 mm) without impairing the MRS measurement. In addition, navigators are easy to apply to the measurement, because no additional hardware and just a minor additional user effort are needed. Moreover, no additional scan time is required, because navigators can be applied in the deadtime of the MRS sequence. Furthermore, in this work, retrospective motion correction combined with frequency stabilization is presented by combining navigators with non-water-suppressed (1)H-MRS, resulting in an improved spectral quality of the spinal cord measurements.

Neurochemical profile of the human cervical spinal cord determined by MRS.

MRS enables insight into the chemical composition of central nervous system tissue. However, technical challenges degrade the data quality when applied to the human spinal cord. Therefore, to date detection of only the most prominent metabolite resonances has been reported in the healthy human spinal cord. The aim of this investigation is to provide an extended metabolic profile including neurotransmitters and antioxidants in addition to metabolites involved in the energy and membrane metabolism of the human cervical spinal cord in vivo. To achieve this, data quality was improved by using a custom-made, cervical detector array together with constructive averaging of a high number of echo signals, which is enabled by the metabolite cycling technique at 3T. In addition, the improved spinal cord spectra were extensively cross-validated, in vivo, post-mortem in situ and ex vivo. Reliable identification of up to nine metabolites was achieved in group analyses for the first time. Distinct features of the spinal cord neurochemical profile, in comparison with the brain neurotransmission system, include decreased concentrations of the sum of glutamate and glutamate and increased concentrations of aspartate, γ-amino-butyric acid, scyllo-inositol and the sum of myo-inositol and glycine.