Spatial Hadamard encoding of J‐edited spectroscopy using slice‐selective editing pulses

A new approach for simultaneous dual‐voxel J‐difference spectral editing is described, which uses spatially selective spectral‐editing pulses and Hadamard encoding. A theoretical framework for spatial Hadamard editing and reconstruction for parallel acquisition (SHERPA) was developed, applying gradient pulses during the frequency‐selective editing pulses. Spectral simulations were performed for either one (gamma‐aminobutyric acid, GABA) or two molecules (glutathione and lactate) simultaneously detected in two voxels. The method was tested in a two‐compartment GABA phantom, and finally applied to the left and right hemispheres of 10 normal control subjects, scanned at 3 T. SHERPA was successfully implemented at 3 T and gave results in close agreement with conventional MEGA‐PRESS scans in both the phantom and in vivo experiments. Simulations for GABA editing for (3 cm)³ voxels in the left and right hemispheres suggest that both editing efficiency losses and contamination between voxels are about 2%. Compared with conventional single‐voxel single‐metabolite J‐difference editing, two‐ or fourfold acceleration is possible without significant loss of SNR using the SHERPA method. Unlike some other dual‐voxel methods, the method can be used with single‐channel receiver coils, and there is no SNR loss due to unfavorable receive‐coil geometry factors.     

Hadamard editing of glutathione and macromolecule‐suppressed GABA

The primary inhibitory neurotransmitter γ‐aminobutyric acid (GABA) and the major antioxidant glutathione (GSH) are compounds of high importance for the function and integrity of the human brain. In this study, a method for simultaneous J‐difference spectral‐edited magnetic resonance spectroscopy (MRS) of GSH and GABA with suppression of macromolecular (MM) signals at 3 T is proposed. MM‐suppressed Hadamard encoding and reconstruction of MEGA (Mescher–Garwood)‐edited spectroscopy (HERMES) consists of four sub‐experiments (TE = 80 ms), with 20‐ms editing pulses applied at: (A) 4.56 and 1.9 ppm; (B) 4.56 and 1.5 ppm; (C) 1.9 ppm; and (D) 1.5 ppm. One Hadamard combination (A + B – C – D) yields GSH‐edited spectra, and another (A – B + C – D) yields GABA‐edited spectra, with symmetric suppression of the co‐edited MM signal. MM‐suppressed HERMES, conventional HERMES and separate Mescher–Garwood point‐resolved spectroscopy (MEGA‐PRESS) data were successfully acquired from a (33 mm)³ voxel in the parietal lobe in 10 healthy subjects. GSH‐ and GABA‐edited MM‐suppressed HERMES spectra were in close agreement with the respective MEGA‐PRESS spectra. Mean GABA (and GSH) estimates were 1.10 ± 0.15 i.u. (0.59 ± 0.12 i.u.) for MM‐suppressed HERMES, and 1.13 ± 0.09 i.u. (0.66 ± 0.09 i.u.) for MEGA‐PRESS. Mean GABA (and GSH) differences between MM‐suppressed HERMES and MEGA‐PRESS were –0.03 ± 0.11 i.u. (–0.07 ± 0.11 i.u.). The mean signal‐to‐noise ratio (SNR) improvement of MM‐suppressed HERMES over MEGA‐PRESS was 1.45 ± 0.25 for GABA and 1.32 ± 0.24 for GSH. These results indicate that symmetric suppression of the MM signal can be accommodated into the Hadamard editing framework. Compared with sequential single‐metabolite MEGA‐PRESS experiments, MM‐suppressed HERMES allows for simultaneous edited measurements of GSH and GABA without MM contamination in only half the scan time, and SNR is maintained.     

Improved efficiency on editing MRS of lactate and γ‐aminobutyric acid by inclusion of frequency offset corrected inversion pulses at high fields

γ‐Aminobutyric acid (GABA) and lactate are metabolites which are present in the brain. These metabolites can be indicators of psychiatric disorders or tumor hypoxia, respectively. The measurement of these weakly coupled spin systems can be performed using MRS editing techniques; however, at high field strength, this can be challenging. This is due to the low available B₁⁺ field at high fields, which results in narrow‐bandwidth refocusing pulses and, consequently, in large chemical shift displacement artifacts. In addition, as a result of the increased chemical shift displacement artifacts and chemical shift dispersion, the efficiency of the MRS method is reduced, even when using adiabatic refocusing pulses. To overcome this limitation, frequency offset corrected inversion (FOCI) pulses have been suggested as a mean to substantially increase the bandwidth of adiabatic pulses. In this study, a Mescher–Garwood semi‐localization by adiabatic selection and refocusing (MEGA‐sLASER) editing sequence with refocusing FOCI pulses is presented for the measurement of GABA and lactate in the human brain. Metabolite detection efficiencies were improved by 20% and 75% for GABA and lactate, respectively, when compared with editing techniques that employ adiabatic radiofrequency refocusing pulses. The highly efficient MEGA‐sLASER sequence with refocusing FOCI pulses is an ideal and robust MRS editing technique for the measurement of weakly coupled metabolites at high field strengths. Copyright © 2013 John Wiley & Sons, Ltd.     

Age-related gene-specific changes of A-to-I mRNA editing in the human brain

A-to-I editing is an adenosine-to-inosine modification of mRNA particularly widespread in the human brain, where it affects thousands of genes. A growing body of evidence suggests that A-to-I RNA editing is necessary for normal development and maintenance in mammals and that its deficiencies contribute to a number of pathological states. In this study, we examined whether mRNA editing levels of two mRNA species, CYFIP2 and GABRA3, change with aging. CYFIP2 has been implicated in synaptic maintenance, while GABRA3 is a GABA receptor subunit, a part of the major inhibitory neurotransmitter system in the CNS. The levels of mRNA editing were assessed in cortex samples of 20 subjects 22-102 years old. The data show an age-dependent statistically significant decrease in editing in CYFIP2. GABRA3 editing remained much more stable with age, implying that age-related decline of RNA editing is gene-specific. This is the first report of age-dependent decline in A-to-I editing. Further examination of these and other vulnerable genes may reveal specific RNA editing mechanisms that contribute to the aging phenotype.     

Brain GABA editing by localized in vivo (1)H magnetic resonance spectroscopy

Editing of GABA by (1)H MRS in a specific brain area is a unique tool for in vivo non-invasive investigation of neurotransmission disorders. Selective GABA detection is achieved using sequences based on double quantum coherence (DQC). Our pulse sequence makes accurate measurements without artefacts due to spatial localization. The sequence was tested on a phantom solution. The effect of vigabatrin, a specific inhibitor of GABA transaminase, was measured in rat brain and GABA detection was performed in vivo in monkey brain using this procedure. Rats were split into two groups. In the control group, the rats had access to water and, in the other group (vigabatrin, VGB, rats), animals were allowed free access to drinking water containing vigabatrin. After 3 weeks of treatment, rats were anesthetized for in vivo NMR spectroscopy investigation. At the end of the experiment, brains were quickly removed, freeze-clamped and extracted with 4% perchloric acid. One part of the acid extract was used for GABA concentrations assessment by ion exchange chromatography with ninhydrin detection. The second was used for high-resolution NMR analysis. By chromatography measurements, the GABA concentration was 1.23+/-0.06 micromol/g for controls, while for vigabatrin-treated rats the GABA concentration was 4.89+/-1.60 micromol/g. The NMR in vivo results were closely correlated with the NMR ex vivo (r=0.99, p<0.01) and chromatography results (r=0.98, p<0.01). The correlation between ex vivo results and chromatography results was also high (r=0.99, p<0.001). This pulse sequence performed GABA editing from a 376 microl voxel located on the right basal ganglia area in a non-human primate brain. This in vivo GABA editing scheme can thus be proposed for accurate measurement of brain GABA concentrations.     

A detailed analysis of localized J-difference GABA editing: theoretical and experimental study at 4 T

The problem of low signal-to-noise ratio for gamma-aminobutyric acid (GABA) in vivo is exacerbated by inefficient detection schemes and non-optimal experimental parameters. To analyze the mechanisms for GABA signal loss of a MEGA-PRESS J-difference sequence at 4 T, numerical simulations were performed ranging from ideal to realistic experimental implementation, including volume selection and experimental radio frequency (RF) pulse shapes with a macromolecular minimization scheme. The simulations were found to be in good agreement with phantom and in vivo data from human brain. The overall GABA signal intensity for the simulations with realistic conditions for the MEGA-PRESS difference spectrum was calculated to be almost half of the signal simulated under ideal conditions (~43% signal loss). In contrast, creatine was reduced significantly less then GABA (~19% signal loss). The 'four-compartment' distribution due to J-coupling in the PRESS-based localization was one of the most significant sources of GABA signal loss, in addition to imperfect RF profiles for volume selection and editing. An alternative strategy that reduces signal loss due to the four-compartment distribution is suggested. In summary, a detailed analysis of J-difference editing is provided with estimates of the relative amounts of GABA signal losses due to various mechanisms. The numerical simulations presented in this study should facilitate both implementation of the more efficient acquisition and quantification process of J-coupled systems.     

In vivo GABA T2 determination with J‐refocused echo time extension at 7 T

A method to measure the T₂ relaxation time of GABA with spectral editing techniques is proposed. Spectral editing techniques can be used to unambiguously extract signals of low concentration J‐coupled spins such as γ‐aminobutyric acid (GABA) from overlapping resonances such as creatine and macromolecules. These sequences, however, generally have fixed and relatively long echo times. Therefore, for the absolute quantification of the edited spectrum, the T₂ relaxation time must be taken into account. To measure the T₂ relaxation time, the signal intensity has to be obtained at multiple echo times. However, on a coupled spin system such as GABA this is challenging, since the signal intensity of the target resonances is modulated not only by T₂ decay but also by the J‐coupling, which strongly influences the shapes and amplitudes of the edited signals, depending on the echo time. Here, we propose to refocus the J‐modulation of the edited signal at different echo times by using chemical shift selective refocusing. In this way the echo time can be arbitrarily extended while preserving the shape of the edited signal. The method was applied in combination with the MEGA‐sLASER editing technique to measure the in vivo T₂ relaxation time of GABA (87 ± 11 ms, n = 10) and creatine (109 ± 8 ms, n = 10) at 7 T. The T₁ relaxation time of these metabolites in a single subject was also determined (GABA, 1334 ± 158 ms; Cr, 1753 ± 12 ms). The T₂ decay curve of coupled spin systems can be sampled in an arbitrary fashion without the need for signal shape correction. Furthermore, the method can be applied with any spectral editing technique. The shortest echo time of the method is limited by the echo time of the spectral editing technique. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of editors at the novel A-to-I editing positions

A-to-I RNA editing modifies a variety of biologically important mRNAs, and is specifically catalyzed by either adenosine deaminase acting on RNA type 1 (ADAR1) or type 2 (ADAR2) in mammals including human. Recently several novel A-to-I editing sites were identified in mRNAs abundantly expressed in mammalian organs by means of computational genomic analysis, but which enzyme catalyzes these editing sites has not been determined. Using RNA interference (RNAi) knockdowns, we found that cytoplasmic fragile X mental retardation protein interacting protein 2 (CYFIP2) mRNA had an ADAR2-mediated editing position and bladder cancer associated protein (BLCAP) mRNA had an ADAR1-mediated editing position. In addition, we found that ADAR2 forms a complex with mRNAs with ADAR2-mediated editing positions including GluR2, kv1.1 and CYFIP2 mRNAs, particularly when the editing sites were edited in human cerebellum by means of immunoprecipitation (IP) method. CYFIP2 mRNA was ubiquitously expressed in human tissues with variable extents of K/E site editing. Because ADAR2 underactivity may be a causative molecular change of death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), this newly identified ADAR2-mediated editing position may become a useful tool for ALS research.     

Short-echo spectroscopic imaging combined with lactate editing in a single scan

A short-echo spectroscopic imaging sequence extended with a frequency-selective multiple-quantum- coherence technique (Sel-MQC) is presented. The method enables acquisition of a complete water-suppressed proton spectrum with a short echo time and filtering of the J-coupling metabolite, lactate, from co-resonant lipids in one scan. The purpose of the study was to validate this combined pulse sequence in vitro and in vivo. Measurements on phantoms confirmed the feasibility of the method, and, for a practical in vivo application, experiments were carried out on eight tumors from two different tumor models [UT-SCC-8 (n = 4) and SAS (n = 4)]. T(1)- and T(2)-weighted metabolite and lipid ratios were calculated, and the tumors showed different values in the central and outer regions. The ratio of the lipid methylene peak area (1.30 ppm) to choline peak area (3.20 ppm) was significantly (p < 0.01) different in the central tumor area between the two models, and lactate was detected in only three out of four tumors in the SAS tumor line. The present approach of combining short-echo spectroscopic imaging and lactate editing allows the characterization of tumor-specific metabolites such as choline, lipid methylene and methyl resonances as well as lactate in a single scan.     

Volumetric navigated MEGA‐SPECIAL for real‐time motion and shim corrected GABA editing

Mescher–Garwood (MEGA) editing with spin echo full intensity acquired localization (MEGA‐SPECIAL, MSpc) is a technique to acquire γ‐aminobutyric acid (GABA) without macromolecule (MM) contamination at a T_E of 68 ms. However, due to the requirement of multiple shot‐localization, it is often susceptible to subject motion and B₀ inhomogeneity. A method is presented for real‐time shim and motion correction (ShMoCo) using volumetric navigators to correct for motion and motion‐related B₀ inhomogeneity during MSpc acquisition. A phantom experiment demonstrates that ShMoCo restores the GABA peak and improves spectral quality in the presence of motion and zero‐ and first‐order shim changes. The ShMoCo scans were validated in three subjects who performed up–down and left–right head rotations. Qualitative assessment of these scans indicates effective reduction of subtraction artefacts and well edited GABA peaks, while quantitative analysis indicates superior fitting and spectral quality relative to scans with no correction. Copyright © 2015 John Wiley & Sons, Ltd.     

基因编辑技术的研究进展

随着对基因功能研究的不断深入,基因修饰技术逐渐成为基因功能研究的主要方法.基因修饰技术根据不同原理及发展阶段可分为同源重组、RNA干扰和基因编辑.其中基因编辑技术以其能够定点切割DNA,构建简单、特异性高并且适用于各类动植物等特点,近年来引起了科学界的广泛关注.本文就基因定点编辑技术研究的相关进展进行简要综述.     

Maximizing sensitivity for fast GABA edited spectroscopy in the visual cortex at 7 T

The combination of functional MRI (fMRI) and MRS is a promising approach to relate BOLD imaging to neuronal metabolism, especially at high field strength. However, typical scan times for GABA edited spectroscopy are of the order of 6‐30 min, which is long compared with functional changes observed with fMRI. The aim of this study is to reduce scan time and increase GABA sensitivity for edited spectroscopy in the human visual cortex, by enlarging the volume of activated tissue in the primary visual cortex. A dedicated setup at 7 T for combined fMRI and GABA MRS is developed. This setup consists of a half volume multi‐transmit coil with a large screen for visual cortex activation, two high density receive arrays and an optimized single‐voxel MEGA‐sLASER sequence with macromolecular suppression for signal acquisition. The coil setup performance as well as the GABA measurement speed, SNR, and stability were evaluated. A 2.2‐fold gain of the average SNR for GABA detection was obtained, as compared with a conventional 7 T setup. This was achieved by increasing the viewing angle of the participant with respect to the visual stimulus, thereby activating almost the entire primary visual cortex, allowing larger spectroscopy measurement volumes and resulting in an improved GABA SNR. Fewer than 16 signal averages, lasting 1 min 23 s in total, were needed for the GABA fit method to become stable, as demonstrated in three participants. The stability of the measurement setup was sufficient to detect GABA with an accuracy of 5%, as determined with a GABA phantom. In vivo, larger variations in GABA concentration are found: 14‐25%. Overall, the results bring functional GABA detections at a temporal resolution closer to the physiological time scale of BOLD cortex activation.     

The scope of receptor editing and its association with autoimmunity

Random assembly of antibody variable (V), diversity (D) and joining (J) gene segments creates a vast repertoire of antigen receptors, including autoreactive ones. Three ways that are known to reduce autoreactivity in the B-cell compartment include clonal deletion, functional inactivation and receptor editing, a mechanism involving a change in antigen receptor specificity through continued V(D)J recombination. New data suggest that editing can efficiently eliminate autoreactivity, yet, in an autoimmune context, secondary antibody gene rearrangements might also contribute to autoimmunity.     

Developmental- and tissue-specificity of RNA editing in mitochondria of suspension-cultured maize cells and seedlings

C to U editing ofapt9, nad3, andcox2 mRNAs was investigated in maize seedlings at various developmental stages as well as in suspension-cultured cells. Heterogeneïty of mRNAs that result from incomplete editing was analyzed for each gene and from five tissues or developmental conditions. The editing status of approximately 30 cDNA clones was determined by digestion with a restriction enzyme that discriminates between unedited and edited DNA sequences. Theatp9 and splicedcox2 cDNAs were essentially completely edited in all samples examined. Analysis of three editing sites ofnad3 cDNAs indicated that incompletely edited cDNAs were detected in all tissues and treatments with a temporal increase in the overall editing status, from 50% at 3 days to about 75% at 7 days. These results indicate that incompletely edited mRNAs are prevalent for some plant mitochondrial genes, and can change with developmental or growth conditions.