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KEY POINTS - The ClC-3 2Cl /1H exchanger modulates endosome pH and Cl concentration. We investigated the relationships between ClC-3-mediated ion transport (steady-state transport current, I ), gating charge (Q) and cytoplasmic alkalization. ClC-3 transport is functionally unidirectional. ClC-5 and ClC-3 display indistinguishable exchange ratios, but ClC-3 cycling is less "efficient", as reflected by a large Q/I . An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Glu ; E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine -OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and -OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a "closed" inner gate conformation that maintains coupling during cycling.
ABSTRACT - We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (I ), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/I ratio, but an indistinguishable Cl /H coupling ratio. External SCN reduced H transport rate and uncoupled anion/H exchange by 80-90%. Removal of the external gating glutamate ("Glu ") (E224A mutation) reduced Q and abolished H transport. We hypothesized that Methionine 531 (M531) impedes "water wire" H transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/I ratio by 50% and enhanced H transport. External protons (pH 5.0) inhibited I and markedly reduced Q while shifting the Q-voltage (V) relationship positively. The Cl /H coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Glu adopting an outward/open position. Internal "anion gate" removal (Y572S) dramatically increased I and impaired coupling, without slowing H transport rate. Loss of both gates (Y572S/E224A) resulted in a large "open pore" conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/I must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H coupling as well as anion selectivity, and interacts directly with E224. Disruption of this "closed gate" conformation by internal protons may represent a critical step in the ClC-3 transport cycle.
© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.
PURPOSE - To test the ability of a novel pulse sequence applied in vivo at 3 Tesla to separate the contributions to the water signal from amide proton transfer (APT) and relayed nuclear Overhauser enhancement (rNOE) from background direct water saturation and semisolid magnetization transfer (MT). The lack of such signal source isolation has confounded conventional chemical exchange saturation transfer (CEST) imaging.
METHODS - We quantified APT and rNOE signals using a chemical exchange rotation transfer (CERT) metric, MTR . A range of duty cycles and average irradiation powers were applied, and results were compared with conventional CEST analyses using asymmetry (MTR ) and extrapolated magnetization transfer (EMR).
RESULTS - Our results indicate that MTR is more specific than MTR and, because it requires as few as 3 data points, is more rapid than methods requiring a complete Z-spectrum, such as EMR. In white matter, APT (1.5 ± 0.5%) and rNOE (2.1 ± 0.7%) were quantified by using MTR with a 30% duty cycle and a 0.5-µT average power. In addition, our results suggest that MTR is insensitive to B inhomogeneity, further magnifying its speed advantage over CEST metrics that require a separate B measurement. However, MTR still has nontrivial sensitivity to B inhomogeneities.
CONCLUSION - We demonstrated that MTR is an alternative metric to evaluate APT and rNOE, which is fast, robust to B inhomogeneity, and easy to process.
© 2018 International Society for Magnetic Resonance in Medicine.
Chemical exchange saturation transfer (CEST) imaging of amides at 3.5 ppm and fast-exchanging amines at 3 ppm provides a unique means to enhance the sensitivity of detection of, for example, proteins/peptides and neurotransmitters, respectively, and hence can provide important information on molecular composition. However, despite the high sensitivity relative to conventional magnetic resonance spectroscopy (MRS), in practice, CEST often has relatively poor specificity. For example, CEST signals are typically influenced by several confounding effects, including direct water saturation (DS), semi-solid non-specific magnetization transfer (MT), the influence of water relaxation times (T ) and nearby overlapping CEST signals. Although several editing techniques have been developed to increase the specificity by removing DS, semi-solid MT and T influences, it is still challenging to remove overlapping CEST signals from different exchanging sites. For instance, the amide proton transfer (APT) signal could be contaminated by CEST effects from fast-exchanging amines at 3 ppm and intermediate-exchanging amines at 2 ppm. The current work applies an exchange-dependent relaxation rate (R ) to address this problem. Simulations demonstrate that: (1) slowly exchanging amides and fast-exchanging amines have distinct dependences on irradiation powers; and (2) R serves as a resonance frequency high-pass filter to selectively reduce CEST signals with resonance frequencies closer to water. These characteristics of R provide a means to isolate the APT signal from amines. In addition, previous studies have shown that CEST signals from fast-exchanging amines have no distinct features around their resonance frequencies. However, R gives Lorentzian lineshapes centered at their resonance frequencies for fast-exchanging amines and thus can significantly increase the specificity of CEST imaging for amides and fast-exchanging amines.
Copyright © 2017 John Wiley & Sons, Ltd.
PURPOSE - To evaluate the magnitude of chemical exchange effects and R dispersion in muscle and their relationship to tissue sodium levels with aging.
METHODS - Seven healthy volunteers (aged 24 to 87years, median age 47) underwent MRI to assess tissue sodium levels and water T values at different spin-locking frequencies in calf muscles. T values at each locking field were computed based on a three-parameter mono-exponential model to fit signals obtained at different locking times, and R (=1/T) rates were compared at different locking fields. In particular, the dispersion of R (ΔR=R(0Hz)-R(500Hz)) was examined as a function of subject age. Muscle sodium content was calculated by comparing signal intensities between tissues and reference standards within the same image. The variations of ΔR with age and sodium were analyzed by linear regression.
RESULTS - T values and sodium content both increased with age. R dispersion also increased with age and showed a strong linear correlation (correlation coefficient r=0.98, P=0.000578) with sodium content.
CONCLUSION - ΔR reports on the contribution of labile protons such as hydroxyls which may be associated with macromolecule accumulation in the extracellular matrix (ECM). An increase of sodium signal suggests an enlarged ECM volume fraction and/or an increase in sodium concentration, which occurs during normal aging. The strong correlation between ΔR and sodium is likely the consequence of increased ECM and density of total charged sites within the matrix from molecules such as collagens and proteoglycans. The results from this study show the potential use of R dispersion and sodium imaging in the assessment of pathological changes in muscle such as fibrosis.
Copyright © 2017 Elsevier Inc. All rights reserved.
The goal is to develop an imaging method where contrast reflects amide-water magnetization exchange, with minimal signal contributions from other sources. Conventional chemical exchange saturation transfer (CEST) imaging of amides (often called amide proton transfer, or APT, and quantified by the metric MTR) is confounded by several factors unrelated to amides, such as aliphatic protons, water relaxation, and macromolecular magnetization transfer. In this work, we examined the effects of combining our previous chemical exchange rotation (CERT) approach with the non-linear AREX method while using different duty cycles (DC) for the label and reference scans. The dependencies of this approach, named AREX, on tissue parameters, including T, T, semi-solid component concentration (f), relayed nuclear Overhauser enhancement (rNOE), and nearby amines, were studied through numerical simulations and control sample experiments at 9.4T and 1μT irradiation. Simulations and experiments show that AREX is sensitive to amide-water exchange effects, but is relatively insensitive to T, T, f, nearby amine, and distant aliphatic protons, while the conventional metric MTR as well as several other APT imaging methods, are significantly affected by at least some of these confounding factors.
Copyright © 2017 Elsevier Inc. All rights reserved.
With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: ()-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against and , pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PURPOSE - To evaluate the accuracy and reproducibility of quantitative chemical shift-encoded (CSE) MRI to quantify proton-density fat-fraction (PDFF) in a fat-water phantom across sites, vendors, field strengths, and protocols.
METHODS - Six sites (Philips, Siemens, and GE Healthcare) participated in this study. A phantom containing multiple vials with various oil/water suspensions (PDFF:0%-100%) was built, shipped to each site, and scanned at 1.5T and 3T using two CSE protocols per field strength. Confounder-corrected PDFF maps were reconstructed using a common algorithm. To assess accuracy, PDFF bias and linear regression with the known PDFF were calculated. To assess reproducibility, measurements were compared across sites, vendors, field strengths, and protocols using analysis of covariance (ANCOVA), Bland-Altman analysis, and the intraclass correlation coefficient (ICC).
RESULTS - PDFF measurements revealed an overall absolute bias (across sites, field strengths, and protocols) of 0.22% (95% confidence interval, 0.07%-0.38%) and R > 0.995 relative to the known PDFF at each site, field strength, and protocol, with a slope between 0.96 and 1.02 and an intercept between -0.56% and 1.13%. ANCOVA did not reveal effects of field strength (P = 0.36) or protocol (P = 0.19). There was a significant effect of vendor (F = 25.13, P = 1.07 × 10 ) with a bias of -0.37% (Philips) and -1.22% (Siemens) relative to GE Healthcare. The overall ICC was 0.999.
CONCLUSION - CSE-based fat quantification is accurate and reproducible across sites, vendors, field strengths, and protocols. Magn Reson Med 77:1516-1524, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
© 2016 International Society for Magnetic Resonance in Medicine.
OBJECTIVES - To determine the added value of amide proton transfer (APT) imaging to conventional and perfusion MRI for differentiating tumour progression (TP) from the treatment-related effect (TE) in patients with post-treatment glioblastomas.
METHODS - Sixty-five consecutive patients with enlarging contrast-enhancing lesions following concurrent chemoradiotherapy were assessed using contrast-enhanced T1-weighted MRI (CE-T1WI), 90th percentile histogram parameters of normalized cerebral blood volume (nCBV90) and APT asymmetry value (APT90). Diagnostic performance was determined using the area under the receiver operating characteristic curve (AUC) and cross validations.
RESULTS - There were statistically significant differences in the mean APT90 between the TP and the TE groups (3.87-4.01 % vs. 1.38-1.41 %; P < .001). Compared with CE-T1WI alone, the addition of APT90 to CE-T1WI significantly improved cross-validated AUC from 0.58-0.74 to 0.89-0.91 for differentiating TP from TE. The combination of CE-T1WI, nCBV90 and APT90 resulted in greater diagnostic accuracy for differentiating TP from TE than the combination of CE-T1WI and nCBV90 (cross-validated AUC, 0.95-0.97 vs. 0.84-0.91). The inter-reader agreement between the expert and trainee was excellent for the measurements of APT90 (intraclass correlation coefficient, 0.94).
CONCLUSION - Adding APT imaging to conventional and perfusion MRI improves the diagnostic performance for differentiating TP from TE.
KEY POINTS - • APT imaging could provide a reliable distinction between TP and TE • Adding APT imaging to CE-T1WI improved the diagnostic accuracy versus CE-T1WI alone • Multimodal imaging using CE-T1WI, perfusion and APT imaging led to accurate diagnosis • The inter-reader agreement of APT histogram parameters was excellent.
The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.
PURPOSE - To correlate and compare diagnostic performance with amide proton transfer (APT) imaging as a tumor proliferation index with that with magnetic resonance (MR) spectroscopy in subgroups of patients with pre- and posttreatment glioma.
MATERIALS AND METHODS - This retrospective study was approved by the institutional review board. In 40 patients with pretreatment glioma and 25 patients with posttreatment glioma, correlation between APT asymmetry and the choline-to-creatine and choline-to-N-acetylaspartate ratios in corresponding voxels of interest was determined, and the 90% histogram cutoff of APT asymmetry values (APT90) for the entire solid portion of gliomas was calculated for diagnostic performance. Area under the receiver operating characteristic curve (AUC), leave-one-out cross validation, and intraclass correlation coefficients were analyzed.
RESULTS - The APT asymmetry values showed a moderate correlation (r = 0.49, P < .001) with the choline-to-creatine ratios and a mild correlation with the choline-to-N-acetyl-aspartate ratios (r = 0.32, P = .011) in the corresponding lesions. The APT90 showed comparable diagnostic accuracy for grading of gliomas (AUC, 0.81-0.84 vs 0.86; P = .582-.864) and superior accuracy for differentiation of tumor progression from treatment-related change (AUC, 0.89-0.90 vs 0.60; P = .031-.046) compared with those with MR spectroscopy. The cross-validated area under the curve and accuracy of the APT90 in posttreatment gliomas were 0.89-0.90 and 72%, respectively. The interreader agreement for APT90 was excellent in both pretreatment and posttreatment gliomas (intraclass correlation coefficient, 0.95 and 0.96, respectively).
CONCLUSION - APT imaging used as a tumor proliferation index showed moderate correlation with MR spectroscopic values and is a superior imaging method to MR spectroscopy, particularly for assessment of posttreatment gliomas.
© RSNA, 2015.