Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

Improved Resting-State Functional MRI Using Multi-Echo Echo-Planar Imaging on a Compact 3T MRI Scanner with High-Performance Gradients.

In blood-oxygen-level-dependent (BOLD)-based resting-state functional (RS-fMRI) studies, usage of multi-echo echo-planar-imaging (ME-EPI) is limited due to unacceptable late echo times when high spatial resolution is used. Equipped with high-performance gradients, the compact 3T MRI system (C3T) enables a three-echo whole-brain ME-EPI protocol with smaller than 2.5 mm isotropic voxel and shorter than 1 s repetition time, as required in landmark fMRI studies. The performance of the ME-EPI was comprehensively evaluated with signal variance reduction and region-of-interest-, seed- and independent-component-analysis-based functional connectivity analyses and compared with a counterpart of single-echo EPI with the shortest TR possible. Through the multi-echo combination, the thermal noise level is reduced. Functional connectivity, as well as signal intensity, are recovered in the medial orbital sulcus and anterior transverse collateral sulcus in ME-EPI. It is demonstrated that ME-EPI provides superior sensitivity and accuracy for detecting functional connectivity and/or brain networks in comparison with single-echo EPI. In conclusion, the high-performance gradient enabled high-spatial-temporal resolution ME-EPI would be the method of choice for RS-fMRI study on the C3T.

Improved Brain MR Imaging from a Compact, Lightweight 3T Scanner with High-Performance Gradients.

BACKGROUND: A low-cryogen, compact 3T (C3T) MRI scanner with high-performance gradients capable of simultaneously achieving 80 mT/m gradient amplitude and 700 T/m/second slew rate has been in use to study research patients since March 2016 but has not been implemented in the clinical practice. PURPOSE: To compare head MRI examinations obtained with the C3T system and a conventional whole-body 3T (WB3T) scanner in seven parameters across five commonly used brain imaging sequences. STUDY TYPE: Prospective. SUBJECTS: Thirty patients with a clinically indicated head MRI. SEQUENCE: 3T; T1 FLAIR, T1 MP-RAGE, 3D T2 FLAIR, T2 FSE, and DWI. ASSESSMENT: All patients tolerated the scans well. Three board-certified neuroradiologists scored the comparative quality of C3T and WB3T images in blinded fashion using a five-point Likert scale in terms of: signal-to-noise ratio, lesion conspicuity, motion artifact, gray/white matter contrast, cerebellar folia, susceptibility artifact, and overall quality. STATISTICAL TEST: Left-sided, right-sided, and two-sided Wilcoxon signed rank test; Fisher's method. A P value <0.05 was considered statistically significant. RESULTS: The C3T system performed better than the WB3T in virtually all comparisons, except for motion artifacts for the T1 FLAIR and T1 MP-RAGE sequences, where the WB3T system was deemed better. When combining all sequences together, the C3T system outperformed the WB3T system in all image quality parameters evaluated, except for motion artifact (P = 0.13). DATA CONCLUSION: The C3T scanner provided better overall image quality for all sequences, and performed better in all individual categories, except for motion artifact on the T1 FLAIR and T1 MP-RAGE. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 1.

Application of Adaptive Image Receive Coil Technology for Whole-Brain Imaging.

OBJECTIVE. The Adaptive Image Receive (AIR) radiofrequency coil is an emergent technology that is lightweight and flexible and exhibits electrical characteristics that overcome many of the limitations of traditional rigid coil designs. The purpose of this study was to apply the AIR coil for whole-brain imaging and compare the performance of a prototype AIR coil array with the performance of conventional head coils. SUBJECTS AND METHODS. A phantom and 15 healthy adult participants were imaged. A prototype 16-channel head AIR coil was compared with conventional 8-and 32-channel head coils using clinically available MRI sequences. During consensus review, two board-certified neuroradiologists graded the AIR coil compared with an 8-channel coil and a 32-channel coil on a 5-point ordinal scale in multiple categories. One- and two-sided Wilcoxon signed rank tests were performed. Noise covariance matrices and geometry factor (g-factor) maps were calculated. RESULTS. The signal-to-noise ratio, structural sharpness, and overall image quality scores of the prototype 16-channel AIR coil were better than those of the 8-channel coil but were not as good as those of the 32-channel coil. Noise covariance matrices showed stable performance of the AIR coil across participants. The median g-factors for the 16-channel AIR coil were, overall, less than those of the 8-channel coil but were greater than those of the 32-channel coil. CONCLUSION. On average, the prototype 16-channel head AIR coil outperformed a conventional 8-channel head coil but did not perform as well as a conventional 32-channel head coil. This study shows the feasibility of the novel AIR coil technology for imaging the brain and provides insight for future coil design improvements.

Disruption of GpI mGluR-Dependent Cav2.3 Translation in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is an inherited intellectual impairment that results from the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates mRNA translation at synapses. The absence of FMRP leads to neuronal and circuit-level hyperexcitability that is thought to arise from the aberrant expression and activity of voltage-gated ion channels, although the identification and characterization of these ion channels have been limited. Here, we show that FMRP binds the mRNA of the R-type voltage-gated calcium channel Cav2.3 in mouse brain synaptoneurosomes and represses Cav2.3 translation under basal conditions. Consequently, in hippocampal neurons from male and female FMRP KO mice, we find enhanced Cav2.3 protein expression by western blotting and abnormally large R currents in whole-cell voltage-clamp recordings. In agreement with previous studies showing that FMRP couples Group I metabotropic glutamate receptor (GpI mGluR) signaling to protein translation, we find that GpI mGluR stimulation results in increased Cav2.3 translation and R current in hippocampal neurons which is disrupted in FMRP KO mice. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and in response to GpI mGluR stimulation. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies.SIGNIFICANCE STATEMENT Patients with fragile X syndrome (FXS) exhibit signs of neuronal and circuit hyperexcitability, including anxiety and hyperactive behavior, attention deficit disorder, and seizures. FXS is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein, and the neuronal hyperexcitability observed in the absence of FMRP likely results from its ability to regulate the expression and activity of voltage-gated ion channels. Here we find that FMRP serves as a key translational regulator of the voltage-gated calcium channel Cav2.3 under basal conditions and following activity. Cav2.3 impacts cellular excitability and calcium signaling, and the alterations in channel translation and expression observed in the absence of FMRP could contribute to the neuronal hyperactivity that underlies FXS.

The effect of spiral trajectory correction on pseudo-continuous arterial spin labeling with high-performance gradients on a compact 3T scanner.

PURPOSE: To demonstrate the feasibility of pseudo-continuous arterial-spin-labeled (pCASL) imaging with 3D fast-spin-echo stack-of-spirals on a compact 3T scanner (C3T), to perform trajectory correction for eddy-current-induced deviations in the spiral readout of pCASL imaging, and to assess the correction effect on perfusion-related images with high-performance gradients (80 mT/m, 700T/m/s) of the C3T. METHODS: To track eddy-current-induced artifacts with Archimedean spiral readout, the spiral readout in pCASL imaging was performed with 5 different peak gradient slew rate (Smax ) values ranging from 70 to 500 T/m/s. The trajectory for each Smax was measured using a dynamic field camera and applied in a density-compensated gridding image reconstruction in addition to the nominal trajectory. The effect of the trajectory correction was assessed with perfusion-weighted (ΔM) images and proton-density-weighted images as well as cerebral blood flow (CBF) maps, obtained from 10 healthy volunteers. RESULTS: Blurring artifact on ΔM images was mitigated by the trajectory correction. CBF values on the left and right calcarine cortices showed no significant difference after correction. Also, the signal-to-noise ratio of ΔM images improved, on average, by 7.6% after correction (P < .001). The greatest improvement of 12.1% on ΔM images was achieved with a spiral readout using Smax of 300~400 T/m/s. CONCLUSION: Eddy currents can cause spiral trajectory deviation, which leads to deformation of the CBF map even in cases of low value Smax . The trajectory correction for spiral-readout-based pCASL produces more reliable results for perfusion imaging. These results suggest that pCASL is feasible on C3T with high-performance gradients.

Distortion-free imaging: A double encoding method (DIADEM) combined with multiband imaging for rapid distortion-free high-resolution diffusion imaging on a compact 3T with high-performance gradients.

BACKGROUND: Distortion-free, high-resolution diffusion imaging using DIADEM (Distortion-free Imaging: A Double Encoding Method), proposed recently, has great potential for clinical applications. However, it can suffer from prolonged scan times and its reliability for quantitative diffusion imaging has not been evaluated. PURPOSE: To investigate the clinical feasibility of DIADEM-based high-resolution diffusion imaging on a novel compact 3T (C3T) by evaluating the reliability of quantitative diffusion measurements and utilizing both the high-performance gradients (80 mT/m, 700 T/m/s) and the sequence optimization with the navigator acquisition window reduction and simultaneous multislice (multiband) imaging. STUDY TYPE: Prospective feasibility study. PHANTOM/SUBJECTS: Diffusion quality control phantom scans to evaluate the reliability of quantitative diffusion measurements; 36 normal control scans for B0 -field mapping; six healthy and two patient subject scans with a brain tumor for comparisons of diffusion and anatomical imaging. FIELD STRENGTH/SEQUENCE: 3T; the standard single-shot echo-planar-imaging (EPI), multishot DIADEM diffusion, and anatomical (2D-FSE [fast-spin-echo], 2D-FLAIR [fluid-attenuated-inversion-recovery], and 3D-MPRAGE [magnetization prepared rapid acquisition gradient echo]) imaging. ASSESSMENT: The scan time reduction, the reliability of quantitative diffusion measurements, and the clinical efficacy for high-resolution diffusion imaging in healthy control and brain tumor volunteers. STATISTICAL TEST: Bland-Altman analysis. RESULTS: The scan time for high in-plane (0.86 mm2 ) resolution, distortion-free, and whole brain diffusion imaging were reduced from 10 to 5 minutes with the sequence optimizations. All of the mean apparent diffusion coefficient (ADC) values in phantom were within the 95% confidence interval in the Bland-Altman plot. The proposed acquisition with a total off-resonance coverage of 597.2 Hz wider than the expected bandwidth of 500 Hz in human brain could yield a distortion-free image without foldover artifacts. Compared with EPI, therefore, this approach allowed direct image matching with the anatomical images and enabled improved delineation of the tumor boundaries. DATA CONCLUSION: The proposed high-resolution diffusion imaging approach is clinically feasible on C3T due to a combination of hardware and sequence improvements. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 1 J. Magn. Reson. Imaging 2020;51:296-310.

The effect of concomitant fields in fast spin echo acquisition on asymmetric MRI gradient systems.

PURPOSE: To investigate the effect of the asymmetric gradient concomitant fields (CF) with zeroth and first-order spatial dependence on fast/turbo spin-echo acquisitions, and to demonstrate the effectiveness of their real-time compensation. METHODS: After briefly reviewing the CF produced by asymmetric gradients, the effects of the additional zeroth and first-order CFs on these systems are investigated using extended-phase graph simulations. Phantom and in vivo experiments are performed to corroborate the simulation. Experiments are performed before and after the real-time compensations using frequency tracking and gradient pre-emphasis to demonstrate their effectiveness in correcting the additional CFs. The interaction between the CFs and prescan-based correction to compensate for eddy currents is also investigated. RESULTS: It is demonstrated that, unlike the second-order CFs on conventional gradients, the additional zeroth/first-order CFs on asymmetric gradients cause substantial signal loss and dark banding in fast spin-echo acquisitions within a typical brain-scan field of view. They can confound the prescan correction for eddy currents and degrade image quality. Performing real-time compensation successfully eliminates the artifacts. CONCLUSIONS: We demonstrate that the zeroth/first-order CFs specific to asymmetric gradients can cause substantial artifacts, including signal loss and dark bands for brain imaging. These effects can be corrected using real-time compensation. Magn Reson Med 79:1354-1364, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Lightweight, compact, and high-performance 3T MR system for imaging the brain and extremities.

PURPOSE: To build and evaluate a small-footprint, lightweight, high-performance 3T MRI scanner for advanced brain imaging with image quality that is equal to or better than conventional whole-body clinical 3T MRI scanners, while achieving substantial reductions in installation costs. METHODS: A conduction-cooled magnet was developed that uses less than 12 liters of liquid helium in a gas-charged sealed system, and standard NbTi wire, and weighs approximately 2000 kg. A 42-cm inner-diameter gradient coil with asymmetric transverse axes was developed to provide patient access for head and extremity exams, while minimizing magnet-gradient interactions that adversely affect image quality. The gradient coil was designed to achieve simultaneous operation of 80-mT/m peak gradient amplitude at a slew rate of 700 T/m/s on each gradient axis using readily available 1-MVA gradient drivers. RESULTS: In a comparison of anatomical imaging in 16 patients using T2 -weighted 3D fluid-attenuated inversion recovery (FLAIR) between the compact 3T and whole-body 3T, image quality was assessed as equivalent to or better across several metrics. The ability to fully use a high slew rate of 700 T/m/s simultaneously with 80-mT/m maximum gradient amplitude resulted in improvements in image quality across EPI, DWI, and anatomical imaging of the brain. CONCLUSIONS: The compact 3T MRI system has been in continuous operation at the Mayo Clinic since March 2016. To date, over 200 patient studies have been completed, including 96 comparison studies with a clinical 3T whole-body MRI. The increased gradient performance has reliably resulted in consistently improved image quality.

Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia.

Diverse molecules induce general anesthesia with potency strongly correlated with both their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma-membrane-derived vesicles by lowering the critical temperature (Tc) for phase separation. Here, we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First, we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol-induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described "intoxication reversers" raise Tc and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that elevated hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

Nucleophilic (Radio)Fluorination of α-Diazocarbonyl Compounds Enabled by Copper-Catalyzed H-F Insertion.

The copper-catalyzed H-F insertion into α-diazocarbonyl compounds is described using potassium fluoride (KF) and hexafluoroisopropanol. Access to complex α-fluorocarbonyl derivatives is achieved under mild conditions, and the method is readily adapted to radiofluorination with [(18)F]KF. This late-stage strategy provides an attractive route to (18)F-labeled biomolecules.

Partial fourier and parallel MR image reconstruction with integrated gradient nonlinearity correction.

PURPOSE: To describe how integrated gradient nonlinearity (GNL) correction can be used within noniterative partial Fourier (homodyne) and parallel (SENSE and GRAPPA) MR image reconstruction strategies, and demonstrate that performing GNL correction during, rather than after, these routines mitigates the image blurring and resolution loss caused by postreconstruction image domain based GNL correction. METHODS: Starting from partial Fourier and parallel magnetic resonance imaging signal models that explicitly account for GNL, noniterative image reconstruction strategies for each accelerated acquisition technique are derived under the same core mathematical assumptions as their standard counterparts. A series of phantom and in vivo experiments on retrospectively undersampled data were performed to investigate the spatial resolution benefit of integrated GNL correction over conventional postreconstruction correction. RESULTS: Phantom and in vivo results demonstrate that the integrated GNL correction reduces the image blurring introduced by the conventional GNL correction, while still correcting GNL-induced coarse-scale geometrical distortion. Images generated from undersampled data using the proposed integrated GNL strategies offer superior depiction of fine image detail, for example, phantom resolution inserts and anatomical tissue boundaries. CONCLUSION: Noniterative partial Fourier and parallel imaging reconstruction methods with integrated GNL correction reduce the resolution loss that occurs during conventional postreconstruction GNL correction while preserving the computational efficiency of standard reconstruction techniques. Magn Reson Med 75:2534-2544, 2016. © 2015 Wiley Periodicals, Inc.

Finding safety: a pilot study of managed alcohol program participants' perceptions of housing and quality of life.

BACKGROUND: There is a higher prevalence of alcohol use and severe alcohol dependence among homeless populations. The combination of alcohol use and lack of housing contributes to increased vulnerability to the harms of substance use including stigma, injury, illness, and death. Managed alcohol programs (MAPs) administer prescribed doses of alcohol at regular intervals to people with severe and chronic alcohol dependence and homelessness. As a pilot for a larger national study of MAPs, we conducted an in-depth evaluation of one program in Ontario, Canada. In this paper, we report on housing and quality of life outcomes and experiences of the MAP participants and staff. METHODS: We conducted a pilot study using mixed methods. The sample consisted of 38 people enrolled in or eligible for entry into a MAP who completed a structured quantitative survey that included measures related to their housing and quality of life. All of the participants self-identified as Indigenous. In addition, we conducted 11 in-depth qualitative interviews with seven MAP residents and four program staff and analyzed the interviews using constant comparative analysis. The qualitative analysis was informed by Rhodes' risk environment framework. RESULTS: When compared to controls, MAP participants were more likely to retain their housing and experienced increased safety and improved quality of life compared to life on the streets, in jails, shelters, or hospitals. They described the MAP as a safe place characterized by caring, respect, trust and a nonjudgmental approach with a sense of family and home as well as opportunities to reconnect with family members. CONCLUSIONS: The MAP was, as described by participants, a safer environment and a home with feelings of family and a sense of community that countered stigma, loss, and dislocation with potential for healing and recovery. The MAP environment characterized by caring, respect, trust, a sense of home, "feeling like family", and the opportunities for family and cultural reconnections is consistent with First Nations principles for healing and recovery and principles of harm reduction.

Do managed alcohol programs change patterns of alcohol consumption and reduce related harm? A pilot study.

BACKGROUND: Managed alcohol programs (MAPs) are a harm reduction strategy for people with severe alcohol dependence and unstable housing. MAPs provide controlled access to alcohol usually alongside accommodation, meals, and other supports. Patterns of alcohol consumption and related harms among MAP participants and controls from a homeless shelter in Thunder Bay, Ontario, were investigated in 2013. METHODS: Structured interviews were conducted with 18 MAP and 20 control participants assessed as alcohol dependent with most using non-beverage alcohol (NBA). Qualitative interviews were conducted with seven participants and four MAP staff concerning perceptions and experiences of the program. Program alcohol consumption records were obtained for MAP participants, and records of police contacts and use of health services were obtained for participants and controls. Some participants' liver function test (LFT) results were available for before and after MAP entry. RESULTS: Compared with periods off the MAP, MAP participants had 41 % fewer police contacts, 33 % fewer police contacts leading to custody time (x (2) = 43.84, P < 0.001), 87 % fewer detox admissions (t = -1.68, P = 0.06), and 32 % fewer hospital admissions (t = -2.08, P = 0.03). MAP and control participants shared similar characteristics, indicating the groups were broadly comparable. There were reductions in nearly all available LFT scores after MAP entry. Compared with controls, MAP participants had 43 % fewer police contacts, significantly fewer police contacts (-38 %) that resulted in custody time (x (2) = 66.10, P < 0.001), 70 % fewer detox admissions (t = -2.19, P = 0.02), and 47 % fewer emergency room presentations. NBA use was significantly less frequent for MAP participants versus controls (t = -2.34, P < 0.05). Marked but non-significant reductions were observed in the number of participants self-reporting alcohol-related harms in the domains of home life, legal issues, and withdrawal seizures. Qualitative interviews with staff and MAP participants provided additional insight into reductions of non-beverage alcohol use and reductions of police and health-care contacts. It was unclear if overall volume of alcohol consumption was reduced as a result of MAP participation. CONCLUSIONS: The quantitative and qualitative findings of this pilot study suggest that MAP participation was associated with a number of positive outcomes including fewer hospital admissions, detox episodes, and police contacts leading to custody, reduced NBA consumption, and decreases in some alcohol-related harms. These encouraging trends are being investigated in a larger national study.

Inhibitory interactions between phosphorylation sites in the C terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor GluA1 subunits.

The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca(2+) sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca(2+) influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function.

Oxygen depletion speeds and simplifies diffusion in HeLa cells.

Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

Roles of brca2 (fancd1) in oocyte nuclear architecture, gametogenesis, gonad tumors, and genome stability in zebrafish.

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.

FISH-Flow to quantify nascent and mature ribosomal RNA in mouse and human cells.

FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis. For complete details on the use and execution of this protocol, please refer to Antony et al. (2022).1.

Enhanced clinical task-based fMRI metrics through locally low-rank denoising of complex-valued data.

OBJECTIVE: This study investigates a locally low-rank (LLR) denoising algorithm applied to source images from a clinical task-based functional MRI (fMRI) exam before post-processing for improving statistical confidence of task-based activation maps. METHODS: Task-based motor and language fMRI was obtained in eleven healthy volunteers under an IRB approved protocol. LLR denoising was then applied to raw complex-valued image data before fMRI processing. Activation maps generated from conventional non-denoised (control) data were compared with maps derived from LLR-denoised image data. Four board-certified neuroradiologists completed consensus assessment of activation maps; region-specific and aggregate motor and language consensus thresholds were then compared with nonparametric statistical tests. Additional evaluation included retrospective truncation of exam data without and with LLR denoising; a ROI-based analysis tracked t-statistics and temporal SNR (tSNR) as scan durations decreased. A test-retest assessment was performed; retest data were matched with initial test data and compared for one subject. RESULTS: fMRI activation maps generated from LLR-denoised data predominantly exhibited statistically significant (p = 4.88×10-4 to p = 0.042; one p = 0.062) increases in consensus t-statistic thresholds for motor and language activation maps. Following data truncation, LLR data showed task-specific increases in t-statistics and tSNR respectively exceeding 20 and 50% compared to control. LLR denoising enabled truncation of exam durations while preserving cluster volumes at fixed thresholds. Test-retest showed variable activation with LLR data thresholded higher in matching initial test data. CONCLUSION: LLR denoising affords robust increases in t-statistics on fMRI activation maps compared to routine processing, and offers potential for reduced scan duration while preserving map quality.

Evaluation of a compact 3 T MRI scanner for patients with implanted devices.

Access to high-quality MR exams is severely limited for patients with some implanted devices due to labeled MR safety conditions, but small-bore systems can overcome this limitation. For example, a compact 3 T MR scanner (C3T) with high-performance gradients can acquire exams of the head, extremities, and infants. Because of its reduced bore size and the patient being advanced only partially into the bore, the associated electromagnetic (EM) fields drop off rapidly caudal to the head, compared to whole-body systems. Therefore, some patients with MR conditional implanted devices can safely receive 3 T brain exams on the C3T using its strong gradients and a multiple-channel receive coil, while a corresponding exam on whole-body MR is precluded. The purpose of this study is to evaluate the performance of a small-bore scanner for subjects with MR conditional spinal or sacral nerve stimulators, or abandoned cardiac implantable electronic device (CIED) leads. The spatial dependence of specific absorption rate (SAR) on the C3T was compared to whole-body scanners. A device assessment tool was developed and applied to evaluate MR safety individually on the C3T for 12 subjects with implanted devices or abandoned CIED leads. Once MR safety was established, the subjects received a C3T brain exam along with their clinical, 1.5 T exam. The resulting images were graded by three board-certified neuroradiologists. The C3T exams were well-tolerated with no adverse events, and significantly outperformed the whole-body 1.5 T exams in terms of overall image quality.