Single Gating Research Articles

Extended phase-correlated rescanning irradiation to improve dose homogeneity in carbon-ion beam liver treatment

We previously reported that an adequate number of simple layered phase-correlated rescanning (PCR) irradiations improve dose conformation to a moving target. However, if PCR is not completed within a single gating window due to the particular irradiation specifications selected in advance and/or layer size, the iso-energy layer is completed using next gating window, and the effect of rescanning is effectively nullified. To solve this problem, we developed extended PCR irradiation.Extended PCR is conceptually based on simple PCR. It differs in that the dose rate is adjusted to irradiate the number of rescans within multiple gating windows when simple PCR is not finished within a single gating window. This process is repeated until the total prescribed dose is given. Here, 4DCT imaging was performed under free-breathing conditions in 10 patients with hepatocellular carcinoma. 4D dose distributions for a single field approach were performed with simple PCR and extended PCR using 4DCT. A total dose of 11.25 Gy(RBE) was administered to the field-specific target volume. The number of rescans was changed from 1 to 10. Respiratory gating of a 30% duty cycle around exhalation was applied.Dose assessment metrics (CTV-D95, CTV-Dmin, CTV-Dmax) with PCR improved with an increasing number of rescans, and largely plateaued at five or more rescans. Minimum D95 and Dmin values, and maximum Dmax and HI values fluctuated at odd and even numbers of rescans. Extended PCR improved all dose assessment metrics whereas PCR did not. Values with extended PCR were mostly constant with three or more rescans, or in other words independent of the number of rescans.Extended PCR improved dose conformation to a moving target with greater efficacy than simple PCR when more than two gating windows were necessary. Extended PCR can be applied to irradiation machines having a low maximum dose rate.

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated

Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several of these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by the transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20–31% of wild type levels. After the inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition).

Synaptic vesicle tethering and the CaV2.2 distal C-terminal

Evidence that synaptic vesicles (SVs) can be gated by a single voltage sensitive calcium channel (CaV2.2) predict a molecular linking mechanism or “tether” (Stanley, 1993). Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel (Kaeser et al., 2011; Wong et al., 2013) while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site (Kaeser et al., 2011). Using a novel in vitro SV pull down binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137–2357; Wong et al., 2013). Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137–2299). To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349–2357) and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or “grabbed,” from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be “locked” close to the channel by a second attachment mechanism in preparation for single channel domain gating.

Dynamic inversion time for improved 3D late gadolinium enhancement imaging in patients with atrial fibrillation

High resolution three-dimensional (3D) late gadolinium enhancement (LGE) imaging is performed with single R-wave gating to minimize lengthy acquisition durations. In patients with atrial fibrillation (AF), heart rate variability results in variable magnetization recovery between sequence repeats, and image quality is often poor. In this study, we implemented and tested a dynamic inversion time (dynamic-TI) scheme designed to reduce sequence sensitivity to heart rate variations. An inversion-prepared 3D segmented gradient echo sequence was modified so that the TI varied automatically from beat-to-beat (dynamic-TI) based on the time since the last sequence repeat. 3D LGE acquisitions were performed in 17 patients prior to radio frequency ablation of persistent AF both with and without dynamic-TI. Qualitative image quality scores, blood signal-to-ghosting ratios (SGRs). and blood-myocardium contrast-to-ghosting ratios (CGRs) were compared. Image quality scores were higher with dynamic-TI than without dynamic-TI (2.2 ± 0.9 vs. 1.8 ± 1.1, P = 0.008), as were blood-myocardium CGRs (13.8 ± 7.6 vs. 8.3 ± 6.1, P = 0.003) and blood SGRs (19.6 ± 8.5 vs. 13.1 ± 8.0, P = 0.003). The dynamic-TI algorithm improves image quality of 3D LGE imaging in this difficult patient population by reducing the sequence sensitivity to RR interval variations

GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABAAR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABAARs consistently deactivating ∼10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABAARs was due to longer burst durations at low GABA concentrations, corresponding to a ∼4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABAARs containing γ1 and γ2 use different mechanisms for synaptic clustering.

Improved 3D late gadolinium enhancement (LGE) imaging with dynamic-TI in patients with persistent atrial fibrillation

Background High resolution 3D late gadolinium enhancement (LGE) imaging prior to RF ablation of atrial fibrillation (AF) is performed with single R-wave gating to reduce lengthy acquisition times [1]. However, this increases the sequence sensitivity to RR interval variations and missed cardiac triggers which cause variable magnetization recovery between sequence repeats, leading to ghosting of blood pool and unsuppressed fat, together with poor myocardial nulling. 3D LGE image quality in AF is consequently often poor. We have developed a dynamic inversion recovery (dynamic-TI) 3D LGE sequence which minimises variations in the longitudinal magnetisation (Mz) of myocardium throughout the acquisition [2] and have performed a study to assess its efficacy in 17 patients in persistent AF.

Single polymer gating of channels under a solvent gradient

We study the effect of a gradient of solvent quality on the coil-globule transition for a polymer in a narrow pore. A simple self-attracting, self-avoiding walk model of a polymer in solution shows that the variation in the strength of the interaction across the pore leads the system to go from one regime (good solvent) to the other (poor solvent) across the channel. This may be thought to be analogous to thermophoresis, where the polymer goes from the hot region to the cold region under the temperature gradient. The behavior of short chains is studied using exact enumeration while the behavior of long chains is studied using transfer matrix techniques. The distribution of the monomer density across the layer suggests that a gatelike effect can be created, with potential applications as a sensor.

Modeling transition diffusive–nondiffusive transport in a turbid media and application to time-resolved reflectance

In this work a generalized solution for the photon density, Φgen(r,t), is applied to two types of experiments in turbid media carried out in the last years. Both involve small typical distances, where it is known that the diffusion approximation ceases to be valid. In one case, the use of time-resolved reflectance at small or null source-detector separation using fast single photon gating to localize small inhomogeneities embedded in diffusive media has been proposed. In other type of experiments, it is addressed the transition between the ballistic and the diffusive regimes, measuring the transmitted light within a relatively narrow solid angle. The model proposed here corroborates the importance of the solid angle of the measurement device in order to see the ballistic photons, and the given generalized solution provides valid answers to problems posed by the mentioned experiments. Furthermore, it permits the description of diffusive photons when the absorption coefficient is relatively high, where the diffusion approximation is not valid.

The Nature of Ca2+-Controlled Gating in the Human Cardiac Ryanodine Receptor (hRyR2): A Zero Ca2+ Approach

RyR2-mediated intracellular Ca2+ release is pivotal in maintaining the rhythmic contraction of cardiac myocytes. The precise mechanism by which binding of cytosolic Ca2+ triggers gating is unknown. Functional studies of single RyR2s in lipid bilayers provide insights into gating behaviour as detailed structural information regarding the nature of the Ca2+-dependent gate or Ca2+ binding site(s) is as yet unavailable.This study aims to uncover the properties of the Ca2+-controlled ‘gate' by examining changes in single channel gating in the virtual absence of Ca2+. To visualize these changes, ryanodine was employed to keep the channel open in a subconductance state in the presence and absence of Ca2+, while K+ was used as the permeant ion for better resolution of single channel currents. On reducing the Ca2+ from contaminant levels to nominally zero on both cytosolic and luminal sides of the ryanodine-modified hRyR2 (using 3.5mM EGTA), a significant decrease in channel Po is seen at positive (current direction: cytosolic-luminal) holding potentials but not in the physiological direction of current flow (0.99±0.002 to 0.75±0.05 at +40mV (p<0.0005) and 0.99 to 0.98±0.009 at −40mV; n=9). Analysis of dwell times demonstrates that Po decreases by increasing the frequency of closings (∼170 fold increase at +40mV, ∼8-fold at −40mV). Further examination of the voltage sensitivity of the modified channel reveals a sharp dependence of activity on potential. High cytosolic Ba2+ had a direct inhibitory effect on the gating kinetics induced by the removal of Ca2+. This study demonstrates that ryanodine modifies RyR2 by acting on a Ca2+-controlled gate and gating of the modified channel is voltage-sensitive. HMM-based modeling of this data provides further insights into this fundamental process of Ca2+-induced channel opening. This study is supported by the BHF.

Dynamic TI for late gadolinium enhancement imaging in atrial fibrillation

Background There has been considerable interest in high resolution 3D late gadolinium enhancement (LGE) imaging in the atrial fibrillation (AF) population, both pre and post RF ablation. Unlike conventional studies, these are performed with single R-wave gating to reduce the acquisition duration and the effects of gadolinium wash-out. For AF, the heart rate variability results in variable magnetization recovery between sequence repeats which, with a fixed inversion time (TI), causes ghosting and poor nulling of normal tissue. This is exacerbated by missed triggers and the number of poor quality studies is high. An adaptive inversion recovery preparation has previously been demonstrated in a 2D phantom acquisition [1]. The purpose of this study is to implement such a technique (dynamic_TI) for improved imaging in the AF population.

Light emitting diodes: Light engines to simplify and economize advanced microscopy

THE old adage, ‘‘A picture is worth a thousand words,’’ is especially true in biological research, with microscopic images serving to mark the development of key concepts in biology since Hooke first described ‘‘cells’’ in thin slices of cork (1). More than 400 years later, microscopic imaging still serves as a primary data source for biological research, with increasingly advanced techniques that provide information on molecular scale interactions and dynamics. Such advanced molecular imaging techniques take advantage of ever more sophisticated light sources, optics, detectors, and image processing methods, many of which can be quite complex and expensive. At the same time, new technologies and production methods, many driven by the consumer market, have resulted in key components of imaging systems becoming smaller, simpler, and cheaper. The convergence of these effects is having profound effects on our options for light sources for microscopic imaging. The properties of the illumination source are critical to many advanced optical microscopy methods. Lasers play a key role in many of these owing to the monochromatic and coherent nature of their light, plus their ability to be pulsed and otherwise controlled. However, illumination with high-pressure mercury gas discharge or xenon lamps is still the standard for most imaging applications. These light sources provide bright, broad-spectrum excitation (300–900 nm) that can be easily selected by excitation filters for fluorescent imaging applications. However, there are quite a few disadvantages that limit the wide-spread of mercury or xenon lamps-based microscopy system in biological applications. First, sometimes these traditional light sources are too bright and harmful for the living organisms being studied. Excessive brightness can cause rapid photo-bleaching of the dyes used to stain the cells/ tissues. UV light especially, if not blocked, can have significant deleterious effects on live cells. Second, there are substantial costs involved in using mercury or xenon lamps because of their relatively short lifespan. The life of a mercury lamp is usually several hundred hours, and the intensity of these lamps decays progressively during this time. In addition, gas discharge lamps require some time to warm up before lamp intensity reaches steady state. Therefore, once mercury lamps are turned on, they have to be left on for hours to enable fluorescent measurements as needed without delay, thereby further shortening the lamp lifetime. Third, there are also some hazards issues of using mercury or xenon lamps. These lamps generate a significant amount of heat and therefore introduce complications when used in a confined space. When mercury lamps die, they can explode and damage the lenses or mirrors within the lamp housing. These factors combine to make gas discharge lamps inconvenient for lab-based applications and impractical for field applications, including use in portable, miniature, or low cost imaging devices. By contrast, light-emitting diodes (LEDs) show great pro

2SH-01 KcsAカリウムイオンチャネルの溶液条件変化応答1分子開閉ダイナミクスの解析(2SH 先端顕徴計測が照らす生命の輝き,シンポジウム,日本生物物理学会第50回年会(2012年度))

2SH-01 KcsAカリウムイオンチャネルの溶液条件変化応答1分子開閉ダイナミクスの解析(2SH 先端顕徴計測が照らす生命の輝き,シンポジウム,日本生物物理学会第50回年会(2012年度))

Gating Properties and Voltage Sensing in Kv1.2

Kv1.2 is a voltage gated potassium channel whose crystal structure has been solved. There is markedly little electrophysiological information about it, which limits the vast potential usefulness of the structure. In an effort to deepen our functional understanding of the Kv1.2 channel, we set out to characterize its properties through recordings of macroscopic and single ionic and gating currents and develop models for activation based on established models for other Shaker channels. Preliminary data indicate that even though Shaker and Kv1.2 are closely related, subtle differences exist in their mechanisms of voltage sensing. As an example of these differences, limiting slope analysis reveals that the apparent charge coupled to gating of Kv1.2 is only 10 e0 compared to Shaker's 13 e0. Also, substitution of Kv1.2's tryptophan at position 366, the analogue of W434 in Shaker, for the other two aromatic residues does not abolish conduction as in Shaker. Our macroscopic data are best fitted by a 32 state model involving three independent transitions for each of the subunits and two final, voltage independent, concerted transitions to the open state.These findings make apparent the need for more detailed electrophysiological analysis of Kv1.2 and will certainly prove useful to clarify the structure-function relationship of voltage gated potassium channels.

Laser-Triggered Single Molecular Gating Motions of the KcsA Potassium Channels Recorded in a Sub-Millisecond Time Resolution

We previously revealed that the KcsA potassium channel exhibited global twisting motions upon gating (Shimizu, H., et al Cell 132, 67-78, 2008). The method we used was the diffracted X-ray tracking (DXT), in which the conformational changes were recorded under equilibrium conditions at video rate. To trace the whole picture of the gating motions, further development of the method is required. We recently have succeeded in developing a laser-triggered measurement system, while the X-ray spectrum was optimized for fast recordings with a high-speed camera. The channel was fixed on a glass plate at the extracellular loop and a gold nanocrystal was attached to the C-terminal end of the channel molecule as a probe. The sample was irradiated with an optimized white-beam from a synchrotron, and the motions of the Laue diffraction spots from the nanocrystal were tracked on a CMOS detector at the speed of 5000 frames/s. During the measurements the pH of the solution was changed from neutral to acidic by laser-photolysis of the caged proton, which triggers the gating motions of the pH-gated KcsA channel. Trajectories of the diffraction spots on the detector plane were translated as movements of the channel in the real space. This modified DXT method now enables us to detect the initial opening processes of the conformational changes in the time resolution comparable to that of the single channel current recordings.

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

Synaptic AMPA receptors are greatly influenced by a family of transmembrane AMPA receptor regulatory proteins (TARPs) which control trafficking, channel gating and pharmacology. The prototypical TARP, stargazin (or γ2), shifts the blocking ability of several AMPAR-selective compounds including the commonly used quinoxalinedione antagonists, CNQX and NBQX. Stargazin's effect on CNQX is particularly intriguing as it not only apparently lowers the potency of block, as with NBQX, but also renders it a partial agonist. Given this, agonist behaviour by CNQX has been speculated to account for its weaker blocking effect on AMPAR-TARP complexes. Here we show that this is not the case. The apparent effect of stargazin on CNQX antagonism can be almost entirely explained by an increase in the apparent affinity for l-glutamate (l-Glu), a full agonist and neurotransmitter at AMPAR synapses. Partial agonism at best plays a minor role but not through channel gating per se but rather because CNQX elicits AMPAR desensitization. Our study reveals that CNQX is best thought of as a non-competitive antagonist at glutamatergic synapses due to the predominance of non-equilibrium conditions. Consequently, CNQX primarily reports the proportion of AMPARs available for activation but may also impose additional block by receptor desensitization.

ISHAGE protocol: Are we doing it correctly?

Flow cytometric CD34(+) stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD34(+) stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance. In send out 0801, where two stabilized samples were issued, the EQA center surveyed 255 participants with flow analysis data and subsequent results collected. One hundred and ninety-six laboratories returned results with 103 returning dot plots. Eighty-three out of one hundred and three stated that they used the ISHAGE protocol gating strategy but 43% (36/83) were incorrectly set-up. Analysis of the data showed those incorrectly using single platform ISHAGE gating strategy were twice as likely to fail an EQA exercise compared to those using the protocol correctly. This failure rate increased two fold when incorrect ISHAGE protocol was used in a dual platform setting. This study suggests a widespread fundamental lack of understanding of the ISHAGE protocol and the need to deploy it correctly, potentially having significant clinical implications and highlights the need to monitor participants rigorously in their deployment of the ISHAGE protocol. It is hoped that once these findings have been disseminated, performance can be improved.

High‐resolution 3D coronary vessel wall imaging with near 100% respiratory efficiency using epicardial fat tracking: Reproducibility and comparison with standard methods

To quantitatively assess the performance and reproducibility of 3D spiral coronary artery wall imaging with beat-to-beat respiratory-motion-correction (B2B-RMC) compared to navigator gated 2D spiral and turbo-spin-echo (TSE) acquisitions. High-resolution (0.7 × 0.7 mm) cross-sectional right coronary wall acquisitions were performed in 10 subjects using four techniques (B2B-RMC 3D spiral with alternate (2RR) and single (1RR) R-wave gating, navigator-gated 2D spiral (2RR) and navigator-gated 2D TSE (2RR)) on two occasions. Wall thickness measurements were compared with repeated measures analysis of variance (ANOVA). Reproducibility was assessed with the intraclass correlation coefficient (ICC). In all, 91% (73/80) of acquisitions were successful (failures: four TSE, two 3D spiral (1RR) and one 3D spiral (2RR)). Respiratory efficiency of the B2B-RMC was less variable and substantially higher than for navigator gating (99.6 ± 1.2% vs. 39.0 ± 7.5%, P < 0.0001). Coronary wall thicknesses (± standard deviation [SD]) were not significantly different: 1.10 ± 0.14 mm (3D spiral (2RR)), 1.20 ± 0.16 mm (3D spiral (1RR)), 1.14 ± 0.15 mm (2D spiral), and 1.21 ± 0.17 mm (TSE). Wall thickness reproducibility ranged from good (ICC = 0.65, 3D spiral (1RR)) to excellent (ICC = 0.87, 3D spiral (2RR)). High-resolution 3D spiral imaging with B2B-RMC permits coronary vessel wall assessment over multiple thin contiguous slices in a clinically feasible duration. Excellent reproducibility of the technique potentially enables studies of disease progression/regression.

Pulse shape measurements using single shot-frequency resolved optical gating for high energy (80 J) short pulse (600 fs) laser

Relevant to laser based electron/ion accelerations, a single shot second harmonic generation frequency resolved optical gating (FROG) system has been developed to characterize laser pulses (80 J, ∼600 fs) incident on and transmitted through nanofoil targets, employing relay imaging, spatial filter, and partially coated glass substrates to reduce spatial nonuniformity and B-integral. The device can be completely aligned without using a pulsed laser source. Variations of incident pulse shape were measured from durations of 613 fs (nearly symmetric shape) to 571 fs (asymmetric shape with pre- or postpulse). The FROG measurements are consistent with independent spectral and autocorrelation measurements.

PERSPECTIVES: Silencing vascular smooth muscle ATP-sensitive K+channels with caveolin-1

PERSPECTIVES: Silencing vascular smooth muscle ATP-sensitive K⁺channels with caveolin-1

Perception of temporally interrupted speech: Effects of two concurrent gating rates on intelligibility.

Perception of temporally interrupted speech was investigated with either one or two concurrent square-wave gating functions. Sentences were interrupted at a single rate of 1, 2, 4, 8, 16, 24, or 32 Hz with a 50% duty cycle. For each rate between 1 to 8 Hz, stimuli were additionally gated at a secondary rate ranging from twice the primary rate up to 32 Hz. The secondary gating rate thus interrupted only the segments of speech which were left intact after application of the primary gating function to reduce by half the remaining speech content independent of secondary rate. With a single gating function, intelligibility scores increased with rate, reaching perfect accuracy at 8 Hz. Application of the second gating function led to a decrease in intelligibility which was greatest when the secondary rate was twice the primary rate, a condition equivalent to a 25% duty cycle of the primary rate. Further increase in the secondary rate generally led to an improvement in intelligibility, although never to the level achieved with the primary rate alone. The incomplete recovery of intelligibility scores at fast secondary gating rates suggest disruption of separate perceptual processes by each of the two concurrent interruption rates.