Ms Resolution Research Articles

A single molecule approach to mRNA transport by a class V myosin

mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. We show that an in vitro single molecule approach, using purified recombinant full-length proteins and synthesized mRNA, provides insight into the mechanism by which localizing mRNAs are carried to their destination. A messenger ribonucleoprotein (mRNP) complex was reconstituted from a budding yeast class V myosin motor complex (Myo4p–She3p), an mRNA-binding adaptor protein (She2p), and a localizing mRNA (ASH1). The motion of the mRNP was tracked with high spatial (∼10 nm) and temporal (70 ms) resolution. Using this “bottom-up” methodology, we show that mRNA triggers the assembly of a high affinity double-headed motor-mRNA complex that moves continuously for long distances on actin filaments at physiologic ionic strength. Without mRNA, the myosin is monomeric and unable to move continuously on actin. This finding reveals an elegant strategy to ensure that only cargo-bound motors are activated for transport. Increasing the number of localization elements (“zip codes”) in the mRNA enhanced both the frequency of motile events and their run length, features which likely enhance cellular localization. Future in vitro reconstitution of mRNPs with kinesin and dynein motors should similarly yield mechanistic insight into mRNA transport by microtubule-based motors.

Measurement of spatial and temporal behavior of H(α) emission from Aditya tokamak using a diagnostic based on a photomultiplier tube array.

A photo multiplier tube (PMT) array based spectroscopic diagnostic with fast time response of 10 μs and spatial resolution ∼3 cm has been developed and installed on Aditya tokamak to study the spatial and temporal behavior of Hα emissions from typical discharges. Collimated light has been collected from the plasma along 16 lines of sight passing through entire plasma poloidal cross section of Aditya and detected by two 8 channels PMT arrays after selecting Hα emission using interference filter. The studies are carried out during plasma formation phase of Aditya by changing vertical field and its delay with respect to loop voltage. It is observed that plasma initiated in the high field side in typical discharges of Aditya. The plasma formation position is matched with null field location estimated through simulation.

Real-time phase-contrast flow MRI of haemodynamic changes in the ascending aorta and superior vena cava during Mueller manoeuvre

To evaluate the potential of real-time phase-contrast flow magnetic resonance imaging (MRI) at 40 ms resolution for the simultaneous determination of blood flow in the ascending aorta (AA) and superior vena cava (SVC) in response to reduced intrathoracic pressure (Mueller manoeuvre). Through-plane flow was assessed in 20 healthy young subjects using real-time phase-contrast MRI based on highly undersampled radial fast low-angle shot (FLASH) with image reconstruction by regularized non-linear inversion. Haemodynamic alterations (three repetitions per subject = 60 events) were evaluated during normal breathing (10 s), inhalation with nearly closed epiglottis (10 s), and recovery (20 s). Relative to normal breathing and despite interindividual differences, reduced intrathoracic pressure by at least 30 mmHg significantly decreased the initial peak mean velocity (averaged across the lumen) in the AA by -24 ± 9% and increased the velocity in the SVC by +28 ± 25% (p < 0.0001, n = 23 successful events). Respective changes in flow volume per heartbeat were -25 ± 9% in the AA and +49 ± 44% in the SVC (p < 0.0001, n = 23). Flow parameters returned to baseline during sustained pressure reduction, while the heart rate was elevated by 10% (p < 0.0001) after the start (n = 24) and end (n = 17) of the manoeuvre. Real-time flow MRI during low intrathoracic pressure non-invasively revealed quantitative haemodynamic adjustments in both the AA and SVC.

Capturing thermal, mechanical, and acoustic effects of the diode (980 nm) laser in stapedotomy.

The diode laser, with a wavelength of 980 nm, has promising characteristics for being used for the fenestration during stapedotomy. It is known that at this wavelength absorption in pigmented tissues is high, and absorption in water is relatively low compared with medical lasers in the infrared, making it theoretically an applicable laser for stapes surgery in patients with otosclerosis. Another important advantage is that, with respect to other lasers, this device is relatively inexpensive. Despite the potential advantages, the available literature only shows limited reports of this laser being used in stapes surgery. The present article evaluates the thermal, mechanical, and acoustic properties of the diode laser during stapes surgery. For the mechanical effects, high-speed imaging with a frame rate up to 4000 f/s (=250 μs resolution) was performed in an inner ear model. For thermal effects, the high-speed Schlieren technique was used. Acoustics were recorded by a hydrophone, incorporated in the model. Pulse settings were 100 ms, 3 W, which are the same settings used during stapes surgery. The application of the diode laser resulted in limited mechanical and thermal effects. Impulse noise was low with an average of 52 (SD, 7.8) dB (A). Before carbonization of the tip of the delivery laser, fiber enhances ablation of the footplate. The 980-nm diode laser is a useful tool for laser-assisted stapedotomy in patients with otosclerosis. Mechanical, thermal, and acoustic effects are limited and well within the safety limits.

P643: Safety and characterization of a novel multi-channel TMS stimulator

Background: Currently available TMS stimulators have a single channel operating a single coil. Objective: To outline and present physical and physiological benefits of a novel convenient multi-channel stimulator, comprising five channels, where the stimulation parameters of each channel are independently controllable. Methods: Simultaneous and sequential operation of various channels was tested in healthy volunteers. Paired pulses schemes with various inter-stimulus intervals (ISIs) were studied for the hand APB and the leg AH muscles. Energy consumption and coil heating rates with simultaneous operation of 4 channels was compared to a single figure-8 coil. Results: Repetitive operation of separate channels with different stimulation parameters is demonstrated. The operations of various channels can be combined simultaneously or sequentially to induce multiple pulses with ISIs of ms resolution. A universal pattern of inhibition and facilitation as a function of ISI was found, with some dependence on coils configurations and on pulse widths. A strong dependence of the induced inhibition on the relative orientation of the conditioning and test pulses was discovered. The ability of this method to induce inhibition in shallow brain region but not in deeper region, thus focusing the effect in the deep brain region, is demonstrated. A significant saving in energy consumption and a reduction in coil heating were demonstrated for several channels operated simultaneously compared to a standard single channel figure-8 coil. Conclusions: The multi-channel stimulator enables the synchronized induction of different excitability modulations to different brain regions using different stimulation patterns in various channels. Multiple pulses operation with coils with various depth profiles can increase the focality of TMS effect in deep brain regions.

Mechanism of inhibition of the GluA1 AMPA receptor channel opening by the 2,3-benzodiazepine compound GYKI 52466 and a N-methyl-carbamoyl derivative.

2,3-Benzodiazepine derivatives, also known as GYKI compounds, represent a group of the most promising synthetic inhibitors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Here we investigate the mechanism of inhibition of the GluA1 channel opening and the site of inhibition by GYKI 52466 and its N-3 methyl-carbamoyl derivative, which we term as BDZ-f. GluA1 is a key AMPA receptor subunit involved in the brain function. Excessive activity and elevated expression of GluA1, however, has been implicated in a number of neurological disorders. Using a laser-pulse photolysis technique, which provides ∼60 μs resolution, we measured the effect of these inhibitors on the rate of GluA1 channel opening and the amplitude of the glutamate-induced whole-cell current. We found that both compounds inhibit GluA1 channel noncompetitively. Addition of an N-3 methyl-carbamoyl group to the diazepine ring with the azomethine feature (i.e., GYKI 52466) improves the potency of the resulting compound or BDZ-f without changing the site of binding. This site, which we previously termed as the “M” site on the GluA2 AMPA receptor subunit, therefore favorably accommodates an N-3 acylating group. On the basis of the magnitude of the inhibition constants for the same inhibitors but different receptors, the “M” sites on GluA1 and GuA2 are different. Overall, the “M” site or the binding environment on GluA2 accommodates the same compounds better, or the same inhibitors show stronger potency on GluA2, as we have reported previously [Wang et al. Biochemistry (2011) 50, 7284−729321751782]. However, acylating the N-3 position to occupy the N-3 side pocket of the “M” site can significantly narrow the difference and improve the potency of a resulting compound on GluA1.

A Nano Ultra-Performance Liquid Chromatography–High Resolution Mass Spectrometry Approach for Global Metabolomic Profiling and Case Study on Drug-Resistant Multiple Myeloma

Global metabolomics relies on highly reproducible and sensitive detection of a wide range of metabolites in biological samples. Here we report the optimization of metabolome analysis by nanoflow ultraperformance liquid chromatography coupled to high-resolution orbitrap mass spectrometry. Reliable peak features were extracted from the LC-MS runs based on mandatory detection in duplicates and additional noise filtering according to blank injections. The run-to-run variation in peak area showed a median of 14%, and the false discovery rate during a mock comparison was evaluated. To maximize the number of peak features identified, we systematically characterized the effect of sample loading amount, gradient length, and MS resolution. The number of features initially rose and later reached a plateau as a function of sample amount, fitting a hyperbolic curve. Longer gradients improved unique feature detection in part by time-resolving isobaric species. Increasing the MS resolution up to 120000 also aided in the differentiation of near isobaric metabolites, but higher MS resolution reduced the data acquisition rate and conferred no benefits, as predicted from a theoretical simulation of possible metabolites. Moreover, a biphasic LC gradient allowed even distribution of peak features across the elution, yielding markedly more peak features than the linear gradient. Using this robust nUPLC-HRMS platform, we were able to consistently analyze ~6500 metabolite features in a single 60 min gradient from 2 mg of yeast, equivalent to ~50 million cells. We applied this optimized method in a case study of drug (bortezomib) resistant and drug-sensitive multiple myeloma cells. Overall, 18% of metabolite features were matched to KEGG identifiers, enabling pathway enrichment analysis. Principal component analysis and heat map data correctly clustered isogenic phenotypes, highlighting the potential for hundreds of small molecule biomarkers of cancer drug resistance.

Comparison of Numerical and Experimental Time-Resolved Near-Field Hall Thruster Plasma Properties

Breathing mode oscillations of a xenon 600 W Hall effect thruster have been studied using temporally resolved experimental data and numerical modeling. Fluctuations in xenon neutral near infrared (810-835 nm) emission in the near field thruster plume have been measured at 1- μs resolution using a high speed, phase-matched intensified charge coupled device. Oscillations in electron temperature, 3-9 eV, have been inferred using a collisional-radiative model and a two-line ratio method. The time-resolved emission and electron temperature measurements are then used to assess the accuracy of the numerical model HPHall. Although simulations were able to accurately predict the time averaged thruster behavior, the model greatly under predicts the magnitude of the oscillations. General phase trends between the discharge current and emission as well as electron temperature are consistent with observations, suggesting that the model is capable of capturing some of the oscillatory behavior despite the dampening of the oscillations.

Fast Pyrolysis of Wood for Biofuels: Spatiotemporally Resolved Diffuse Reflectance In situ Spectroscopy of Particles.

Fast pyrolysis of woody biomass is a promising process capable of producing renewable transportation fuels to replace gasoline, diesel, and chemicals currently derived from nonrenewable sources. However, biomass pyrolysis is not yet economically viable and requires significant optimization before it can contribute to the existing oil-based transportation system. One method of optimization uses detailed kinetic models for predicting the products of biomass fast pyrolysis, which serve as the basis for the design of pyrolysis reactors capable of producing the highest value products. The goal of this work is to improve upon current pyrolysis models, usually derived from experiments with low heating rates and temperatures, by developing models that account for both transport and pyrolysis decomposition kinetics at high heating rates and high temperatures (>400 °C). A new experimental technique is proposed herein: spatiotemporally resolved diffuse reflectance in situ spectroscopy of particles (STR-DRiSP), which is capable of measuring biomass composition during fast pyrolysis with high spatial (10 μm) and temporal (1 ms) resolution. Compositional data were compared with a comprehensive 2D single-particle model, which incorporated a multistep, semiglobal reaction mechanism, prescribed particle shrinkage, and thermophysical properties that varied with temperature, composition, and orientation. The STR-DRiSP technique can be used to determine the transport-limited kinetic parameters of biomass decomposition for a wide variety of biomass feedstocks.

On the Temporal Fidelity of Nonlinear Inverse Reconstructions for Real- Time MRI – The Motion Challenge

Purpose: To evaluate the temporal accuracy of a self-consistent nonlinear inverse reconstruction method (NLINV) for real-time MRI using highly undersampled radial gradient-echo sequences and to present an open source framework for the motion assessment of real-time MRI methods. Methods: Serial image reconstructions by NLINV combine a joint estimation of individual frames and corresponding coil sensitivities with temporal regularization to a preceding frame. The temporal fidelity of the method was determined with a phantom consisting of water-filled tubes rotating at defined angular velocity. The conditions tested correspond to real-time cardiac MRI using SSFP contrast at 1.5 T (40 ms resolution) and T1 contrast at 3.0 T (33 ms and 18 ms resolution). In addition, the performance of a post-processing temporal median filter was evaluated. Results: NLINV reconstructions without temporal filtering yield accurate estimations as long as the speed of a small moving object corresponds to a spatial displacement during the acquisition of a single frame which is smaller than the object itself. Faster movements may lead to geometric distortions. For small objects moving at high velocity, a median filter may severely compromise the spatiotemporal accuracy. Conclusion: NLINV reconstructions offer excellent temporal fidelity as long as the image acquisition time is short enough to adequately sample (“freeze”) the object movement. Temporal filtering should be applied with caution. The motion framework emerges as a valuable tool for the evaluation of real-time MRI methods.

Safety and Characterization of a Novel Multi-channel TMS Stimulator

BackgroundCurrently available TMS stimulators have a single channel operating a single coil. ObjectiveTo outline and present physical and physiological benefits of a novel convenient multi-channel stimulator, comprising five channels, where the stimulation parameters of each channel are independently controllable. MethodsSimultaneous and sequential operation of various channels was tested in healthy volunteers. Paired pulses schemes with various inter-stimulus intervals (ISIs) were studied for the hand APB and the leg AH muscles. Energy consumption and coil heating rates with simultaneous operation of 4 channels was compared to a single figure-8 coil. ResultsRepetitive operation of separate channels with different stimulation parameters is demonstrated. The operations of various channels can be combined simultaneously or sequentially to induce multiple pulses with ISIs of μs resolution. A universal pattern of inhibition and facilitation as a function of ISI was found, with some dependence on coils configurations and on pulse widths. A strong dependence of the induced inhibition on the relative orientation of the conditioning and test pulses was discovered. The ability of this method to induce inhibition in shallow brain region but not in deeper region, thus focusing the effect in the deep brain region, is demonstrated. A significant saving in energy consumption and a reduction in coil heating were demonstrated for several channels operated simultaneously compared to a standard single channel figure-8 coil. ConclusionsThe multi-channel stimulator enables the synchronized induction of different excitability modulations to different brain regions using different stimulation patterns in various channels. Multiple pulses operation with coils with various depth profiles can increase the focality of TMS effect in deep brain regions.

Glucose utilization rates regulate intake levels of artificial sweeteners

It is well established that animals including humans attribute greater reinforcing value to glucose-containing sugars compared to their non-caloric counterparts, generally termed 'artificial sweeteners'. However, much remains to be determined regarding the physiological signals and brain systems mediating the attribution of greater reinforcing value to sweet solutions that contain glucose. Here we show that disruption of glucose utilization in mice produces an enduring inhibitory effect on artificial sweetener intake, an effect that did not depend on sweetness perception or aversion. Indeed, such an effect was not observed in mice presented with a less palatable, yet caloric, glucose solution. Consistently, hungry mice shifted their preferences away from artificial sweeteners and in favour of glucose after experiencing glucose in a hungry state. Glucose intake was found to produce significantly greater levels of dopamine efflux compared to artificial sweetener in dorsal striatum, whereas disrupting glucose oxidation suppressed dorsal striatum dopamine efflux. Conversely, inhibiting striatal dopamine receptor signalling during glucose intake in sweet-naïve animals resulted in reduced, artificial sweetener-like intake of glucose during subsequent gluco-deprivation. Our results demonstrate that glucose oxidation controls intake levels of sweet tastants by modulating extracellular dopamine levels in dorsal striatum, and suggest that glucose utilization is one critical physiological signal involved in the control of goal-directed sweetener intake.

Spiral tissue phase velocity mapping in a breath-hold with non-cartesian SENSE.

Tissue phase velocity mapping (TPVM) is capable of reproducibly measuring regional myocardial velocities. However acquisition durations of navigator gated techniques are long and unpredictable while current breath-hold techniques have low temporal resolution. This study presents a spiral TPVM technique which acquires high resolution data within a clinically acceptable breath-hold duration. Ten healthy volunteers are scanned using a spiral sequence with temporal resolution of 24 ms and spatial resolution of 1.7 × 1.7 mm. Retrospective cardiac gating is used to acquire data over the entire cardiac cycle. The acquisition is accelerated by factors of 2 and 3 by use of non-Cartesian SENSE implemented on the Gadgetron GPU system resulting in breath-holds of 17 and 13 heartbeats, respectively. Systolic, early diastolic, and atrial systolic global and regional longitudinal, circumferential, and radial velocities are determined. Global and regional velocities agree well with those previously reported. The two acceleration factors show no significant differences for any quantitative parameter and the results also closely match previously acquired higher spatial resolution navigator-gated data in the same subjects. By using spiral trajectories and non-Cartesian SENSE high resolution, TPVM data can be acquired within a clinically acceptable breath-hold.

Range resolution improvement by a reversed m-sequence phase coded lidar system

The traditional phase coded lidar systems require the collection of every returned laser pulse and are restricted in range resolution by sampling frequency and subpulse width. This paper proposes a reversed m-sequence acquisition method for signal detection. The detector accumulates N − 1 bits of the emitted laser sequence to deduce the Nth bit of the sequence. The indoor experiment achieved 2 μs resolution with the sampling period of 28 μs by employing a 15-bit m-sequence. This method achieves the acquisition of an m-sequence with narrow subpulse width whereas the sampling frequency is kept low. This paper also presents the phase coded imaging lidar simulation, which achieved 1.5 m range resolution with a 50 kHz sampling frequency by employing a 2047-bit m-sequence. The simulation showed an approach to implement phase coded imaging lidar in practical application.

Mapping fine-grained power measurements to HPC application runtime characteristics on IBM POWER7

Optimization of energy consumption is a key issue for future HPC. Evaluation of energy consumption requires a fine-grained power measurement. Additional useful information is obtained when performing these measurements at component level. In this paper we describe a setup which allows to perform fine-grained power measurements up to a 1 ms resolution at component level on IBM POWER (IBM and POWER are trademarks of IBM in USA and/or other countries.) machines. We further developed a plug-in for VampirTrace that allows us to correlate these power measurements with application performance characteristics, e.g. obtained by hardware performance counters. This environment enables us to generate both power and performance profiles. Such profiles provide valuable input to develop future strategies for improving workload-driven energy usage per performance. We show in comparison with power profiles of coarser granularity that these fine-grained measurements are necessary to capture the dynamics of power switching.

Gradual Disordering of the Native State on a Slow Two-State Folding Protein Monitored by Single-Molecule Fluorescence Spectroscopy and NMR

Theory predicts that folding free energy landscapes are intrinsically malleable and as such are expected to respond to perturbations in topographically complex ways. Structural changes upon perturbation have been observed experimentally for unfolded ensembles, folding transition states, and fast downhill folding proteins. However, the native state of proteins that fold in a two-state fashion is conventionally assumed to be structurally invariant during unfolding. Here we investigate how the native and unfolded states of the chicken α-spectrin SH3 domain (a well characterized slow two-state folder) change in response to chemical denaturants and/or temperature. We can resolve the individual properties of the two end-states across the chemical unfolding transition employing single-molecule fluorescence spectroscopy (SM-FRET) and across the thermal unfolding transition by NMR because SH3 folds-unfolds in the slow chemical exchange regime. Our results demonstrate that α-spectrin SH3 unfolds in a canonical way in the sense that it converts between the native state and an unfolded ensemble that expands in response to chemical denaturants. However, as conditions become increasingly destabilizing, the native state also expands gradually, and a large fraction of its native intramolecular hydrogen bonds break up. This gradual disordering of the native state takes place in times shorter than the 100 μs resolution of our SM-FRET experiments. α-Spectrin SH3 thus showcases the extreme plasticity of folding landscapes, which extends to the native state of slow two-state proteins. Our results point to the idea that folding mechanisms under physiological conditions might be quite different from those obtained by linear extrapolation from denaturing conditions. Furthermore, they highlight a pressing need for re-evaluating the conventional procedures for analyzing and interpreting folding experiments, which may be based on too-simplistic assumptions.

Chemometric Resolution for Rapid Determination of Prometryn in Leek Samples Using GC–MS

Much effort has been made to analyze the pesticides in foods or vegetables due to the interferences in the sample matrix. In this work, taking the determination of prometryn in leek samples as an example, a simple method is proposed for fast analysis of the components in real samples using GC–MS and chemometric resolution. The purification step in preparing the samples was simplified, and a short capillary column and fast temperature program were employed in GC–MS measurement. Although the measured signal is composed of overlapped peaks with the interferences and background, the signal of prometryn can be extracted by chemometric resolution. Six leek samples from different markets were analyzed within an elution time of 6 min. Compared with the results by the standard method, the results by the proposed method were found to be reliable.

Observations of ionic species produced in an atmospheric pressure pulse-modulated RF plasma needle

Pulse-modulated generation of atmospheric pressure plasma promises lower operating temperatures and enhanced energy efficiencies compared with those generated using continuous excitation. In this study, the effect of pulse modulation on the ionic content of a radio-frequency helium plasma needle was explored using molecular beam mass spectrometry (MBMS). In all cases reported the pulse repetition frequency was held constant at 6 kHz while the duty cycle and distance between the plasma needle and MBMS were varied from 30% to 90% and 4 mm to 10 mm, respectively. Decreasing the duty cycle was seen to enhance the negative ion flux to the MBMS and yields a clear shift to higher masses for positive ion species. Time-resolved ion intensity measurements (5 µs resolution) demonstrated that negative ions were created almost exclusively in the pulse off-time while positive ions were generated mainly in the on-time. The ions detected were also seen to change as the distance between the sampling orifice and tip of the plasma needle increased, highlighting the effect of different ion mobilities in the flowing gas stream and the presence of an extended cooler afterglow region. The results suggest that there is a possibility of tailoring the ionic plasma chemistry through pulsed modulation, increasing the versatility of the already widely studied plasma needle device.

FMRI hemodynamics accurately reflects neuronal timing in the human brain measured by MEG

Neuronal activation sequence information is essential for understanding brain functions. Extracting such timing information from blood oxygenation level dependent (BOLD) fMRI is confounded by interregional neurovascular differences and poorly understood relations between BOLD and electrophysiological response delays. Here, we recorded whole-head BOLD fMRI at 100 ms resolution and magnetoencephalography (MEG) during a visuomotor reaction-time task. Both methods detected the same activation sequence across five regions, from visual towards motor cortices, with linearly correlated interregional BOLD and MEG response delays. The smallest significant interregional BOLD delay was 100 ms; all delays ≥400 ms were significant. Switching the order of external events reversed the sequence of BOLD activations, indicating that interregional neurovascular differences did not confound the results. This may open new avenues for using fMRI to follow rapid activation sequences in the brain.

Nocapyrones H–J, 3,6-Disubstituted α-Pyrones from the Marine Actinomycete &lt;i&gt;Nocardiopsis&lt;/i&gt; sp. KMF-001

Three new 3,6-disubstituted α-pyrones, nocapyrones H-J (1-3), were isolated from the marine actinomycete Nocardiopsis sp. KMF-001. Their structures were assigned to be 3-alkylated 6-(1-methyl-1-propenyl)-2H-pyran-2-ones on the basis of UV, MS, NMR, and high resolution (HR)-FAB-MS analyses. Nocapyrone H (1) reduced the pro-inflammatory factor such as nitric oxide (NO), prostaglandin E(2) (PGE(2)) and interleukin-1β (IL-1β). Moreover, nocapyrone H showed 5.82% stronger inhibitory effect on NO production than chrysin at a concentration of 10 µM in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.