Single-channel Measurements Research Articles

ZAR1 resistosome and helper NLRs: Bringing in calcium and inducing cell death

ZAR1 resistosome and helper NLRs: Bringing in calcium and inducing cell death

Small PN-Code Lidar for Asteroid and Comet Missions—Receiver Processing and Performance Simulations

Space missions to study small solar system bodies, such as asteroids and comet cores, are enhanced by lidar that can provide global mapping and serve as navigation sensors for landing and surface sampling. A small swath-mapping lidar using a fiber laser modulated by pseudo-noise (PN) codes is well-suited to small space missions and can provide contiguous measurements of surface topography with <10 cm precision. Here, we report the design and simulation of receiver signal processing of such a lidar using the small all-range lidar (SALi) as a design example. We simulated its performance in measuring the lidar range and surface reflectance by using instrument and target parameters, noise sources, and the receiver correlation processing method under various conditions. In single-beam Reconnaissance mode, the simulation predicted a maximum range of 440 km under sunlit conditions with a range precision as small as 8 cm. In its multi-pixel Mapping mode, the lidar can provide measurements out to 110 km with range precision of 5 cm. The effects of Doppler shift were quantified. From these results, we discuss the need for Doppler compensation via the receiver clock rate. We also describe a novel reflectance measurement method using active laser control, which allows the receiver to use simple comparators for analog-to-digital conversion. This method was simulated with surface reflectance values from 4% to 36% resulting in an RMS precision of 3% and a bias of 1% of the surface reflectance. We also performed an orbital ranging simulation using a shape model of 101955 Bennu for target surface elevation. The range residuals showed a sub-mm bias with a standard deviation of 5 cm. We implemented the receiver processor design on a Xilinx Ultrascale field-programmable gate array (FPGA). It was able to process received signals and retrieve accurate ranges at a single-channel measurement rate of 3050 Hz with a latency of 1.07 ms.

127-OR: In Vivo Genetic Evidence That the Pyruvate Kinase Isoforms PKM1 and PKM2 Differentially Control Beta-Cell Fuel Sensing

Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we examined the requirements of pyruvate kinase (PK) isoforms in fuel sensing through the β-cell specific deletion of consititutively active PKM1 and allosterically recruitable PKM2 (PKM1- βKO, PKM2-βKO). β-cell deletion of PEP carboxykinase (PCK2-βKO) was used to block mitochondrial PEP production, leaving glycolytic PEP production intact. In vivo, only the PKM1-βKO mice showed glucose intolerance, and ex vivo insulin secretion was correspondingly reduced. Glucose-dependent Ca2+ oscillations were comparable in PKM1 βKO and PKM2-βKO islets, while the oscillatory frequency and duty cycle in PCK2-βKO islets increased relative to controls, suggesting redundancy in the PK response to glycolytic but not mitochondrial PEP. Using direct single-channel measurements of KATP in intact β-cells, amino acids were sufficient to stimulate KATP closure in control and PKM2-βKO cells, but failed to do so in the PCK2-βKO and PKM1-βKO models. PK activators (PKa) rescued KATP closure in PKM1- but not PCK2-deficient β-cells, arguing that PKM1 and PCK2 mediate membrane depolarization in response to mitochondrial fuels. The dependence of PKM2 on glucose was confirmed by the ability of PKa to rescue KATP closure in excised membrane patches from PKM1-βKO mice. Consistently, calcium influx stimulated by amino acids at low glucose was deficient in islets from PKM1-βKO and PCK2-βKO mice. PKa was sufficient to rescue the calcium defect in the PKM1-βKO islets but did not fully rescue the PCK2-βKO. Our findings provide strong genetic and electrophysiological evidence that the PCK2-PKM1 pathway is uniquely suited to direct the active (secretory) phase of calcium oscillations through direct effects on KATP, when FBP is low and PKM2 is inactive. While more limited in function, PKM2 can be therapeutically targeted, endowing it with the ability to respond to mitochondrial PEP and boost insulin secretion. Disclosure H. R. Foster: None. T. Ho: None. E. Potapenko: None. S. L. Lewandowski: None. S. Sdao: None. H. R. Vandeusen: None. R. L. Cardone: None. R. Kibbey: Research Support; Self; Agios, Inc. M. J. Merrins: None. Funding Health Resources and Services Administration (T32HP10010); National Institute on Aging (T32AG000213); National Institutes of Health (R01DK113103)

129-OR: Glycolytic Enzymes and Creatine Kinase Locally Regulate KATP Channel Activity in Both ß- and a-Cells

The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. In ß-cells, it has been previously showed that the local activity of pyruvate kinase (PK) can sufficiently raise ATP/ADP to induce KATP closure, thus providing a another route to trigger membrane depolarization parallel to mitochondrial oxidative phosphorylation. Here, using direct single-channel measurements of KATP in both ß-and the lesser studied α-cells, we provide evidence for a larger network of metabolic enzymes that operate in the vicinity of KATP channels to fine-tune their activity. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in ß-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally PK. Strikingly, while withholding the substrates at GAPDH, we observed that FBP can independently inhibit KATP channels by direct action. In addition, we further provide evidence that creatine kinase (CK) activity can either oppose or enhance KATP closure, depending on the reaction direction. Finally, we demonstrated that lactate has an activating effect on KATP. In summary, these data provide evidence for the direct regulation of KATP by two metabolites, FBP and lactate, and identify two local ATP/ADP controllers, the glycolytic complex and the creatine kinase system in both ß-cells and α-cells. Disclosure T. Ho: None. E. Potapenko: None. M. J. Merrins: None.

Mechanistic link between CaM-RyR2 interactions and the genesis of cardiac arrhythmia

In this study, we develop a computational model of the interaction between ryanodine receptor type 2 (RyR2) and calmodulin (CaM) to explore the mechanistic link between CaM-RyR2 interactions and cardiac arrhythmia. Our starting point is a biophysically based computational model of CaM binding to a single RyR2 subunit, which reproduces single-channel RyR2 measurements in lipid bilayers. We then integrate this CaM-RyR2 model into a spatially distributed whole-cell model of Ca cycling, which is used to investigate the relationship between CaM and Ca cycling homeostasis. We show that a reduction in CaM concentration leads to a substantial increase in the rate of spontaneous Ca sparks, and this induces a marked reduction in sarcoplasmic reticulum Ca load during steady-state pacing. Also, we show that a reduction in CaM modifies the RyR2 open probability, which makes the cell more prone to Ca wave propagation. These results indicate that aberrant Ca cycling activity during pacing is determined by the interplay between sarcoplasmic reticulum load reduction and the threshold for Ca wave propagation. Based on these results, we show that when CaM is reduced, Ca waves can occur in a cell and induce action potential perturbations that are arrhythmogenic. Thus, this study outlines a novel, to our knowledge, mechanistic link between CaM-RyR2 binding kinetics and the induction of arrhythmias in the heart.

Extracting Robust Biomarkers From Multichannel EEG Time Series Using Nonlinear Dimensionality Reduction Applied to Ordinal Pattern Statistics and Spectral Quantities

In this study, ordinal pattern analysis and classical frequency-based EEG analysis methods are used to differentiate between EEGs of different age groups as well as individuals. As characteristic features, functional connectivity as well as single-channel measures in both the time and frequency domain are considered. We compare the separation power of each feature set after nonlinear dimensionality reduction using t-distributed stochastic neighbor embedding and demonstrate that ordinal pattern-based measures yield results comparable to frequency-based measures applied to preprocessed data, and outperform them if applied to raw data. Our analysis yields no significant differences in performance between single-channel features and functional connectivity features regarding the question of age group separation.

Alpha-Hemolysin Channel Interaction with Neutral Polymers

Polyethylene glycols (PEG) or dextran are among the neutral polymers used in single-channel conductance measurements to size the aqueous pore, to evaluate access resistance or to induce osmotic stress in polymer-excluded regions. The use of polymer partitioning into biological nanopores to size the aqueous pore of protein ion channels has become a common approach. While the basic principles of polymer behavior in restricted volumes are well established, little is known about the actual interaction of neutral polymer molecules with charged sites in membranes and channel proteins.

PEG Equilibrium Partitioning in the α-Hemolysin Channel: Neutral Polymer Interaction with Channel Charges.

We study the interaction of neutral polyethylene glycol (PEG) molecules of different molecular weights (MWs) with the charged residues of the α-hemolysin channel secreted by Staphylococcus aureus. Previously reported experiments of PEG equilibrium partitioning into this nanopore show that the charge state of the channel changes the ability of PEG entry in an MW-dependent manner. We explain such an effect by parameter-free calculations of the PEG self-energy from the channel 3D atomic structure that include repulsive dielectrophoretic and hydrostatic forces on the polymer. We found that the pH-induced shift in the measured free energy of partitioning ΔΔGexp from single-channel conductance measurements agrees with calculated energy changes ΔΔEcalc. Our results show that the PEG-sizing technique may need corrections in the case of charged biological pores.

Evidence of cadmium and mercury involvement in the Aβ42 aggregation process

Aβ42 is a small peptide formed from 42 aminoacids that presents a great propensity to aggregate until it forms fibrils. Aβ42 aggregation and fibrillation are very complex processes whose molecular mechanisms seem to depend on characteristics intrinsic to the peptide molecule, as well as extrinsic factors. Peptide concentration, mean pH and several substances, including metal ions, are principal extrinsic factors for the oligomerization process. Different metals affect the aggregation of the Aβ42 molecule, and their toxicity favours the misfolding and aggregation of the peptide. In this study, we evaluate the effect of different concentrations of Cd2+ and Hg2+ on the Aβ42 peptide in solution by different methods. The toxicity of Aβ42 was evaluated with the MTT assay, while the aggregation process was monitored by single-channel measurements, electrophoresis and western blot. Cd2+ and Hg2+ seem to favour the formation of high-molecular-weight aggregates, to decrease ion channel turnover inside the membrane and to significantly increase Aβ42 toxicity.

Single-channel EEG measurement of engagement in virtual rehabilitation: a validation study

Stroke rehabilitation suffers from low levels of patient engagement, impeding recovery. Virtual rehabilitation (VR) approaches can improve patient outcomes; however, there is limited understanding of the participant’s user experience and the field lacks a validated, objective measure of VR engagement. A neurophysiological measure of engagement in healthy adults was therefore examined, to inform future clinical studies. Twenty-four participants (Mage 26.7 years, range 18–47) interacted with a tabletop VR system (Elements DNA, or EDNA), after which they rated their experience on the presence questionnaire (PQ). Separately, participants completed tasks eliciting low (resting eyes-open and -closed) and high (EDNA VR and roller coaster simulation) levels of engagement while continuous electroencephalogram (EEG) was recorded from a single, left pre-frontal electrode. EEG differences between the resting and simulation conditions included an increase in theta power (p < 0.01) and a decrease in alpha power (p < 0.01). Importantly, theta power in simulation conditions correlated with PQ scores expressing the hands-on EDNA VR experience (rs = 0.38–0.48). In conclusion, the current results provide proof of concept that increased frontal theta power in healthy adults provides a valid measure of user engagement in VR simulation and participation. As the practical potential of VR is increasingly realised in stroke rehabilitation, objective EEG-based measures of engagement may provide a convenient and sensitive technique to assist in evaluating these interventions.

Bifurcated binding of the OmpF receptor underpins import of the bacteriocin colicin N into Escherichia coli

Colicins are Escherichia coli–specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A colicins typically parasitize the proton-motive force–linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A colicins.

Volumetric acoustic holography and its application to self-positioning by single channel measurement

We generated dense mid-air volumetric acoustic holography using a newly developed computation algorithm and experimentally verified its utility for three-dimensional self-positioning via monaural amplitude measurements. We computed the holography by solving an inverse problem where complex amplitudes of ultrasound emissions are to be determined so that the given acoustic amplitude field at a finite set of control points in the workspace could be generated as faithfully as possible. With a one-directional gradient pattern of a 40 kHz ultrasonic field that stretched uniformly toward the depth direction, numerical simulations showed that positioning with an average error less than 3 mm is ideally possible in a 100 mm-sided cubic workspace. We experimentally verified that this error was approximately 8 mm with our custom-made phased array. Our work shows the first example of information systems, where a position-dependent information field is embedded in the environment as a form of holographic wave field that can be sensed by standalone mobile devices.

The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport

Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed.

The problem of accuracy in single-channel open probability measurements

The function of ion channels to mediate the flux of ions through membranes of living cells depends on their number, conductance, and open probability. The open probability, PO, characterizes gating of channels that is sensitive to experimental conditions and that can be determined in single-channel experiments. Individual experimental records and even whole series of single-channel activity measurements represent random samples of the stochastic gating continuous in time. The aim of this study was to understand the relationship between the accuracy (trueness and precision) of PO determination and the method of single-channel activity data collection. We used simulated single-channel experiments with variable settings of data collection for a range of open probability values. We found that at low PO, the trueness of PO determination depends on the average number of channel openings per record, while the precision of PO determination depends on the total number of channel openings in the whole dataset and on the distribution of open and closed times. We derived relationships that allow planning of single-channel experiments for the required accuracy of PO determination over a large span of open probabilities.

A Wearable EEG Instrument for Real-Time Frontal Asymmetry Monitoring in Worker Stress Analysis

A highly wearable single-channel instrument, conceived with off-the-shelf components and dry electrodes, is proposed for detecting human stress in real time by electroencephalography (EEG). The instrument exploits EEG robustness to movement artifacts with respect to other biosignals for stress assessment. The single-channel differential measurement aims at analyzing the frontal asymmetry, a well-claimed EEG feature for stress assessment. The instrument was characterized metrologically on human subjects. As triple metrological references, standardized stress tests, observational questionnaires given by psychologists, and performance measurements were exploited. Four standard machine learning classifiers (SVM, k-NN, random forest, and ANN), trained on 50% of the data set, reached more than 90% accuracy in classifying each 2-s epoch of EEG acquired from the stressed subjects.

Single-channel properties of skeletal muscle ryanodine receptor pore Δ4923FF4924 in two brothers with a lethal form of fetal akinesia

Ryanodine receptor ion channels (RyR1s) release Ca2+ ions from the sarcoplasmic reticulum to regulate skeletal muscle contraction. By whole-exome sequencing, we identified the heterozygous RYR1 variant c.14767_14772del resulting in the in-frame deletion p.(Phe4923_Phe4924del) in two brothers with a lethal form of the fetal akinesia deformation syndrome (FADS). The two deleted phenylalanines (RyR1-Δ4923FF4924) are located in the S6 pore-lining helix of RyR1. Clinical features in one of the two siblings included severe hypotonia, thin ribs, swallowing inability, and respiratory insufficiency that caused early death. Functional consequences of the RyR1-Δ4923FF4924 variant were determined using recombinant 2,200-kDa homotetrameric and heterotetrameric RyR1 channel complexes that were expressed in HEK293 cells and characterized by cellular, electrophysiological, and computational methods. Cellular Ca2+ release in response to caffeine indicated that the homotetrameric variant formed caffeine-sensitive Ca2+ conducting channels in HEK293 cells. In contrast, the homotetrameric channel complex was not activated by Ca2+ and did not conduct Ca2+ based on single-channel measurements. The computational analysis suggested decreased protein stability and loss of salt bridge interactions between RyR1-R4944 and RyR1-D4938, increasing the electrostatic interaction energy of Ca2+ in a region 20 Å from the mutant site. Co-expression of wild-type and mutant RyR1s resulted in Ca2+-dependent channel activities that displayed intermediate Ca2+ conductances and suggested maintenance of a reduced Ca2+ release in the two patients. Our findings reveal that the RYR1 pore variant p.(Phe4923_Phe4924del) attenuates the flow of Ca2+ through heterotetrameric channels, but alone was not sufficient to cause FADS, indicating additional genetic factors to be involved.

Removing Cardiac Artifacts From Single-Channel Respiratory Electromyograms

Electromyographic (EMG) measurements of the respiratory muscles provide a convenient and noninvasive way to assess respiratory muscle function and detect patient activity during assisted mechanical ventilation. However, surface EMG measurements of the diaphragm and intercostal muscles are substantially contaminated by cardiac activity due to the vicinity of the cardiac muscles. Many algorithmic solutions to this problem have been proposed, yet a conclusive performance comparison of the most promising candidates currently is missing. The objective of this work is to provide a quantitative performance comparison of six previously proposed cardiac artifact removal algorithms operating on single-channel EMG measurements, and two newly proposed, improved versions of these algorithms. Algorithmic performance is evaluated quantitatively based on four different measures of separation success, using both synthetic validation signals and electromyographic measurements of the respiratory muscles in eight subjects. The compared algorithms are two versions of the empirical template subtraction algorithm, two model-based Bayesian filtering procedures, a wavelet denoising approach, an empirical mode decomposition (EMD) based approach, and classical high-pass filtering. Different algorithms perform well with respect to different performance measures. Template subtraction algorithms yield the best results if the characteristics of the raw signal are of interest, while filtering algorithms like simple high-pass filtering, wavelet denoising, and EMD-based denoising show superior performance for calculating a cleaned envelope signal. No algorithm completely removes the cardiac interference, but the characteristic errors introduced by the considered algorithms differ. Hence, the choice of the algorithm to use should be made depending on the target application. Finally, we also demonstrate that our empirical SNR measure, which can be calculated without knowledge of the true, undisturbed signals, correlates strongly with the exact reconstruction error. Thus, it represents a reliable indicator for algorithm performance on real measurement data.

Adaptive smartphone-based sensor fusion for estimating competitive rowing kinematic metrics.

Competitive rowing highly values boat position and velocity data for real-time feedback during training, racing and post-training analysis. The ubiquity of smartphones with embedded position (GPS) and motion (accelerometer) sensors motivates their possible use in these tasks. In this paper, we investigate the use of two real-time digital filters to achieve highly accurate yet reasonably priced measurements of boat speed and distance traveled. Both filters combine acceleration and location data to estimate boat distance and speed; the first using a complementary frequency response-based filter technique, the second with a Kalman filter formalism that includes adaptive, real-time estimates of effective accelerometer bias. The estimates of distance and speed from both filters were validated and compared with accurate reference data from a differential GPS system with better than 1 cm precision and a 5 Hz update rate, in experiments using two subjects (an experienced club-level rower and an elite rower) in two different boats on a 300 m course. Compared with single channel (smartphone GPS only) measures of distance and speed, the complementary filter improved the accuracy and precision of boat speed, boat distance traveled, and distance per stroke by 44%, 42%, and 73%, respectively, while the Kalman filter improved the accuracy and precision of boat speed, boat distance traveled, and distance per stroke by 48%, 22%, and 82%, respectively. Both filters demonstrate promise as general purpose methods to substantially improve estimates of important rowing performance metrics.

A Quantitative Ultrasonic Travel-Time Tomography to Investigate Liquid Elaborations in Industrial Processes.

This work presents an ultrasound tomography imaging system and method for quantitative mapping of the sound speed in liquid masses. It is highly desirable to be able to inspect vessel fluid mass distribution, notably in the chemical and food industrial operations. Optimization of industrial reactors has been crucial to the improvement of industrial processes. There is a great need to investigate how and if tomographic imaging sensors could aid the automatic control of these process tanks. Single-measurement ultrasound techniques and especially spectrometric methods have been a subject of study of industrial applications. Tomographic systems provide key multi-dimensional and spatial information when compared to the well-established single-channel measurement system. Recently, ultrasound tomography has attracted a great deal of interest in a wide spectrum of industrial applications. The system has been designed as 32 piezoelectric ring-array positioned in a 30 cm tank, with an excitation frequency of 40 kHz. Two-dimensional transmission travel-time tomography was developed to reconstruct the fluid mass distributions. Prior experiments are mainly based on inclusions of a few centimetres and on a liquid solution of different concentrations. They have been conducted to test the spatial and quantitative resolution of the ultrasound imaging device. Analysing the reconstructed images, it is possible to provide accurate spatial resolution with low position errors. The system also demonstrated inclusion movement with a temporal resolution of 4 frames per second (fps) in dynamical imaging sense. Sound velocity quantitative imaging was developed for the investigation of ultrasonic propagation in different liquids. This work, for the first time, shows how quantitative sound velocity imaging using transmission mode time of flight data could be used to characterize liquid density distribution of industrial reactors. The results suggest that ultrasound tomography can be used to quantitatively monitor important process parameters.

QRStree: A prefix tree-based model to fetal QRS complexes detection.

Non-invasive fetal electrocardiography (NI-FECG) plays an important role in fetal heart rate (FHR) measurement during the pregnancy. However, despite the large number of methods that have been proposed for adult ECG signal processing, the analysis of NI-FECG remains challenging and largely unexplored. In this study, we propose a prefix tree-based framework, called QRStree, for FHR measurement directly from the abdominal ECG (AECG). The procedure is composed of three stages: Firstly, a preprocessing stage is employed for noise elimination. Secondly, the proposed prefix tree-based method is used for fetal QRS complexes (FQRS) detection. Finally, a correction stage is applied for false positive and false negative correction. The novelty of the framework relies on using the range of FHR to establish the connections between the FQRS. The consecutive FQRS can be considered as strings composed of alphabet items, thus we can use the prefix tree to store them. A vertex of the tree contains an alphabet, thus a path of the tree gives a string. Such that, by storing the connections of the FQRS into the prefix tree structure, the problem of FQRS detection converts to a problem of optimal path selection. Specifically, after selecting the optimal path of the tree, the nodes in the optimal path are collected as detected FQRS. Since the prefix tree can cover every possible combination of the FQRS candidates, it has the potential to reduce the occurrence of miss detections. Results on two different databases show that the proposed method is effective in FHR measurement from single-channel AECG. The focus on single-channel FHR measurement facilitates the long-term monitoring for healthcare at home.