Fast processing of data from Sneg-2MP experiment

The following subjects are covered: Basic stages during computer processing of data from Sneg-2MP instrument, basic modes during separation and fast processing (separation of data during satellite flight, separation of burst data segments, sampling and analysis of initial burst data segment). Experimental results obtained on the basis of fast processed data are reported

Electroporation of Mammalian Cells by Nanosecond Electric Field Oscillations and it\u27s Inhibition by the Electric Field Reversal

The present study compared electroporation efficiency of bipolar and unipolar nanosecond electric field oscillations (NEFO). Bipolar NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2-4 decreased to 35%, 12%, and 7% of the first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4. Membrane permeabilization was quantified in CHO and GH3 cells by uptake of a membrane integrity marker dye YO-PRO-1 (YP) and by the membrane conductance increase measured by patch clamp. For treatments with 1-20 unipolar NEFO, at 9.6-24 kV/cm, 10 Hz, the rate and amount of YP uptake were consistently 2-3-fold higher than after bipolar NEFO treatments, despite delivering less energy. However, the threshold amplitude was about 7 kV/cm for both NEFO waveforms. A single 14.4 kV/cm unipolar NEFO caused a 1.5-2 times greater increase in membrane conductance (p \u3c 0.05) than bipolar NEFO, along with a longer and less frequent recovery. The lower efficiency of bipolar NEFO was preserved in Ca2+ free conditions and thus cannot be explained by the reversal of electrophoretic flows of Ca2+. Instead, the data indicate that the electric field polarity reversals reduced the pore yield

Stobe Photography Mapping of Cell Membrane Potential with Nanosecond Resolution

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid

Systematic NLTE study of the -2.6 < [Fe/H] < 0.2 F and G dwarfs in the solar neighbourhood. I. Stellar atmosphere parameters

We present atmospheric parameters for 51 nearby FG dwarfs uniformly distributed over the -2.60 < [Fe/H] < +0.20 metallicity range that is suitable for the Galactic chemical evolution research. Lines of iron, Fe I and Fe II, were used to derive a homogeneous set of effective temperatures, surface gravities, iron abundances, and microturbulence velocities. We used high-resolution (R>60000) Shane/Hamilton and CFHT/ESPaDOnS observed spectra and non-local thermodynamic equilibrium (NLTE) line formation for Fe I and Fe II in the classical 1D model atmospheres. The spectroscopic method was tested with the 20 benchmark stars, for which there are multiple measurements of the infrared flux method (IRFM) Teff and their Hipparcos parallax error is < 10%. We found NLTE abundances from lines of Fe I and Fe II to be consistent within 0.06 dex for every benchmark star, when applying a scaling factor of S_H = 0.5 to the Drawinian rates of inelastic Fe+H collisions. The obtained atmospheric parameters were checked for each program star by comparing its position in the log g-Teff plane with the theoretical evolutionary track in the Yi et al. (2004) grid. Our final effective temperatures lie in between the T_IRFM scales of Alonso et al. (1996) and Casagrande et al. (2011), with a mean difference of +46 K and -51 K, respectively. NLTE leads to higher surface gravity compared with that for LTE. The shift in log g is smaller than 0.1 dex for stars with either [Fe/H] > -0.75, or Teff 4.20. NLTE analysis is crucial for the VMP turn-off and subgiant stars, for which the shift in log g between NLTE and LTE can be up to 0.5 dex. The obtained atmospheric parameters will be used in the forthcoming papers to determine NLTE abundances of important astrophysical elements from lithium to europium and to improve observational constraints on the chemo-dynamical models of the Galaxy evolution.Comment: 18 pages, 14 figures, accepted for publication in Ap

Disassembly of Actin Structures by Nanosecond Pulsed Electric Field is a Downstream Effect of Cell Swelling

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance

Bipolar Nanosecond Electric Pulses are Less Efficient at Electropermeabilization and Killing Cells than Monopolar Pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF

Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

In this study, we determined the LD50 (50% lethal dose) for cell death, and the ED50 (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD50 varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD50 lower than the ED50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue

Control of the Electroporation Efficiency of Nanosecond Pulses by Swinging the Electric Field Vector Direction

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as “bipolar cancellation,” enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing the electric field in the pairs (180° direction change) caused 2-fold (1 Hz) or 20-fold (833 kHz) weaker electroporation than the train of single nsEPs. Reducing the angle between pulse directions in the pairs weakened cancellation and replaced it with facilitation at angles \u3c160° (1 Hz) and \u3c130° (833 kHz). Facilitation plateaued at about three-fold stronger electroporation compared to single pulses at 90–100° angle for both nsEP frequencies. The profound dependence of the efficiency on the angle enables novel protocols for highly selective focal electroporation at one electrode in a three-electrode array while avoiding effects at the other electrodes. Nanosecond-resolution imaging of cell membrane potential was used to link the selectivity to charging kinetics by co- and counter-directional nsEPs

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns–10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15–20% higher LD50, p \u3c 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30–40% lower LD50 (p \u3c 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation