Two-dimensional nanosecond electric field mapping based on cell electropermeabilization

Nanosecond, megavolt-per-meter electric pulses cause permeabilization of cells to small molecules, programmed cell death (apoptosis) in tumor cells, and are under evaluation as a treatment for skin cancer. We use nanoelectroporation and fluorescence imaging to construct two-dimensional maps of the electric field associated with delivery of 15 ns, 10 kV pulses to monolayers of the human prostate cancer cell line PC3 from three different electrode configurations: single-needle, five-needle, and flat-cut coaxial cable. Influx of the normally impermeant fluorescent dye YO-PRO-1 serves as a sensitive indicator of membrane permeabilization. The level of fluorescence emission after pulse exposure is proportional to the applied electric field strength. Spatial electric field distributions were compared in a plane normal to the center axis and 15-20 μm from the tip of the center electrode. Measurement results agree well with models for the three electrode arrangements evaluated in this study. This live-cell method for measuring a nanosecond pulsed electric field distribution provides an operationally meaningful calibration of electrode designs for biological applications and permits visualization of the relative sensitivities of different cell types to nanoelectropulse stimulation. PACS Codes: 87.85.M

MIPAS detection of cloud and aerosol particle occurrence in the UTLS with comparison to HIRDLS and CALIOP

Satellite infrared emission instruments require efficient systems that can separate and flag observations which are affected by clouds and aerosols. This paper investigates the identification of cloud and aerosols from infrared, limb sounding spectra that were recorded by the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS), a high spectral resolution Fourier transform spectrometer on the European Space Agency's (ESA) ENVISAT (Now inoperative since April 2012 due to loss of contact). Specifically, the performance of an existing cloud and aerosol particle detection method is simulated with a radiative transfer model in order to establish, for the first time, confident detection limits for particle presence in the atmosphere from MIPAS data. The newly established thresholds improve confidence in the ability to detect particle injection events, plume transport in the upper troposphere and lower stratosphere (UTLS) and better characterise cloud distributions utilising MIPAS spectra. The method also provides a fast front-end detection system for the MIPClouds processor; a processor designed for the retrieval of macro- and microphysical cloud properties from the MIPAS data. <br><br> It is shown that across much of the stratosphere, the threshold for the standard cloud index in band A is 5.0 although threshold values of over 6.0 occur in restricted regimes. Polar regions show a surprising degree of uncertainty at altitudes above 20 km, potentially due to changing stratospheric trace gas concentrations in polar vortex conditions and poor signal-to-noise due to cold atmospheric temperatures. The optimised thresholds of this study can be used for much of the time, but time/composition-dependent thresholds are recommended for MIPAS data for the strongly perturbed polar stratosphere. In the UT, a threshold of 5.0 applies at 12 km and above but decreases rapidly at lower altitudes. The new thresholds are shown to allow much more sensitive detection of particle distributions in the UTLS, with extinction detection limits above 13 km often better than 10<sup>−4</sup> km<sup>−1</sup>, with values approaching 10<sup>−5</sup> km<sup>−1</sup> in some cases. <br><br> Comparisons of the new MIPAS results with cloud data from HIRDLS and CALIOP, outside of the poles, establish a good agreement in distributions (cloud and aerosol top heights and occurrence frequencies) with an offset between MIPAS and the other instruments of 0.5 km to 1 km between 12 km and 20 km, consistent with vertical oversampling of extended cloud layers within the MIPAS field of view. We conclude that infrared limb sounders provide a very consistent picture of particles in the UTLS, allowing detection limits which are consistent with the lidar observations. Investigations of MIPAS data for the Mount Kasatochi volcanic eruption on the Aleutian Islands and the Black Saturday fires in Australia are used to exemplify how useful MIPAS limb sounding data were for monitoring aerosol injections into the UTLS. It is shown that the new thresholds allowed such events to be much more effectively derived from MIPAS with detection limits for these case studies of 1 × 10<sup>−5</sup> km<sup>−1</sup> at a wavelength of 12 μm

Overshooting of Clean Tropospheric Air in the Tropical Lower Stratosphere as Seen by the CALIPSO Lidar

The evolution of aerosols in the tropical upper troposphere/lower stratosphere between June 2006 and October 2009 is examined using the observations of the space borne CALIOP lidar aboard the CALIPSO satellite. Superimposed on several volcanic plumes and soot from an extreme biomass-burning event in 2009, the measurements reveal the existence of fast cleansing episodes of the lower stratosphere to altitudes as high as 20 km. The cleansing of the full 14-20km layer takes place within 1-4 months. Its coincidence with the maximum of convective activity in the southern tropics, suggests that the cleansing is the result of a large number of overshooting towers, injecting aerosol-poor tropospheric air into the lower stratosphere. The enhancements of aerosols at the tropopause level during the NH summer may be due to the same transport process but associated with intense sources of aerosols at the surface. Since, the tropospheric air flux derived from CALIOP observations during North Hemisphere winter is 5 20 times larger than the slow ascent by radiative heating usually assumed, the observations suggest that convective overshooting is a major contributor to troposphere-to-stratosphere transport with concommitant implications to the Tropical Tropopause Layer top height, chemistry and thermal structure

DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns

Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected

Nanoelectropulse-driven membrane perturbation and small molecule permeabilization

BACKGROUND: Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization. RESULTS: In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FM1-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X(7 )receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts. CONCLUSION: Megavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane

Manipulating ultracold atoms with a reconfigurable nanomagnetic system of domain walls

The divide between the realms of atomic-scale quantum particles and lithographically-defined nanostructures is rapidly being bridged. Hybrid quantum systems comprising ultracold gas-phase atoms and substrate-bound devices already offer exciting prospects for quantum sensors, quantum information and quantum control. Ideally, such devices should be scalable, versatile and support quantum interactions with long coherence times. Fulfilling these criteria is extremely challenging as it demands a stable and tractable interface between two disparate regimes. Here we demonstrate an architecture for atomic control based on domain walls (DWs) in planar magnetic nanowires that provides a tunable atomic interaction, manifested experimentally as the reflection of ultracold atoms from a nanowire array. We exploit the magnetic reconfigurability of the nanowires to quickly and remotely tune the interaction with high reliability. This proof-of-principle study shows the practicability of more elaborate atom chips based on magnetic nanowires being used to perform atom optics on the nanometre scale.Comment: 4 pages, 4 figure

Role of catestatin as such as slowly released by fibronectin-coated pharmacologically active microcarriers (Fn-Pam) in limiting hypoxicinduced cell death

Objectives: Catestatin (CST), a 21-amino acid derivate of Chromogranin A, exerts several biological functions, including inhibition of catecholamine release and cardioprotective role. Moreover positive effect of CST on monocyte migration in vitro and the induction of angiogenesis, arteriogenesis and vasculogenesis in the mouse hind limb ischemia model have been demonstrated. Collateral arteries may provide a biological bypass for occluded atherosclerotic vessels, increasing blood flow to ischemic tissue. In such a prospective, CST is a very promising agent for revascularization purposes, in “NO-OPTION” patients. However, proteins have a very short half-life after administration and must be conveniently protected. FN-PAMs, biodegradable and biocompatible polymeric microspheres, have ideal characteristic for this purpose: besides to convey peptides and allow in situ prolonged/controlled delivery, they may also convey cells on their biomimetic surface and may favor their survival and engraftment after cell transplantation. In this study, we show that CST can be incorporated within FN-PAM and aim to demonstrate that CST may be released in a slowly/prolonged manner by FN-PAM. We also aim to demonstrate that CST released by FN-PAM may reduce cell death under different stress conditions. Materials and methods: CST has to be precipitated to ensure its stability upon subsequent encapsulation. Protein precipitate is formed from aqueous solution by the addition of a watermiscible organic solvent. PLGA–P188–PLGA (triblock) copolymeric microspheres are prepared using solid/oil/water emulsion solvent evaporation–extraction technique. PAMs are coated with Fibronectin and characterized by Immunofluroscence (confocal microscopy). Mesenchymal stem cells (MSC) are exposed to hypoxia (72 h in 1–2%O2) and reoxygenation (6 h in 21% O2) in a hypoxic chamber with or without CTS, FN-PAMs or CTS-FN-PAMs. The protective effects of treatments are detected by MTT assay. Results: To define the optimum condition of nanoprecipitation we used an experimental design, modifying parameters influencing protein precipitation: ionic strength, mixing and centrifugation time. Nanoprecipitation of CST was found to be 72%. Controlled release of CST from CTS-FNPAM greatly limits hypoxic MSC death and enhances MSC survival in post-hypoxic environment. Conclusions: FN-PAMs are successfully formulated with CST. By an experimental design, we found optimal conditions to obtain a good CTS nanoprecipitation yield. MSC readily adhere to the FN-PAM and CST-FN-PAMs reduce MSC death enhancing survival in post-hypoxic environment. Data suggest that CST-FN-PAMs are promising tools for therapeutic purpose