Evaluation of Viral Inactivation on Dry Surface by High Peak Power Microwave (HPPM) Exposure.

Previous research has shown that virus infectivity can be dramatically reduced by radio frequency exposure in the gigahertz (GHz) frequency range. Given the worldwide SARS-CoV-2 pandemic, which has caused over 1 million deaths and has had a profound global economic impact, there is a need for a noninvasive technology that can reduce the transmission of virus among humans. RF is a potential wide area-of-effect viral decontamination technology that could be used in hospital rooms where patients are expelling virus, in grocery and convenience stores where local populations mix, and in first responder settings where rapid medical response spans many potentially infected locations within hours. In this study, we used bovine coronavirus (BCoV) as a surrogate of SARS-CoV-2 and exposed it to high peak power microwave (HPPM) pulses at four narrowband frequencies: 2.8, 5.6, 8.5, and 9.3 GHz. Exposures consisted of 2 µs pulses delivered at 500 Hz, with pulse counts varied by decades between 1 and 10,000. The peak field intensities (i.e. the instantaneous power density of each pulse) ranged between 0.6 and 6.5 MW/m2 , depending on the microwave frequency. The HPPM exposures were delivered to plastic coverslips containing BCoV dried on the surface. Hemagglutination (HA) and cytopathic effect analyses were performed 6 days after inoculation of host cells to assess viral infectivity. No change in viral infectivity was seen with increasing dose (pulse number) across the tested frequencies. Under all conditions tested, exposure did not reduce infectivity more than 1.0 log10. For the conditions studied, high peak power pulsed RF exposures in the 2-10 GHz range appear ineffective as a virucidal approach for hard surface decontamination. © 2023 Bioelectromagnetics Society.

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 < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30-40% lower LD50 (p < 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.

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 <160° (1 Hz) and <130° (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.

Caveolin-1 is Involved in Regulating the Biological Response of Cells to Nanosecond Pulsed Electric Fields.

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC1) and inositol trisphosphate receptor (IP3R)) after exposure to nsPEFs. Release of TRPC1 and IP3R from Cav1 would activate downstream signaling cascades, including store-operated calcium entry, which could explain the influx in calcium after nsPEF exposure. Results of the current study establish a significant relationship between Cav1 and the activation of cell signaling pathways in response to nsPEFs.

Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Visualization of Dynamic Sub-microsecond Changes in Membrane Potential.

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ∼25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.

Nanosecond pulsed electric field exposure does not induce the unfolded protein response in adult human dermal fibroblasts.

Cell-circuit models have suggested that nanosecond pulsed electric fields (nsPEFs) can disrupt intracellular membranes including endoplasmic reticulum (ER), mitochondria, and/or nucleus thereby inducing intrinsic apoptotic pathways. Therefore, we hypothesized that the unfolded protein response (UPR) would be activated, due to the fluctuations of ionic concentrations, upon poration of the ER membrane. Quantitative real-time polymerase chain reaction was utilized to measure changes in messenger RNA (mRNA) expression of specific ER stress genes in adult human dermal fibroblast (HDFa) cells treated with tunicamycin (TM) (known ER stress inducer) and cells exposed to nsPEFs (100, 10-ns pulses at 150 kV/cm delivered at a repetition rate of 1 Hz). For HDFa cells, results showed time-dependent UPR activation to TM; however, when HDFa cells were exposed to nsPEFs, no significant changes in mRNA expression of ER stress genes, and/or caspase gene were observed. These results indicate that although cell death can be observed under these exposure parameters, it is most likely not initiated through activation of the UPR. Bioelectromagnetics. 2018;39:491-499, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

The biological response of cells to nanosecond pulsed electric fields is dependent on plasma membrane cholesterol.

Previous work from our laboratory demonstrated nanopore formation in cell membranes following exposure to nanosecond pulsed electric fields (nsPEF). We observed differences in sensitivity to nsPEF in both acute membrane injury and 24h lethality across multiple cells lines. Based on these data, we hypothesize that the biological response of cells to nsPEF is dependent on the physical properties of the plasma membrane (PM), including regional cholesterol content. Results presented in this paper show that depletion of membrane cholesterol disrupts the PM and increases the permeability of cells to small molecules, including propidium iodide and calcium occurring after fewer nsPEF. Additionally, cholesterol depletion concurrently decreases the "dose" of nsPEF required to induce lethality. In summary, the results of the current study suggest that the PM cholesterol composition is an important determinant in the cellular response to nsPEF.

Quantifying pulsed electric field-induced membrane nanoporation in single cells.

Plasma membrane disruption can trigger a host of cellular activities. One commonly observed type of disruption is pore formation. Molecular dynamic (MD) simulations of simplified lipid membrane structures predict that controllably disrupting the membrane via nano-scale poration may be possible with nanosecond pulsed electric fields (nsPEF). Until recently, researchers hoping to verify this hypothesis experimentally have been limited to measuring the relatively slow process of fluorescent markers diffusing across the membrane, which is indirect evidence of nanoporation that could be channel-mediated. Leveraging recent advances in nonlinear optical microscopy, we elucidate the role of pulse parameters in nsPEF-induced membrane permeabilization in live cells. Unlike previous techniques, it is able to directly observe loss of membrane order at the onset of the pulse. We also develop a complementary theoretical model that relates increasing membrane permeabilization to membrane pore density. Due to the significantly improved spatial and temporal resolution possible with our imaging method, we are able to directly compare our experimental and theoretical results. Their agreement provides substantial evidence that nanoporation does occur and that its development is dictated by the electric field distribution.

Nanosecond pulsed electric field induced dose dependent phosphatidylinositol-4,5-bisphosphate signaling and intracellular electro-sensitization.

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose-dependent PIP2 hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP2 hydrolysis: PLCδ-PH-EGFP and GFP-C1-PKCγ-C1a. The 50% maximum response occurs with a single 600ns pulse achieving an effective dose (ED50) of EF~8kV/cm within our model cell system. At 16.2kV/cm, the ED50 for the pulse width was 484ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60ns 16.2kV/cm pulses produced an effect similar to a single 600ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP2 depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions.

Probe beam deflection optical imaging of thermal and mechanical phenomena resulting from nanosecond electric pulse (nsEP) exposure in-vitro.

Electric-field induced physical phenomena, such as thermal, mechanical and electrochemical dynamics, may be the driving mechanism behind bioeffects observed in mammalian cells during exposure to nanosecond-duration electric pulses (nsEP) in-vitro. Correlating a driving mechanism to a biological response requires the experimental measurement and quantification of all physical dynamics resulting from the nsEP stimulus. A passive and electromagnetic interference (EMI) immune sensor is required to resolve these dynamics in high strength electric fields. The probe beam deflection technique (PBDT) is a passive and EMI immune optical method for quantifying and imaging refractive index gradients in liquids and gases, both dynamic and static, with nanosecond temporal resolution. In this work, a probe beam deflection imaging system was designed to acquire 2-D time-lapse images of thermal/mechanical dynamics resulting from monopolar and bipolar nsEP stimulus.

Cellular response to high pulse repetition rate nanosecond pulses varies with fluorescent marker identity.

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1-43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz-500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd(3+)), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend.

Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure.

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus.

Cellular effects of acute exposure to high peak power microwave systems: Morphology and toxicology.

Electric fields produced by advanced pulsed microwave transmitter technology now readily exceed the Institute of Electrical and Electronic Engineers (IEEE) C.95.1 peak E-field limit of 100 kV/m, highlighting a need for scientific validation of such a specific limit. Toward this goal, we exposed Jurkat Clone E-6 human lymphocyte preparations to 20 high peak power microwave (HPPM) pulses (120 ns duration) with a mean peak amplitude of 2.3 MV/m and standard deviation of 0.1 with the electric field at cells predicted to range from 0.46 to 2.7 MV/m, well in excess of current standard limit. We observed that membrane integrity and cell morphology remained unchanged 4 h after exposure and cell survival 24 h after exposure was not statistically different from sham exposure or control samples. Using flow cytometry to analyze membrane disruption and morphological changes per exposed cell, no changes were observed in HPPM-exposed samples. Current IEEE C95.1-2005 standards for pulsed radiofrequency exposure limits peak electric field to 100 kV/m for pulses shorter than 100 ms [IEEE (1995) PC95.1-Standard for Safety Levels with Respect to Human Exposure to Electric, Magnetic and Electromagnetic Fields, 0 Hz to 300 GHz, Institute of Electrical and Electronic Engineers: Piscataway, NJ, USA]. This may impose large exclusion zones that limit HPPM technology use. In this study, we offer evidence that maximum permissible exposure of 100 kV/m for peak electric field may be unnecessarily restrictive for HPPM devices. Bioelectromagnetics. 37:141-151, 2016. © 2016 Wiley Periodicals, Inc.

Activation of autophagy in response to nanosecond pulsed electric field exposure.

Previous work demonstrated significant changes in cellular membranes following exposure of cells to nanosecond pulsed electric fields (nsPEF), including nanoporation and increases in intracellular calcium concentration. While it is known that nsPEF exposure can cause cell death, how cells repair and survive nsPEF-induced cellular damage is not well understood. In this paper, we investigated whether autophagy is stimulated following nsPEF exposure to repair damaged membranes, proteins, and/or organelles in a pro-survival response. We hypothesized that autophagy is activated to repair nsPEF-induced plasma membrane damage and overwhelming this compensatory mechanism results in cell death. Activation of autophagy and subsequent cell death pathways were assessed measuring toxicity, gene and protein expression of autophagy markers, and by monitoring autophagosome formation and maturation using fluorescent microscopy. Results show that autophagy is activated at subtoxic nsPEF doses, as a compensatory mechanism to repair membrane damage. However, prolonged exposure results in increased cell death and a concomitant decrease in autophagic markers. These results suggest that cells take an active role in membrane repair, through autophagy, following exposure to nsPEF.

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity.

Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca(2+) mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and amplitudes (15-60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-µs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.

Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.

Nanosecond pulsed electric fields modulate the expression of Fas/CD95 death receptor pathway regulators in U937 and Jurkat Cells.

In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60% in the knockdown population while Jurkat survival improved ~40%. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way to control or avoid unintended cell death during nsPEF exposure.

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.

Disruption of the actin cortex contributes to susceptibility of mammalian cells to nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10 ns duration and 150 kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10 min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24 h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects.