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Electropermeabilization of biomembranes often is measured by microscopic imaging of a membrane-impermeable fluorophore that penetrates the cells following pulsed electric field (PEF) exposure. PEF exposure subsequently changes physiological properties of tissue. One way to probe these changes in tissue is measuring electrical properties by way of electrochemical impedance spectroscopy (EIS). In this study, we analyze impedance and conductivity of bovine annulus fibrosus (AF) cartilage before and after exposures to PEF of 100 µs duration. Two PEF parameters – electric field amplitude and number of pulses – are varied, and total specific dose of PEF is calculated. AF tissue conductivity increases with both amplitude and number of pulses, indicating electropermeabilization of the annulus fibrosus cells. A Live/Dead cell imaging assay validates the EIS measurements, indicating intratissue cell permeabilization by µsPEF exposure. These results support the extension of EIS to monitor extent of electropermeabilization of cells within cartilage tissue.

Epigenetic modifications derived from changes in sub-cellular localization and activity of the post-translational histone modifying enzymes, histone deacetylases (HDACs) are one of the strategies to treat various types of cancer. Aberrant HDAC activity is an indicator of glioblastoma multiforme (GBM), generally leading to hypoacetylation of histones and a transcriptionally repressed chromatin state. Most noticeably, HDAC4 expression increases 61,000% in brain tumors!1 Upregulation of HDAC4 in human glioma cells (U87-MG) stimulates proliferation and invasiveness, making HDAC4 a therapeutic target. The effectiveness of pharmacological HDAC inhibitors is hampered by non-specific targeting and low selectivity, especially in solid tumors like GBM, and by side-effects with systemic delivery, e.g. cardiac toxicity.2 Existing electroporation-based approaches to GBM aim to induce cell death either directly3 or in combination with adjuvants such as excess extracellular calcium4 or chemotherapy drugs5.A cytostatic approach is introduced in which the initial electroporation from non-thermal, localized µsPEF exposure elicits downstream epigenetic responses that decrease cell proliferation, circumventing obstacles experienced by pharmacological agents. Accumulation of HDAC4 in the nucleus of U87-MG cells following exposure to µsPEF is hypothesized to lead in a dose-dependent manner to cell death via apoptosis and decreased proliferation.A BTX GeminiX2 electroporator delivered square-wave µsPEF of 100 µs and 1.45 kV/cm, while the number of pulses (0, 1, 10, 20 pulses, delivered at 1 Hz) were varied to determine thresholds for HDAC4 translocation and proliferation reduction of U87-MG cells. For immunofluorescence assay to track HDAC4 location at 3 h after exposure, cells were cultured and exposed on glass-bottom dishes. For MTT and ATP kinase assays, cells were exposed in electroporation cuvettes and transferred to 96-well plates to measure proliferation at 6, 24 and 72 h after exposure. The ratio of HDAC4 in the nucleus compared to the cytoplasm (N/C ratio) more than doubles after 20 pulses, compared to sham control with no µsPEF exposure. Cell concentration relative to control drops over 90% given 20 pulses. Our data reflect the inhibition of cell proliferation and HDAC4 accumulation in nuclei. Although electroporation-based therapies for GBM have been studied pre-clinically, this study delves further into fundamental mechanisms and optimization of the energy delivered by µsPEF exposure that induces cell death with respect to epigenetic modification. 1. Lee P. et al. Anticancer Res, 35:615, 2015.2. Slingerland M. et al. Anticancer Drugs, 25:140, 2014.3. Rossmeisl J.H. et al. J Neurosurg, 123:1008, 2015.4. Wasson E.M. et al. Ann Biomed Eng, 45:2535, 2017.5. Sharabi S. et al. Sci Rep, 10:2178, 2020. Citation Format: Zahra Safaei, Gary L Thompson. Glioblastoma treatment using epigenetic modification induced by microsecond pulsed electric field (µsPEF) exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3314.

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.

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.

Harnessing electrical bias-induced mechanical motion on the nanometer and molecular scale is a critical step towards understanding the fundamental mechanisms of redox processes and implementation of molecular electromechanical machines. Probing these phenomena in biomolecular systems requires electromechanical measurements be performed in liquid environments. Here we demonstrate the use of band excitation piezoresponse force microscopy for probing electromechanical coupling in amyloid fibrils. The approaches for separating the elastic and electromechanical contributions based on functional fits and multivariate statistical analysis are presented. We demonstrate that in the bulk of the fibril the electromechanical response is dominated by double-layer effects (consistent with shear piezoelectricity of biomolecules), while a number of electromechanically active hot spots possibly related to structural defects are observed.

Coupling between electrical and mechanical phenomena is extremely common in inorganic materials, and nearly ubiquitous in biological systems, underpinning phenomena and devices ranging from SONAR to cardiac activity and hearing. This paper briefly summarizes the Scanning Probe Microscopy (SPM) approach, referred to as Piezoresponse Force Microscopy (PFM), for probing electromechanical coupling on the nanometer scales, and delineates some existing and emerging applications to probe local structure and functionality in inorganic ferroelectrics, calcified and connective tissues, and complex biosystems based on electromechanical detection.

This study reports the effect of exposure to liquid carbon dioxide on the mechanical properties of selected medical polymers. The tensile strengths and moduli of fourteen polymers are reported. Materials were exposed to liquid CO(2), or CO(2) + trace amounts of aqueous H(2)O(2), at 6.5 MPa and ambient temperature. Carbon dioxide uptake, swelling, and distortion were observed for the more amorphous polymers while polymers with higher crystallinity showed little effect from CO(2) exposure. Changes in tensile strength were not statistically significant for most plastics, and most indicated good tolerance to liquid CO(2). These results are relevant to evaluating the potential of liquid CO(2)-based sterilization technology.

The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (2 nm) are created in the plasma membrane in contrast to larger diameter pores (2 nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

Electromechanical coupling is ubiquitous in biological systems with examples ranging from simple piezoelectricity in calcified and connective tissues to voltage-gated ion channels, energy storage in mitochondria, and electromechanical activity in cardiac myocytes and outer hair cell stereocilia. Piezoresponse force microscopy (PFM) has originally emerged as a technique to study electromechanical phenomena in ferroelectric materials, and in recent years, has been employed to study a broad range of non-ferroelectric polar materials, including piezoelectric biomaterials. At the same time, the technique has been extended from ambient to liquid imaging on model ferroelectric systems. Here, we present results on local electromechanical probing of several model cellular and biomolecular systems, including insulin and lysozyme amyloid fibrils, breast adenocarcinoma cells, and bacteriorhodopsin in a liquid environment. The specific features of SPM operation in liquid are delineated and bottlenecks on the route towards nanometer-resolution electromechanical imaging of biological systems are identified., 37 pages (including refs), 8 figures

d General Dynamics Information Technology, Naval Medical Research Unit-San Antonio, Fort Sam Houston, Texas 78234 Abstract. The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca 2þ ), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca 2þ overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimu- lation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca 2þ influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca 2þ , was 1.76 kV∕cm for NG108 and 0.84 kV∕cm for PHN. At 16.2 kV∕cm, the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca 2þ channel blocker, failed to prevent Ca 2þ uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca 2þ influx that may be capable of controllably modulating neurological function. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of different types of bacteria in pure water. Here, the BEPFM method is performed for the first time in physiologically-relevant electrolyte media, such as Dulbecco’s phosphate-buffered saline (DPBS) and Dulbecco’s modified Eagle’s medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria are demonstrated in DPBS. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

A semidestructive method has been developed for determining the principal residual stresses and directions in rectangularly orthotropic materials. The reduction equations are based upon a set of functions that describe the surface strain-relaxation field about a hole drilled to a limited depth into the material. Three constants contained in the strain functions have to be determined by calibration tests; they are related to three general constants and the elastic material constants to establish applicability to an orthotropic material. Expressions for the planar residual-stress components in the material-symmetry directions are then devel oped, and from Mohr's stress circle, the principal residual stresses and directions are determined.

Cell swelling and blebbing has been commonly observed following nanosecond pulsed electric field (nsPEF) exposure. The hypothesized origin of these effects is nanoporation of the plasma membrane (PM) followed by transmembrane diffusion of extracellular fluid and disassembly of cortical actin structures. This investigation will provide evidence that shows passive movement of fluid into the cell through nanopores and increase of intracellular osmotic pressure are not solely responsible for this observed phenomena. We demonstrate that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion and hydrolysis are critical steps in the chain reaction leading to cellular blebbing and swelling. PIP2 is heavily involved in osmoregulation by modulation of ion channels and also serves as an intracellular membrane anchor to cortical actin and phospholipase C (PLC). Given the rather critical role that PIP2 depletion appears to play in the response of cells to nsPEF exposure, it remains unclear how its downstream effects and, specifically, ion channel regulation may contribute to cellular swelling, blebbing, and unknown mechanisms of the lasting “permeabilization” of the PM., Highlights • Nanosecond electric pulses (nsEPs) of high amplitude induce hydrolysis of PIP2. • PLC activation is leading to post-exposure cellular swelling and blebbing. • Ion channels modulation and nanoporation are responsible for cellular swelling. • Cortical actin dissociation after PIP2 depletion is critical for cellular blebbing.

contemporary resource management questions, and policies of the federal government toward Native American peoples. The varied facets of the dispute illustrate the difficulties of reaching a settlement which meets the requirements of legality under state and federal law while minimizing the social, economic, and cultural injustices to the parties involved. All three branches of the federal government have been deeply involved in the settlement of the dispute, but the judiciary has been specifically commissioned to end the conflict by partitioning the land.3 When it occurs, this division may result in the forcible re location of as many as 13,000 Navaho Indians to an area outside the present Navaho Reservation,4 thus becoming the first Indian removal program of the twentieth century and the only case of the removal of one tribal group to accommodate the territorial expansion of another. Although one purpose of this paper is to provide a simplified view of the major issues comprising the dispute, the major interest is in providing a case study of a situation which has resulted in the casting

Two high rise poultry houses located in Lancaster Co., PA were used to evaluate residual wall treatments of Ectiban (permerhrin) for controlling adult manure breeding flies. Single applications of 0.1% Ectiban were applied by a hand held compressed air sprayer to the manure pit walls of each house at a rate of 1 gal/^50 sq ft. Alternating 750 sq ft sections were treated. Changes in adult fly populations were monitored by counting the number of flies resting within marked areas on the walls in both treated and untreated sections of the pit and upper level of the building where the birds were housed. The house at Rheems, PA was 400 × 52 ft and housed 60,000 caged layers. The flock had been in place since February and no fly control measures had been taken. A total of 5 gal of spray was applied to 52% of the wall area in the manure pit on Jun 22. The 8 ft high pit walls had cinder blocks on the lower half and unpainted wood paneling above. The upper area where the birds were housed was not sprayed but was treated with 10 to 15 lb of Improved Golden Malrin (Lannate) fly bait at 3, 7, 14 and 21 days post-treatment. The manure pit was cleaned Aug 6-8. House flies comprised 20% and Ophyra sp. 80% of the adult fly population. The house at Ephrata, PA was 500 × 44 ft and housed 45,000 caged layers. The flock had been in place since November and the pit was cleaned in April. Ten days prior to the Ectiban treatment, the aisles in the upper level were baited with Improved Golden Malrin (Lannate) and the manure surface was sprayed with Shell Poultry Spray & Larvicide (Rabon + Vapona) 2-1/2 weeks prior to treatment. On Aug 14, a total of 4 gal of Ectiban spray was applied to 42% of the wall area in the manure pit. The 8 ft high pit walls had cinder blocks on the lower half and plywood above which was painted with white latex paint. House flies comprised 99% an^ Ophyra sp. 1% of the adult fly population.