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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.

Recent advances in three-dimensional (3D) bioprinting technologies have enabled precise patterning of cellular components along with biomimetic constructs for tissue engineering and regenerative medicine. The viscoelasticity of bioinks regulate printability and the smallest feature size in 3D bioprinted constructs. The impact of cellular components is typically neglected when choosing 3D bioprinting parameters. In this short communication, we quantified the effect of cell densities on the printability of hydrogel bioinks. Unexpectedly, our results show that encapsulated cells reduced the steady shear viscosity of gelatin-based bioinks by approximately 50% and the minimum force for onset of flow by approximately 30%. These results may justify the lower spatial resolution in 3D bioprinted cell-laden hydrogels.

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.

This paper investigates thresholds for nonthermal electroporation of cells within an intervertebral disc (IVD) using an in silico model. Simulations run in Comsol Multiphysics 5.3 indicate that electrical bioeffects can be effectively isolated from electrothermal effects by tuning the parameters of pulsed electric field (PEF) exposure. This work specifically tests the effects of applied voltage, pulse width, number of pulses, and IVD geome-tryon local electric field strength and temperature within an isotropic, homogeneous IVD. Results predict that for the modeled IVD, electric field strength depends linearly upon applied voltage, and temperature build-up is determined by the voltage as well as overall exposure time. These relationships are consistent with the fundamental Ohm's Law and Joule heating effect. Finally, a positive linear relationship exists between disc height and the applied voltage necessary to achieve a targeted temperature.

Purpose In response to the opioid epidemic, West Virginia (WV) passed Senate Bill 437 (SB437). This omnibus legislation included measures related to the definition and governance of chronic pain clinics, continuing education requirements related to opioid prescribing, and changes to the prescription drug monitoring program. This analysis assesses the impact of SB437 on opioid-related mortality in WV. Methods All-manner, drug-related resident deaths occurring in WV 2001–2015 and mid-year population estimates from the US Census, were utilised. An interrupted time series analysis compared monthly drug-related mortality rates by type [prescription and/or illicit (heroin and all fentanyl) opioids and non-opioid drugs], pre- and post-enactment of SB437 (June 2012). Level- and slope-change models were fit using segmented quasi-Poisson regression, controlling for seasonal effects. Results Prior to SB437, the mean number of monthly opioid-related deaths was 27.8 (1.52 deaths per 100,000), with an average monthly increase of 1%. When SB437 was enacted, a significant step decrease in any-prescription-opioid-related mortality (RR=0.69, 95% CI: 0.62, 0.76) occurred, while no step change was observed for any-illicit-opioid-related mortality (RR=1.04, 95% CI: 0.83, 1.30). The annual increase in any-prescription-opioid-related mortality ceased after SB437 (pre-implementation RR=1.12, 95% CI: 1.10, 1.13; post-implementation RR=1.04, 95% CI: 0.99, 1.09); whereas, the annual increase in any-illicit-opioid-related mortality became larger (pre-implementation RR=1.11, 95% CI: 1.09, 1.14; post-implementation RR=1.38, 95% CI: 1.28, 1.48). No step (RR=1.01, 95% CI: 0.99, 1.03) or trend changes (RR=1.01, 95% CI: 0.84, 1.21) were observed for non-opioid-drug-related mortality. Conclusions These findings suggest SB437 impacted opioid-related mortality, though differently by opioid type. While prescription-opioid-related mortality declined and levelled off after SB437 (rather than exhibiting sustained decline), illicit-opioid-related mortality accelerated. Significance The shift in the type of opioid sustaining the increase in mortality suggests different approaches ‘“ from those applied to prescription opioids ‘“ are needed to mitigate risk of fatal opioid overdose.

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N -hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.

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.

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways--phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.

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.

Currently, tissue plasminogen activator (tPA) is administered intravenously after myocardial infarction and stroke to ensure blood-clot dissolution. One problem associated with such treatment is systemic toxicity of tPA, which can act upon plasminogen both in circulation and at the clot site, generating systemic plasmin and rendering the patient highly vulnerable to hemorrhaging. Here we report the synthesis and in vitro characterization of prototype nanodevices capable of binding to fibrin clots and initiating their dissolution. The devices consist of tPA and antifibrin antibody covalently attached to a 40 nm polystyrene-latex nanoparticles. These nanoscale devices can directly deliver tPA to the clot site through fibrin-specific antibody. In vitro fibrinolysis assay showed that protein–nanoparticle conjugates were only slightly less potent than free tPA. Moreover, tPA activity in the absence of clots was more than 3-fold less than that of the free enzyme. This property can be critical for therapeutic applications because nanoparticles can be expected to dissolve clots at approximately the same rate as free tPA while simultaneously cleaving much less plasminogen in the circulation, lowering the risk of hemorrhage. This system could therefore become a promising agent for thrombolytic treatment.

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.

Second Harmonic Generation (SHG) imaging is a useful tool for examining the structure of interfaces between bulk materials. Recently, this technique was applied to detecting subtle perturbations in the structure of cellular membranes following nanosecond pulsed electric field (nsPEF) exposure. Monitoring the cell’s outer membrane as it is exposed to nsPEF via SHG has demonstrated that nanoporation is likely the root cause for size-specific, increased cytoplasmic membrane permeabilization. It is theorized that the area of the membrane covered by these pores is tied to pulse intensity or duration. The extent of this effect along the cell’s surface, however, has never been measured due to its temporal brevity and minute pore size. By enhancing the SHG technique developed and elucidated previously, we are able to obtain this information. Further, we vary the pulse width and amplitude of the applied stimulus to explore the mechanical changes of the membrane at various sites around the cell. By using this unique SHG imaging technique to directly visualize the change in order of phospholipids within the membrane, we are able to better understand the complex response of living cells to electric pulses.

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.

Nonlinear optical probes, especially those involving second harmonic generation (SHG), have proven useful as sensors for near-instantaneous detection of alterations to or ientation or energetics within a substance. This has been exploited to some success for observing conformational changes in proteins. SHG probes, therefore, hold promise for reporting rapid and minute changes in lipid membranes. In this report, one of these probes is employed in this regard, using nanosecond electric pulses (nsEPs) as a vehicle for instigating subtle membrane perturbations. The result provides a useful tool and methodology for the observation of minute membrane perturbation, while also providing meaningful information on the phenomenon of electropermeabilization due to nsEP. The SHG probe Di-4-ANEPPDHQ is used in conjunction with a tuned optical setup to demonstrate nanoporation preferential to one hemisphere, or pole, of the cell given a single square shaped pulse. The results also confirm a correlation of pulse width to the amount of poration. Furthermore, the polarity of this event and the membrane physics of both hemispheres, the poles facing either electrode, were tested using bipolar pulses consisting of two pulses of opposite polarity. The experiment corroborates findings by other researchers that these types of pulses are less effective in causing repairable damage to the lipid membrane of cells.

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.

The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.

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.

In cellular electrochemistry, ions respond to stimuli by constantly shuffling across cellular membranes to perform their physiological roles. This flow of ions, the electromotive force, leaves cells vulnerable to exogenous electromagnetic fields that can stimulate and/or modulate cellular activity. An irreparable link exists between changes in ionic concentration and the electric gradient of the cell (or its potential energy). Consequently, we can manipulate the physiology of the cell by altering its permeability to various ions, thereby modulating its electrical gradient. Only a few millivolts in excess of the resting membrane potential can stimulate a dramatic change in ion distribution within the cellular microenvironment. In excitable neural-type cells, electrical-stimulation-induced changes in membrane potential lead to the generation or inactivation of action potentials (AP). These AP trigger activities, such as nerve impulses in

Nanosecond pulsed electric fields (nsPEFs) are known to increase cell membrane permeability to small molecules in accordance with dosages. As previous work has focused on nsPEF exposures in whole cells, electrodeformation may contribute to this induced-permeabilization in addition to other biological mechanisms. Here, we hypothesize that cellular elasticity, based upon the cytoskeleton, affects nsPEF-induced decrease in cellular viability. Young’s moduli of various types of cells have been calculated from atomic force microscopy (AFM) force curve data, showing that CHO cells are stiffer than non-adherent U937 and Jurkat cells, which are more susceptible to nsPEF exposure. To distinguish any cytoskeletal foundation for these observations, various cytoskeletal reagents were applied. Inhibiting actin polymerization significantly decreased membrane integrity, as determined by relative propidium uptake and phosphatidylserine externalization, upon exposure at 150 kV/cm with 100 pulses of 10 ns pulse width. Exposure in the presence of other drugs resulted in insignificant changes in membrane integrity and 24-hour viability. However, Jurkat cells showed greater lethality than latrunculin-treated CHO cells of comparable elasticity. From these results, it is postulated that cellular elasticity rooted in actin-membrane interaction is only a minor contributor to the differing responses of adherent and non-adherent cells to nsPEF insults.

Exposure to nano-second pulsed electrical fields (nsPEFs) has been shown to cause poration of external and internal cell membranes, DNA damage, and blebbing of the plasma membrane. Recovery from nsPEF exposure is likely dependent on multiple factors, including exposure parameters, length of time between pulses, and extent of cellular damage. As cells progress through the cell cycle, variations in DNA and nucleus structure, cytoskeletal arrangement, and elasticity of cell membrane could cause nsPEFs to affect cells differently during different cell cycle phases. To better understand the impact of nsPEF on cell cycle, we investigated CHO cell cycle progression following varying intensities of nsPEFexposures. Cell populations were examined post exposure (10 ns pulse trains at 100, 150, or 200kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis to determine cell cycle phase. Populations exhibited arrest in G2/M phase, but not in G1 phase at 1h post-exposure that increased in severity and duration with increasing exposure dose. Recovery from arrest was complete after 12h, and populations did not exhibit an increase in apoptosis as a result of exposure. Post exposure arrest in G2/M phase may indicate that nsPEF-induced damage is not significant to cause G1 arrest or that mitotic checkpoints are more important regulators of cell cycle progression after nsPEF exposure.

Cellular exposure to nanosecond pulsed electric fields (nsPEF) are believed to cause immediate creation of nanopores in the plasma membrane. These nanopores enable passage of small ions, but remain impermeable to larger molecules like propidium iodide. Previous work has shown that nanopores are stable for minutes after exposure, suggesting that formation of nanopores in excitable cells could lead to prolonged action potential inhibition. Previously, we measured the formation of nanopores in neuroblastoma cells by measuring the influx of extracellular calcium by preloading cells with Calcium Green-AM. In this work, we explored the impact of changing the width of a single nsPEF, at constant amplitude, on uptake of extracellular calcium ions by primary hippocampal neurons (PHN). Calcium Green was again used to measure the influx of extracellular calcium and FM1-43 was used to monitor changes in membrane conformation. The observed thresholds for nanopore formation in PHN by nsPEF were comparable to those measured in neuroblastoma. This work is the first study of nsPEF effects on PHN and strongly suggests that neurological inhibition by nanosecond electrical pulses is highly likely at doses well below irreversible damage.

Numerous studies provide evidence that nanosecond electric pulses (nsEPs) can trigger the formation of nanopores in the plasma membranes of cells. However, the biophysical mechanism responsible for nanopore formation is not well understood. In this study, we hypothesize that membrane damage induced by nsEPs is primarily dependent on the local molecular composition and mechanical strength of the plasma membrane. To test this hypothesis, we positioned metal-coated, nanoscale cantilever tips using an atomic force microscope (AFM) to deliver nsEPs to localized areas on the surface of the plasma membrane. We conducted computational modeling simulations to verify that the electric field provided by the nsEP is concentrated between the tip and the plasma membrane. The results show that we could effectively deliver nsEPs using the AFM tips at very low voltages. Using scanning electron microscopy we analyzed the tips after applying 10V over 5 seconds duration and found no damage to the tip or loss of platinum coating. As a proof of concept, we applied a 1 and 10V, 5 second pulse to HeLa cells causing large morphological changes. We also applied both a mechanical indention and 600ns electrical pulse stimulus and measured positive propidium ion uptake into the cytoplasm suggesting formation of membrane pores. In future studies, we plan to elucidate the effect that specific, local molecular structures and compositions have on efficacy of electroporation using the newly constructed nano-electrode system.

Piezoresponse force microscopy (PFM) is an atomic force microscopy-based approach utilized for measuring local properties of piezoelectric materials. The objective of this study is to propose a practical framework for simultaneous estimation of the local stiffness and piezoelectric properties of materials. For this, the governing equation of motion of a vertical PFM is derived at a given point on the sample. Using the expansion theorem, the governing ordinary differential equations of the system and their state-space representation are derived under applied external voltage. For the proof of the concept, the results obtained from both frequency and step responses of a PFM experiment are utilized to simultaneously identify the microcantilever parameters along with local spring constant and piezoelectric coefficient of a periodically poled lithium niobate sample. In this regard, a new parameter estimation strategy is developed for modal identification of system parameters under general frequency response. Results indicate good agreements between the identified model and the experimental data using the proposed modeling and identification framework. This method can be particularly applied for accurate characterization of mechanical and piezoelectric properties of biological species and cells.

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.

The objective of this study is to propose a practical framework for simultaneous estimation of the local stiffness and piezoelectric properties of materials via piezoresponse force microscopy (PFM). For this, the governing equation of motion of a vertical PFM is derived at a given point on the sample. Using the expansion theorem, the governing ordinary differential equations (ODEs) of the system and their state-space representation are derived under applied external voltage. For the proof of the concept, the results obtained from both frequency and step responses of a PFM experiment are utilized to simultaneously identify the microcantilever parameters along with local spring constant and piezoelectric coefficient of a Periodically Poled Lithium Niobate (PPLN) sample. In this regard, a new parameter estimation strategy is developed for modal identification of system parameters under general frequency response. Results indicate good agreements between the identified model and the experimental data using the proposed modeling and identification framework.

Piezoresponse force microscopy (PFM) is proposed in this article as a new technique for identification of elastically distributed thin layers on top of microcantilever sensors. Using the conventional actuation methods such as base excitation, the ratio of stiffness over the layer mass per unit length affects the resonant frequencies of the system as a single parameter. However, due to tip/sample elastic contact in PFM, these two parameters can be separately identified using the frequency shifts before and after attaching the layer. The concept is theoretically proven here using the modal analysis of the system. For practical verification, three gold-coated AFM microcantilevers were primarily tested for their initial resonant frequencies. The Focused Ion Beam (FIB) technique was then employed to deposit thin layers of Pt-based material in different configurations on the cantilevers’ surfaces. The microcantilevers were then reexamined for their new resonances, and the properties of the deposits were identified using a robust system identification procedure. Results indicate acceptable estimation of the cantilevers’ added mass and stiffness, making the technique suitable for detection of elastically distributed biological species.Copyright © 2009 by ASME

What the authors are calling “Oklahomaness” is meant to evoke that (psychogeographic) feeling of what it is to be Oklahoman, both as experienced by Oklahomans and by those outside Oklahoma as well. While this study will delineate Oklahomaness per se, the authors hope that it will stimulate the reader's imagination so that the reader might take from it a new way of looking at, and feeling into, the human sculpting of natural and social space.

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.

Piezoresponse force microscopy (PFM) is utilized in this paper as a new technique for the identification of elastically distributed thin layers on top of microcantilever sensors. Using the conventional actuation methods such as base excitation, the ratio of stiffness over the layer mass per unit length affects the resonant frequencies of the system as a single parameter. However, due to the tip/sample elastic contact in PFM, these two parameters can be separately identified using the frequency shifts before and after attaching the layer. The concept is theoretically proven here using the modal analysis of the system. For practical verification, three gold-coated AFM microcantilevers, well suited for PFM application, were primarily tested for their initial resonant frequencies using a periodically poled lithium niobate (PPLN) piezoelectric sample. The focused ion beam (FIB) technique was then employed to deposit thin layers of Pt-based material in different configurations on the cantilevers' surfaces. The microcantilevers were then reexamined for their new resonances, and the properties of the deposits were identified using a robust system identification procedure. Results indicate acceptable estimation of the cantilevers' added mass and stiffness, making the technique suitable for detection of elastically distributed biological species. The framework proposed here can serve as an alternative approach for precise characterization of the solutions containing biomolecules (e.g., unknown DNA), their assemblies or small organisms.

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.