Molecular Biology Commons^™

Glycocalyx Mechanotransduction Mechanisms Are Involved In Renal Cancer Metastasis, Heriberto Moran, Limary M. Cancel, Peigen Huang, Sylvie Roberge, Tuoye Xu, John M. Tarbell, Lance L. Munn

Publications and Research

Mammalian cells, including cancer cells, are covered by a surface layer containing cell bound proteoglycans, glycoproteins, associated glycosaminoglycans and bound proteins that is commonly referred to as the glycocalyx. Solid tumors also have a dynamic fluid microenvironment with elevated interstitial flow. In the present work we further investigate the hypothesis that interstitial flow is sensed by the tumor glycocalyx leading to activation of cell motility and metastasis. Using a highly metastatic renal carcinoma cell line (SN12L1) and its low metastatic counterpart (SN12C) we demonstrate in vitro that the small molecule Suberoylanilide Hydroxamic Acid (SAHA) inhibits the heparan sulfate synthesis enzyme …

Sonoporation-Mediated Loading Of Trehalose In Cells For Cryopreservation., Charles W. Shaffer Iv, David F. Grimm, Michael A. Menze, Jonathan A. Kopechek

Undergraduate Research Events

Trehalose, a non-reducing disaccharide, is present in many microorganisms and metazoans. In these organisms, trehalose acts as a stress protectant and helps preserve lipid membranes of cells during states of desiccation and freezing. Trehalose is required on both sides of the cell membrane to achieve a significant cryoprotective effect. Specific loading methods for trehalose are required since this sugar is impermeant to mammalian cells. Trehalose loading in mammalian cells has been achieved by fluid-phase endocytosis and genetic modification for the expression of trehalose transporters, however cryoprotective outcomes are unable to compete with established methods of cryopreservation for mammalian cells. Sonoporation …

Secretion Of Proteins And Antibody Fragments From Transiently Transfected Endothelial Progenitor Cells, Loree Heller, Reynald Thinard, Melanie Chevalier, Sezgi Arpag, Yu Jing, Ruth Greferath, Richard Heller, Claude Nicolau

Bioelectrics Publications

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization.

Rapid Antibiotic Susceptibility Testing Platform For Direct Clinical Samples, Terrance Zhang

Honors Scholar Theses

Infectious diseases and septicemia are two of the major causes of death in the U.S., necessitating rapid treatment of septic patients with proper, efficacious antibiotics. Unfortunately, the emergence and spread of multidrug-resistant bacteria are continuously being aggravated by an abuse in antibiotic prescription at a clinical and agricultural level. It is known that antibiotic resistance evolves through the sequential accumulation of multiple mutations in bacteria, which is accentuated by prolonged exposure of bacteria to ineffective antibiotics when implementing traditional septicemia treatment. The goal of this project is to develop a novel, easy-to-use AST platform for rapid antimicrobial susceptibility profiling to …

Probing Nanoelectroporation And Resealing Of The Cell Membrane By The Entry Of Ca2+ And Ba2+ Ions, Wenfei Bo, Mantas Silkunas, Uma Mangalanathan, Vitalij Novickij, Maura Casciola, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Andrei G. Pakhomov

Bioelectrics Publications

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca²⁺, Cd²⁺, Zn²⁺, and Ba²⁺ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca²⁺ and Ba²⁺ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than >7 …

Bubble Lab Exercise, Peter Beltramo

Science and Engineering Saturday Seminars

The cell membrane is a ubiquitous component in mammalian cells which control many vital biological functions. It consists of a phospholipid bilayer with embedded protein molecules which serve to transport molecules between the interior and exterior of the cell. Understanding what makes cell membranes so important and how they function requires concepts from physics, chemistry, and of course biology, but it is difficult to learn and conceptualize the structure and function of membranes due to their nanoscopic size and dynamic nature which can’t be properly appreciated in a static textbook. This activity draws analogies between the chemistry and structure of …

Electropermeabilization Does Not Correlate With Plasma Membrane Lipid Oxidation, Olga Michel, Andrei G. Pakhomov, Maura Casciola, Jolanta Saczko, Julita Kulbacka, Olga N. Pakhomova

Bioelectrics Publications

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) …

Label-Free Microrna Optical Biosensors, Meimei Lai, Gymama Slaughter

Bioelectrics Publications

MicroRNAs (miRNAs) play crucial roles in regulating gene expression. Many studies show that miRNAs have been linked to almost all kinds of disease. In addition, miRNAs are well preserved in a variety of specimens, thereby making them ideal biomarkers for biosensing applications when compared to traditional protein biomarkers. Conventional biosensors for miRNA require fluorescent labeling, which is complicated, time-consuming, laborious, costly, and exhibits low sensitivity. The detection of miRNA remains a big challenge due to their intrinsic properties such as small sizes, low abundance, and high sequence similarity. A label-free biosensor can simplify the assay and enable the direct detection …

Modulation Of Biological Responses To 2 Ns Electrical Stimuli By Field Reversal, Esin B. Sözer, P. Thomas Vernier

Bioelectrics Publications

Nanosecond bipolar pulse cancellation, a recently discovered Phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (> 100 mu s) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). …

Development Of A Sonically Powered Biodegradable Nanogenerator For Bone Regeneration, Avi S. Patel

University Scholar Projects

Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries.

Objective: Here, we establish a biodegradable, piezoelectric …

Development Of A Sonically Powered Biodegradable Nanogenerator For Bone Regeneration, Avi Patel

Honors Scholar Theses

Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries.

Objective: Here, we establish a biodegradable, piezoelectric …

Human Islet Response To Selected Type 1 Diabetes-Associated Bacteria: A Transcriptome-Based Study, Ahmed M. Abdellatif, Heather Jensen Smith, Robert Z. Harms, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from destruction of pancreatic β-cells. T1D subjects were recently shown to harbor distinct intestinal microbiome profiles. Based on these findings, the role of gut bacteria in T1D is being intensively investigated. The mechanism connecting intestinal microbial homeostasis with the development of T1D is unknown. Specific gut bacteria such as Bacteroides dorei (BD) and Ruminococcus gnavus (RG) show markedly increased abundance prior to the development of autoimmunity. One hypothesis is that these bacteria might traverse the damaged gut barrier, and their constituents elicit a response from human islets that causes …

Tracing Actin Filament Bundles In Three-Dimensional Electron Tomography Density Maps Of Hair Cell Stereocilia, Salim Sazzed, Junha Song, Julio Kovacs, Willi Wriggers, Manfred Auer, Jing He

Computer Science Faculty Publications

Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia. Stereocilia are pivotal to the mechanoelectrical transduction process in inner ear sensory epithelial hair cells. Given the complexity and dense arrangement of actin …

Quantum Confined Peptide Assemblies With Tunable Visible To Near-Infrared Spectral Range, Kai Tao, Zhen Fan, Leming Sun, Pandeeswar Makam, Zhen Tian, Mark Ruegsegger, Shira Shaham-Niv, Derek Hansford, Ruth Aizen, Zui Pan, Scott Galster, Jianjie Ma, Fan Yuan, Mingsu Si, Songnan Qu, Mingjun Zhang, Ehud Gazit, Junbai Li

Faculty & Staff Scholarship

Quantum confined materials have been extensively studied for photoluminescent applica- tions. Due to intrinsic limitations of low biocompatibility and challenging modulation, the utilization of conventional inorganic quantum confined photoluminescent materials in bio- imaging and bio-machine interface faces critical restrictions. Here, we present aromatic cyclo-dipeptides that dimerize into quantum dots, which serve as building blocks to further self-assemble into quantum confined supramolecular structures with diverse morphologies and photoluminescence properties. Especially, the emission can be tuned from the visible region to the near-infrared region (420 nm to 820 nm) by modulating the self-assembly process. Moreover, no obvious cytotoxic effect is observed for …

Graphene Nanoplatelets-Sericin Surface-Modified Gum Alloy For Improved Biological Response, Valentina Mitran, Valentina Dinca, Raluca Ion, Vasile D. Cojocaru, Patricia Neacsu, Cerasela Zoica Dinu, Laurentiu Rusen, Simona Brajnicov, Anca Bonciu, Maria Dinescu, Doina Raducanu, Ioan Dan

Faculty & Staff Scholarship

In this study a “Gum Metal” titanium-based alloy, Ti-31.7Nb-6.21Zr-1.4Fe-0.16O, was synthesized by melting and characterized in order to evaluate its potential for biomedical applications. The results showed that the newly developed alloy presents a very high strength, high plasticity and a low Young's modulus relative to titanium alloys currently used in medicine. For further bone implant applications, the newly synthesized alloy was surface modified with graphene nanoplatelets (GNP), sericin (SS) and graphene nanoplatelets/sericine (GNP–SS) composite films via Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique. The characterization of each specimen was monitored by scanning electron microscopy (SEM), atomic force microscopy (AFM), …

Targeting Ovarian Cancer And Endothelium With An Allosteric Ptp4a3 Phosphatase Inhibitor, Kelley E. Mcqueeney, Joseph M. Salamoun, James C. Burnett, Nektarios Barabutis, Paula Pekic, Sophie L. Lewandowski, Danielle C. Llaneza, Robert Cornelison, Yunpeng Bai, Zhong-Yin Zhang, John D. Catravas

Bioelectrics Publications

Overexpression of protein tyrosine phosphatase PTP4A oncoproteins is common in many human cancers and is associated with poor patient prognosis and survival. We observed elevated levels of PTP4A3 phosphatase in 79% of human ovarian tumor samples, with significant overexpression in tumor endothelium and pericytes. Furthermore, PTP4A phosphatases appear to regulate several key malignant processes, such as invasion, migration, and angiogenesis, suggesting a pivotal regulatory role in cancer and endothelial signaling pathways. While phosphatases are attractive therapeutic targets, they have been poorly investigated because of a lack of potent and selective chemical probes. In this study, we disclose that a potent, …

Wild-Type P53 Enhances Endothelial Barrier Function By Mediating Rac1 Signalling And Rhoa Inhibition, Nektarios Barabutis, Christiana Dimitropoulou, Betsy Gregory, John D. Catravas

Bioelectrics Publications

Inflammation is the major cause of endothelial barrier hyper-permeability, associated with acute lung injury and acute respiratory distress syndrome. This study reports that p53 "orchestrates" the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1 and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1- and P21-activated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and cofilin, in contrast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of phospho-cofilin. 17AAG treatment …

Asymmetric Patterns Of Small Molecule Transport After Nanosecond And Microsecond Electropermeabilization, Esin B. Sözer, C. Florencia Pocetti, P. Thomas Vernier

Bioelectrics Publications

Imaging of fluorescent small molecule transport into electropermeabilized cells reveals polarized patterns of entry, which must reflect in some way the mechanisms of the migration of these molecules across the compromised membrane barrier. In some reports, transport occurs primarily across the areas of the membrane nearest the positive electrode (anode), but in others cathode-facing entry dominates. Here we compare YO-PRO-1, propidium, and calcein uptake into U-937 cells after nanosecond (6 ns) and microsecond (220 µs) electric pulse exposures. Each of the three dyes exhibits a different pattern. Calcein shows no preference for anode- or cathode-facing entry that is detectable with …

Dielectric Properties Of Isolated Adrenal Chromaffin Cells Determined By Microfluidic Impedance Spectroscopy, A. C. Sabuncu, M. Stacey, G. L. Graviso, N. Semenova, P. T. Vernier, N. Leblanc, I. Chatterjee, J. Zaklit

Bioelectrics Publications

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1 kHz and 20 MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner …

Computing Spatiotemporal Heat Maps Of Lipid Electropore Formation: A Statistical Approach, Willy Wriggers, Frederica Castellani, Julio A. Kovacs, P. Thomas Vernier

Mechanical & Aerospace Engineering Faculty Publications

We extend the multiscale spatiotemporal heat map strategies originally developed for interpreting molecular dynamics simulations of well-structured proteins to liquids such as lipid bilayers and solvents. Our analysis informs the experimental and theoretical investigation of electroporation, that is, the externally imposed breaching of the cell membrane under the influence of an electric field of sufficient magnitude. To understand the nanoscale architecture of electroporation, we transform time domain data of the coarse-grained interaction networks of lipids and solvents into spatial heat maps of the most relevant constituent molecules. The application takes advantage of our earlier graph-based activity functions by accounting for …

A Balanced Approach To Adaptive Probability Density Estimation, Julio Kovacs, Cailee Helmick, Willy Wriggers

Mechanical & Aerospace Engineering Faculty Publications

Our development of a Fast (Mutual) Information Matching (FIM) of molecular dynamics time series data led us to the general problem of how to accurately estimate the probability density function of a random variable, especially in cases of very uneven samples. Here, we propose a novel Balanced Adaptive Density Estimation (BADE) method that effectively optimizes the amount of smoothing at each point. To do this, BADE relies on an efficient nearest-neighbor search which results in good scaling for large data sizes. Our tests on simulated data show that BADE exhibits equal or better accuracy than existing methods, and visual tests …

Quantitative Limits On Small Molecule Transport Via The Electropermeome - Measuring And Modeling Single Nanosecond Perturbations, Esin B. Sözer, Zachary A. Levine, P. Thomas Vernier

Bioelectrics Publications

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated …

Engineering A Fluorescent Protease Sensor For In Vivo Protein Detection, Thomas C. Kinard

Honors Scholar Theses

This report details the results of an ongoing project to engineer a mutant form of Red Fluorescent Protein (RFP) variant mCherry that acts as a real-time in vivo protease sensor. The sought-after mutant only becomes fluorescent when exposed to Tobacco Etch Virus (TEV) Protease, this system’s model protease. This will be accomplished via the insertion of the TEV Protease Recognition Site (TEV-PRS) in such a position that, before cleavage, will prevent the protein from folding to fluorescent conformation, but upon cleavage, will allow for fluorescent conformation to occur. The cylindrical structure of the protein, composed of beta-pleated sheets, contains “loops” …

Biochemical And Histological Differences Between Costal And Articular Cartilages, Michael W. Stacey

Bioelectrics Publications

Biologically, costal cartilage is an understudied tissue type and much is yet to be learned regarding underlying mechanisms related to form and function, and how these relate to disease states, specifically chest wall deformity. Chest wall deformities have a component of inheritance, implying underlying genetic causes; however the complexity of inheritance suggests multiple genetic components. At our Centre investigations were performed on gene expression of key select genes from costal cartilage removed at surgery of patients with chest wall deformity to show high expression of decorin, a key player in collagen fiber formation and growth. Also, the degree of tissue …

Hiv Vaccines: Progress, Limitations And A Crispr/Cas9 Vaccine, Omar A. Garcia Martinez

Biology: Student Scholarship & Creative Works

ABSTRACT: The HIV-1 pandemic continues to thrive due to ineffective HIV-1 vaccines. Historically, the world’s most infectious diseases, such as polio and smallpox, have been eradicated or have come close to eradication due to the advent of effective vaccines. Highly active antiretroviral therapy is able to delay the onset of AIDS but can neither rid the body of HIV-1 proviral DNA nor prevent further transmission. A prophylactic vaccine that prevents the various mechanisms HIV-1 has to evade and attack our immune system is needed to end the HIV-1 pandemic. Recent advances in engineered nuclease systems, like the CRISPR/Cas9 system, have …

Stabilizing The Cold Plasma-Stimulated Medium By Regulating Medium’S Composition, Dayun Yan, Niki Nourmohammadi, Ka Bian, Ferid Murad, Jonathan H. Sherman, Michael Keidar

Biochemistry and Molecular Medicine Faculty Publications

Over past several years, the cold plasma-stimulated medium (PSM) has shown its remarkable anti-cancer capacity in par with the direct cold plasma irradiation on cancer cells or tumor tissues. Independent of the cold plasma device, PSM has noticeable advantage of being a flexible platform in cancer treatment. Currently, the largest disadvantage of PSM is its degradation during the storage over a wide temperature range. So far, to stabilize PSM, it must be remained frozen at −80 °C. In this study, we first reveal that the degradation of PSM is mainly due to the reaction between the reactive species and specific …

Quantitative Comparison Of A Nanoengineered Alumina Coated Cnt Arrays To Sio2 Coated Cnts And Solution Based Delivery System, Shree Aier

Undergraduate Research & Mentoring Program

To meet the growing need for nanoengineered biocompatible materials to serve as drug delivery platforms, in this research, carbon nanotube arrays were fabricated by chemical vapor deposition, followed by an alumina coating by the high yielding, tightly controlled atomic layer deposition. This nanoengineered vertically aligned alumina nanowire array serves as a platform for delivering antigens, which act as cancer adjuvants. The physicochemical characteristics of the nanowires (NWs) can significantly influence the delivery of a biomolecule to immune cells. To investigate the material characteristics, the delivery efficiency of the antigen using NWs was quantitatively assessed by flow cytometry. Further, the mechanism …

Effects Of High Voltage Nanosecond Electric Pulses On Eukaryotic Cells (In Vitro): A Systematic Review, Tina Batista Napotink, Matej Reberšek, P. Thomas Vernier, Babara Mali, Damijan Miklavčič

Bioelectrics Publications

For this systematic review, 203 published reports on effects of electroporation using nanosecond high-voltage electric pulses (nsEP) on eukaryotic cells (human, animal, plant) in vitro were analyzed. A field synopsis summarizes current published data in the field with respect to publication year, cell types, exposure configuration, and pulse duration. Published data were analyzed for effects observed in eight main target areas (plasma membrane, intracellular, apoptosis, calcium level and distribution, survival, nucleus, mitochondria, stress) and an additional 107 detailed outcomes. We statistically analyzed effects of nsEP with respect to three pulse duration groups: A: 1–10 ns, B: 11–100 ns and C: …

Altered Cd161bright Cd8+ Mucosal Associated Invariant T (Mait)-Like Cell Dynamics And Increased Differentiation States Among Juvenile Type 1 Diabetics., Robert Z. Harms, Kristina M. Lorenzo, Kevin P. Corley, Monina S. Cabrera, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Type 1A diabetes (T1D) is believed to be caused by immune-mediated destruction of β-cells, but the immunological basis for T1D remains controversial. Microbial diversity promotes the maturation and activation of certain immune subsets, including CD161bright CD8+ mucosal associated invariant T (MAIT) cells, and alterations in gut mucosal responses have been reported in type 1 diabetics (T1Ds). We analyzed T cell populations in peripheral blood leukocytes from juvenile T1Ds and healthy controls. We found that proportion and absolute number of MAIT cells were similar between T1Ds and controls. Furthermore, while MAIT cell proportions increased with age among healthy controls, this trend …

Diffuse, Non-Polar Electropermeabilization And Reduced Propidium Uptake Distinguish The Effect Of Nanosecond Electric Pulses, Iurii Semenov, Christian W. Zemlin, Olga N. Pakhomova, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Ca²⁺ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca²⁺ transients was expectedly higher for nsEP (36 kV/cm)than forms EP (0.09 kV/cm) but the respective doses were similar (190 and460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca²⁺ level by N10 nM in cells with blocked voltage-gated Ca²⁺ channels and depleted Ca²⁺ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These …