Engineering Commons^™

Control Of The Electroporation Efficiency Of Nanosecond Pulses By Swinging The Electric Field Vector Direction, Vitalii Kim, Iurii Semenov, Allen S. Kiester, Mark A. Keppler, Bennett L. Ibey, Joel N. Bixler, Ruben M. L. Colunga Biancatelli, Andrei G. Pakhomov

Bioelectrics Publications

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as “bipolar cancellation,” enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing …

Nanosecond Pulsed Electric Field Modulates Electron Transport And Mitochondrial Structure And Function, Lucas Nelson Potter

Biomedical Engineering Theses & Dissertations

Pulsed power treatment has been used to induce regulated cell death (RCD) in cells or ablate tumors in animals. A subset of pulsed power as electroporation with high voltage and pulse duration of milliseconds is used for biomedical treatment to induce pores in the plasma membrane of cells. Nanosecond Pulsed Electric Fields (nsPEFs)– an extension of electroporation, uses waveforms with pulse durations on the order of 10-900 nanoseconds. nsPEF treatment has demonstrated intracellular effects for potential biomedical applications. In this work, nsPEF treatment is used to demonstrate changes that affect viability, plasma membrane permeability ROS (Reactive Oxygen Species) in the …

E-Cadherin Force Transmission And Stiffness Sensing, Mazen Mezher

Mechanical & Aerospace Engineering Theses & Dissertations

E-cadherin is the chief mediator of cell-cell adhesion between epithelial cells and is a known mechanosensor. Force transmission and stiffness sensing are two crucial aspects of E-cadherin mechanobiology.

E-cadherin has an extracellular adhesive region, a transmembrane region and an intracellular region that binds to adhesion-associated proteins. Here, we assessed how different factors affect the level of force transmission (i) from inside the cell such as adhesion-associated proteins, (ii) on the cell membrane, such as growth factor receptors and (iii) outside the cell, such as different binding partners in adhesion. To study the level of force transmission inside the cell, we …

Type-B Energetic Processes And Their Associated Scientific Implications, James Weifu Lee

Chemistry & Biochemistry Faculty Publications

Recently, our work has identified two thermodynamically distinct types (A and B) of energetic processes naturally occurring on Earth. Type-A energy processes such as the classical heat engines, ATP hydrolysis, and many of the known chemical, electrical, and mechanical processes apparently follow well the second law of thermodynamics; Type-B energy processes, for example, the newly discovered thermotropic function that isothermally utilizes environmental heat energy to do useful work in driving ATP synthesis, follow the first law of thermodynamics (conservation of mass and energy) but do not have to be constrained by the second law, owing to its special asymmetric functions. …

Evaluation Of Horizontal Gene Transfer Between Genetically Engineered Cyanobacteria And Gram-Negative Bacteria, Andriana Chrysovalanti Zourou

Chemistry & Biochemistry Theses & Dissertations

As the world population is increasing and societies become more technology driven, there is an imperative to develop ‘green energy’ sources to protect our planet. Cyanobacteria that have been genetically engineered to produce organic compounds that may be burnt as fuels show great potential, as they are an environmentally friendly and self-renewable, net carbon-neutral option. However, there are potential risks in the development and use of genetically modified organisms (GMOs). We need to understand in advance the risks that GMOs may pose to our environment and to animal and human health. This will enable experimental procedures, containment strategies and policies …

Electrohydrodynamic Simulations Of Capsule Deformation Using A Dual Time-Stepping Lattice Boltzmann Scheme, Charles Leland Armstrong

Mathematics & Statistics Theses & Dissertations

Capsules are fluid-filled, elastic membranes that serve as a useful model for synthetic and biological membranes. One prominent application of capsules is their use in modeling the response of red blood cells to external forces. These models can be used to study the cell’s material properties and can also assist in the development of diagnostic equipment. In this work we develop a three dimensional model for numerical simulations of red blood cells under the combined influence of hydrodynamic and electrical forces. The red blood cell is modeled as a biconcave-shaped capsule suspended in an ambient fluid domain. Cell deformation occurs …

Cyclophilin D Is A Sensor Of Nano-Pulse Stimulation, Brittney Ruedlinger, Bani Hani Maisoun, Lucas Potter, Nicola Lai, Stephen J. Beebe

The Graduate School Posters

Nano-Pulse Stimulation (NPS), a pulsed power-derived technology, stimulates structural and functional changes in plasma membranes and cellular organelles. NPS induces a Ca²⁺ influx and opening of the mitochondrial permeability transition pore (mPTP) that dissipates the mitochondrial membrane potential (ΔΨ_m) and, when sustained, induces regulated cell death. Here we show that in rat cardiomyoblasts (H9C2) cyclophilin D (CypD) is a mitochondrial sensor for NPS as defined by observations that loss of ΔΨ_m is Ca²⁺ and mitochondrial reactive oxygen species (mROS) dependent and cyclosporin A (CsA)-sensitive, which are diagnostic qualities for effects on CypD and the mPTP. …

Biomechanical And Biophysical Properties Of Breast Cancer Cells Under Varying Glycemic Regimens, Diganta Dutta, Xavier-Lewis Palmer, Jose Ortega-Rodas, Vasundhara Balraj, Indrani Ghosh Dastider, Surabhi Chandra

Electrical & Computer Engineering Faculty Publications

Diabetes accelerates cancer cell proliferation and metastasis, particularly for cancers of the pancreas, liver, breast, colon, and skin. While pathways linking the 2 disease conditions have been explored extensively, there is a lack of information on whether there could be cytoarchitectural changes induced by glucose which predispose cancer cells to aggressive phenotypes. It was thus hypothesized that exposure to diabetes/high glucose alters the biomechanical and biophysical properties of cancer cells more than the normal cells, which aids in advancing the cancer. For this study, atomic force microscopy indentation was used through microscale probing of multiple human breast cancer cells (MCF-7, …

Validation Of Nanosecond Pulse Cancellation Using A Quadrupole Exposure System, Hollie A. Ryan

Biomedical Engineering Theses & Dissertations

Nanosecond pulsed electric fields (nsPEFs) offer a plethora of opportunities for developing integrative technologies as complements or alternatives to traditional medicine. Studies on the biological effects of nsPEFs in vitro and in vivo have revealed unique characteristics that suggest the potential for minimized risk of complications in patients, such as the ability of unipolar nsEPs to create permanent or transient pores in cell membranes that trigger localized lethal or non-lethal outcomes without consequential heating. A more recent finding was that such responses could be diminished by applying a bipolar pulse instead, a phenomenon dubbed bipolar cancellation, paving the way …

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.

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 …

Size-Dependent Inhibitory Effects Of Antibiotic Nanocarriers On Filamentation Of E. Coli, Preeyaporn Songkiatisak, Feng Ding, Pavan Kumar Cherukuri, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Multidrug membrane transporters exist in both prokaryotic and eukaryotic cells and cause multidrug resistance (MDR), which results in an urgent need for new and more effective therapeutic agents. In this study, we used three different sized antibiotic nanocarriers to study their mode of action and their size-dependent inhibitory effects against Escherichia coli (E. coli). Antibiotic nanocarriers (AgMUNH–Oflx NPs) with 8.6 × 10², 9.4 × 10³ and 6.5 × 10⁵ Oflx molecules per nanoparticle (NP) were prepared by functionalizing Ag NPs (2.4 ± 0.7, 13.0 ± 3.1 and 92.6 ± 4.4 nm) with a monolayer …

Impedance Analysis Of Tissues In Nspef Treatment For Cancer Therapy, Edwin Ayobami Oshin

Biomedical Engineering Theses & Dissertations

Nanosecond pulsed electric field (nsPEF) for cancer therapy is characterized by applications of high voltage pulses with low pulsed energy to induce non-thermal effects on tissues such as tumor ablation. It nonthermally treats tissues via electroporation. Electroporation is the increase in permeabilization of a cell membrane due to the application of high pulsed electric field. The objective of this study was to investigate the effect of nsPEF on tissue by monitoring the tissue’s impedance in real-time. Potato slices (both untreated and electroporated), and tumors extracted from female BALBc mice were studied. 100ns, 1-10kV pulses were applied to the tissues using …

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 …

Endogenous Force Transmission Between Epithelial Cells And A Role For Α-Catenin, Sandeep Dumbali

Mechanical & Aerospace Engineering Theses & Dissertations

In epithelial tissues, epithelial cells adhere to each other as well as to the underlying extra-cellular matrix (ECM). E-cadherin-based intercellular junctions play an important role in tissue integrity. These junctions experience cell-generated mechanical forces via apparent adaptor proteins such as beta (β) catenin, alpha (α) catenin and vinculin. Abnormalities in these junctions may result in skin related diseases and cancers. Here, I devised methods to determine the endogenous intercellular force within cell pairs as well as in large epithelial islands. I further ascertained the factors that affect the level of inter-cellular tension.

Experiments with pairs of epithelial cells exogenously expressing …

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). …

Lipid Extraction From Spirulina Sp. And Schizochytrium Sp. Using Supercritical Co2 With Methanol, Shihong Liu, Husam A. Abu Hajar, Guy Riefler, Ben J. Stuart

Civil & Environmental Engineering Faculty Publications

Microalgae are one of the most promising feedstocks for biodiesel production due to their high lipid content and easy farming. However, the extraction of lipids from microalgae is energy intensive and costly and involves the use of toxic organic solvents. Compared with organic solvent extraction, supercritical CO₂ (SCCO₂) has demonstrated advantages through lower toxicity and no solvent-liquid separation. Due to the nonpolar nature of SCCO_2, polar organic solvents such as methanol may need to be added as a modifier in order to increase the extraction ability of SCCO₂. In this paper, pilot scale lipid …

Investigation Of Electrolytic Flocculation For Microalga Scenedesmus Sp Using Aluminum And Graphite Electrodes, Shihong Liu, Husam A. Abu Hajar, Guy Riefler, Ben J. Stuart

Civil & Environmental Engineering Faculty Publications

Electrolytic flocculation using non-sacrificial electrodes with flocculants added was studied on harvesting Scenedesmus sp. In order to optimize the operating conditions of the electrolytic flocculation process and to quantify the amount of flocculants added, aluminum electrodes were first used in the process. It was found that under optimal conditions, the microalgae removal efficiency using aluminum electrodes could reach 98.5%, while 34.2 mg L^-1 of aluminum ions were released during the process. Different metal electrodes were also studied, but high microalgae removal efficiency was witnessed only using aluminum electrodes, indicating the influence of the aluminum ion in flocculation. When non-sacrificial …

Accurate Flexible Refinement Of Atomic Models Against Medium-Resolution Cryo-Em Maps Using Damped Dynamics, Julio A. Kovacs, Vitold E. Galkin, Willy Wriggers

Mechanical & Aerospace Engineering Faculty Publications

Background: Dramatic progress has recently been made in cryo-electron microscopy technologies, which now make possible the reconstruction of a growing number of biomolecular structures to near-atomic resolution. However, the need persists for fitting and refinement approaches that address those cases that require modeling assistance.

Methods: In this paper, we describe algorithms to optimize the performance of such medium-resolution refinement methods. These algorithms aim to automatically optimize the parameters that define the density shape of the flexibly fitted model, as well as the time-dependent damper cutoff distance. Atomic distance constraints can be prescribed for cases where extra containment of parts of …

Characterization And Analysis Of Ultrathin Cigs Films And Solar Cells Deposited By 3-Stage Process, Grace Rajan, Krishna Aryal, Shankar Karki, Puruswottam Aryal, Robert W. Collins, Sylvain Marsillac

Electrical & Computer Engineering Faculty Publications

In view of the large-scale utilization of Cu(In,Ga)Se₂ (CIGS) solar cells for photovoltaic application, it is of interest not only to enhance the conversion efficiency but also to reduce the thickness of the CIGS absorber layer in order to reduce the cost and improve the solar cell manufacturing throughput. In situ and real-time spectroscopic ellipsometry (RTSE) has been used conjointly with ex situ characterizations to understand the properties of ultrathin CIGS films. This enables monitoring the growth process, analyzing the optical properties of the CIGS films during deposition, and extracting composition, film thickness, grain size, and surface roughness which …

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 …

Study Of Abc Membrane Transporters In Single Live Cells, Preeyaporn Songkiatisak

Chemistry & Biochemistry Theses & Dissertations

The multidrug ATP-binding cassette (ABC) membrane transporters (efflux pumps) are found in both prokaryotes and eukaryotes and they can extrude diverse structurally unrelated substrates, such as antibiotics and chemotherapeutic agents out of the cells. The efflux pumps are responsible for multidrug resistance (MDR) and the failure of numerous treatments in infections and cancers. All ABC membrane transporters share a common modular topology containing two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The underlying molecular mechanisms regarding how the similar structural ABC membrane transporters could selectively extrude a wide variety of substrates and cause MDR, are not yet fully …

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 …

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, …

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 …

Transport Of Charged Small Molecules After Electropermeabilization - Drift And Diffusion, Esin B. Sözer, C. Florencia Pocetti, P. Thomas Vernier

Bioelectrics Publications

Background: Applications of electric-field-induced permeabilization of cells range from cancer therapy to wastewater treatment. A unified understanding of the underlying mechanisms of membrane electropermeabilization, however, has not been achieved. Protocols are empirical, and models are descriptive rather than predictive, which hampers the optimization and expansion of electroporation-based technologies. A common feature of existing models is the assumption that the permeabilized membrane is passive, and that transport through it is entirely diffusive. To demonstrate the necessity to go beyond that assumption, we present here a quantitative analysis of the post-permeabilization transport of three small molecules commonly used in electroporation research-YO-PRO-1, propidium, …

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 …

Activation Of The Phospholipid Scramblase Tmem16f By Nanosecond Pulsed Electric Field (Nspef) Facilitates Its Diverse Cytophysiological Effects, Claudia Muratori, Andrei G. Pakhomov, Elena Gianulis, Jade Meads, Maura Casciola, Peter A. Mollica, Olga N. Pakhomova

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca²⁺-dependent phospholipid scramblase and Ca²⁺-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, …

Perspective: The Physics, Diagnostics, And Applications Of Atmospheric Pressure Low Temperature Plasma Sources Used In Plasma Medicine, M. Laroussi

Electrical & Computer Engineering Faculty Publications

Low temperature plasmas have been used in various plasma processing applications for several decades. But it is only in the last thirty years or so that sources generating such plasmas at atmospheric pressure in reliable and stable ways have become more prevalent. First, in the late 1980s, the dielectric barrier discharge was used to generate relatively large volume diffuse plasmas at atmospheric pressure. Then, in the early 2000s, plasma jets that can launch cold plasma plumes in ambient air were developed. Extensive experimental and modeling work was carried out on both methods and much of the physics governing such sources …