Molecular, Cellular, and Tissue Engineering Commons^™

A Purification And In Vitro Activity Assay For A (P)Ppgpp Synthetase From Clostridium Difficile, Astha Pokhrel, Asia Poudel, Erin B. Purcell

Chemistry & Biochemistry Faculty Publications

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of γ-³²-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for …

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 …

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 …

A 'Tissue Model' To Study The Barrier Effects Of Living Tissues On The Reactive Species Generated By Surface Air Discharge, Tongtong He, Dingxin Liu, Han Xu, Zhichao Liu, Dehui Xu, Dong Li, Qiosong Li, Mingzhe Rong, Michael G. Kong

Bioelectrics Publications

Gelatin gels are used as surrogates of human tissues to study their barrier effects on incoming reactive oxygen and nitrogen species (RONS) generated by surface air discharge. The penetration depth of nitrite into gelatin gel is measured in real time during plasma treatment, and the permeabilities of nitrite, nitrate, O₃ and H₂O₂ through gelatin gel films are quantified by measuring their concentrations in the water underneath such films after plasma treatment. It is found that the penetration speed of nitrite increases linearly with the mass fraction of water in the gelatin gels, and the permeabilities of …

Membrane Channel Gene Expression In Human Costal And Articular Chondrocytes, A. Asmar, R. Barrett-Jolley, A. Werner, R. Kelly Jr., M. Stacey

Bioelectrics Publications

Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Sch€onberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human …

Temporal Modulation Of Plasma Species In Atmospheric Dielectric Barrier Discharges, Aijun Yang, Xiaohua Wang, Dingxin Liu, Mingzhe Rong, Michael G. Kong

Bioelectrics Publications

The atmospheric pressure dielectric barrier discharge in helium is a pulsed discharge in nature and the moment of maximum species densities is almost consistent with peak discharge current density. In this paper, a one-dimensional fluid model is used to investigate the temporal structure of plasma species in an atmospheric He-N₂ dielectric barrier discharge (DBD). It is demonstrated that there exist microsecond delays of the moments of the maximum electron and ion densities from the peak of discharge current density. These time delays are caused by a competition between the electron impact and Penning ionizations, modulated by the N_{2 …}

The Use Of An In Vitro 3d Melanoma Model To Predict In Vivo Plasmid Transfection Using Electroporation, Benadette Marrero, Richard Heller

Bioelectrics Publications

A large-scale in vitro 3D tumor model was generated to evaluate gene delivery procedures in vivo. This 3D tumor model consists of a "tissue-like" spheroid that provides a micro-environment supportive of melanoma proliferation, allowing cells to behave similarly to cells in vivo. This functional spheroid measures approximately 1 cm in diameter and can be used to effectively evaluate plasmid transfection when testing various electroporation (EP) electrode applicators. In this study, we identified EP conditions that efficiently transfect green fluorescent protein (GFP) and interleukin 15 (IL-15) plasmids into tumor cells residing in the 3D construct. We found that plasmids …

Simulation Of Temperature Distributions In Tissue Upon Laser Pulse Irradiation Including Water Mass Transfer, Deepthi Nagulapally

Electrical & Computer Engineering Theses & Dissertations

Lasers, due to their unique characteristics, have tremendous applications in the medical field. Generally all laser applications in hie-medicine involve the propagation of laser energy through tissue. Energy is deposited giving rise to many physical, hie-chemical, and mechanical effects. Among these are dynamical changes in the local temperature distributions within the tissue. Analysis of the temperature changes requires the creation of an appropriate optical transmission and heat flow model that incorporates user specified laser parameters. Here, a numerical simulation model was developed to study both steady state and time dependent temperature distributions due to pulsed laser radiation within user specified …

Effects Of Non-Equilibrium Plasma On Eukaryotic Cells (Final Report: Grant Fa9550-06-1-0004), Mounir Laroussi, Fred C. Dobbs, Old Dominion University

Electrical & Computer Engineering Faculty Publications

This document is our final report describing the research activities carried out under AFOSR Grant FA9550-06-1-0004. First, descriptions of our cold plasma generation systems are presented. Two systems, developed with past and present AFOSR support, are available in our laboratory. The first is a pulsed device capable of emitting a cold plasma plume in room air. The second is an air plasma generator the core of which is a dielectric barrier discharge excited by a high AC voltage. Following these brief descriptions we first present the effects of an atmospheric pressure air plasma on four different types of eukaryotic microalgae. …

Stochastic Calculations For Computation Of Radiation Effects And Cell Survivability Under Voltage Pulsing, Madhuri Ganapathiraju

Electrical & Computer Engineering Theses & Dissertations

Statistical computations are an important tool for the analysis of stochastic phenomena and processes that are characterized by variability. Biological systems (e.g., cells, tissues etc.) are perfect examples wherein response to a given external stimulus can be varied and needs to be adequately considered. The Monte Carlo method of analysis has now been recognized as the most effective way of treating stochastic variability.

This thesis uses Monte Carlo based simulations to probe two problems that require the quantification and modeling of effects caused by energy deposition onto biological matter from external sources. One problem involves the probabilistic study of the …

Nanoelectropulse-Driven Membrane Perturbation And Small Molecule Permeabilization, P. Thomas Vernier, Yinghua Sun, Martin A. Gundersen

Bioelectrics Publications

Background
Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization.

Results
In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to …

Stimulation Of Capacitative Calcium Entry In Hl-60 Cells By Nanosecond Pulsed Electric Fields, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (similar to100 nM), while the uptake of …

Time Domain Dielectric Spectroscopy Analysis Of Hl-60 Cell Suspensions After Microsecond And Submicrosecond Pulsed Electric Fields, Allen Laurence Garner

Electrical & Computer Engineering Theses & Dissertations

Pulsed electric fields (PEF's) above a certain voltage threshold cause electroporation for microsecond pulses and intracellular effects for submicrosecond pulses. Models describing these effects often depend on the electrical properties of the cell, which are altered by the PEF. Time domain dielectric spectroscopy was used to provide data for these models and to measure changes in the conductivity of HL-60 cell suspensions due to single 50 μs and 10 ns PEF's of the same energy. For 1.1 kV/cm, 50 μs pulses, the conductivity rose within a minute after the pulse and dropped dramatically approximately forty minutes after the pulse. For …

Brownian Dynamics Simulation Studies Of Ion Throughput During Cellular Electroporation, Juan A. Gonzalez-Cuevas

Electrical & Computer Engineering Theses & Dissertations

The purpose of the present research work is to analyze the flow of ions through pores formed in the plasma membrane of biological cells during electroporation. In this regard, simulations have been conducted to track the movement of ions in 3-D space based on a Brownian dynamics model, which is mostly deterministic but has some stochastic properties to account for ionic scattering. From the Brownian dynamics simulations, it has been possible to estimate the pore's conductance as well as transport parameters such as the diffusion coefficient and mobility within the pore region, of which there is no experimental data currently …

Simulation Studies Of Cellular Electroporation Due To Ultra-Fast Electric Pulses, Gayathri R. Barthaki

Electrical & Computer Engineering Theses & Dissertations

Biological systems, from cells to ecosystems, are intrinsically complex information-processing networks. It often becomes difficult to predict their behavior during an experimental investigation, as their behavior is fundamentally chaotic, Hence, building models that mimic the dynamics of such systems and performing computer simulations would help in understanding, predicting, controlling and optimizing these systems. This thesis is aimed at the development of a mathematical model for the process of electroporation in biological cells when they are subjected to ultra-fast electric pulses and at conducting simulations to understand its dynamic behavior with special emphasis on the physics of ultra-short time scales.

With …

Retention Of Cellular Viability And Growth Potential In Cryopreserved Bovine Arterial Tissue: Implications For Clinical Use Of Cryopreserved Vascular Allografts, Kip G. Gardner

Biological Sciences Theses & Dissertations

Segments of bovine calf aortas and pulmonary arteries were cryopreserved at -196° C for two weeks to 18 months and subsequently thawed. Cellular morphology, viability, and growth potential in these segments were then compared with the same attributes in segments from fresh (control) arteries. Scanning electron microscopy revealed no disruption of intimal surfaces attributable to cryopreservation. Endothelial cells were successfully cultured from all cryopreserved arterial segments. Endothelial monolayers grown from cryopreserved arteries had a cobblestone appearance and expressed Factor VIII antigen but not smooth muscle alpha-actin. Plating efficiency in primary cultures of endothelial cells was lower for cryopreserved arteries than …