Life Sciences Commons^™

High-Throughput Single-Molecule Telomere Characterization, Jennifer Mccaffrey, Eleanor Young, Katy Lassahn, Justin Sibert, Steven Pastor, Harold Riethman, Ming Xiao

School of Medical Diagnostics & Translational Sciences Faculty Publications

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG) n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG) n tract length at the end of each …

Delayed Hypersensitivity To Nanosecond Pulsed Electric Field In Electroporated Cells, Sarah D. Jensen, Vera A. Khorokhorina, Claudia Muratori, Andrei G. Pakhomov, Olga N. Pakhomova

Bioelectrics Publications

We demonstrate that conditioning of mammalian cells by electroporation with nanosecond pulsed electric field (nsPEF) facilitates their response to the next nsPEF treatment. The experiments were designed to unambiguously separate the electroporation-induced sensitization and desensitization effects. Electroporation was achieved by bursts of 300-ns, 9 kV/cm pulses (50 Hz, n = 3–100) and quantified by propidium dye uptake within 11 min after the nsPEF exposure. We observed either sensitization to nsPEF or no change (when the conditioning was either too weak or too intense, or when the wait time after conditioning was too short). Within studied limits, conditioning never caused desensitization. …

Damage-Free Peripheral Nerve Stimulation By 12-Ns Pulsed Electric Field, Maura Casciola, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Modern technologies enable deep tissue focusing of nanosecond pulsed electric field (nsPEF) for non-invasive nerve and muscle stimulation. However, it is not known if PEF orders of magnitude shorter than the activation time of voltage-gated sodium channels (VGSC) would evoke action potentials (APs). One plausible scenario requires the loss of membrane integrity (electroporation) and resulting depolarization as an intermediate step. We report, for the first time, that the excitation of a peripheral nerve can be accomplished by 12-ns PEF without electroporation. 12-ns stimuli at 4.1-11 kV (3.3-8.8 kV/cm) evoked APs similarly to conventional stimuli (100-250 mus, 1-5 V, 103-515 V/m), …

Controllable Moderate Heating Enhances The Therapeutic Efficacy Of Irreversible Electroporation For Pancreatic Cancer, Chelsea M. Edelblute, James Hornef, Niculina I. Burcus, Thomas Norman, Stephen J. Beebe, Karl Schoenbach, Richard Heller, Chunqi Jiang, Sigi Guo

Bioelectrics Publications

Irreversible electroporation (IRE) as a non-thermal tumor ablation technology has been studied for the treatment of pancreatic carcinoma and has shown a significant survival benefit. We discovered that moderate heating (MH) at 43°C for 1-2 minutes significantly enhanced ex vivo IRE tumor ablation of Pan02 cells by 5.67-fold at 750 V/cm and by 1.67-fold at 1500 V/cm. This amount of heating alone did not cause cell death. An integrated IRE system with controllable laser heating and tumor impedance monitoring was developed to treat mouse ectopic pancreatic cancer. With this novel IRE system, we were able to heat and maintain the …

Multi-Material Mesh Representation Of Anatomical Structures For Deep Brain Stimulation Planning, Tanweer Rashid

Computational Modeling & Simulation Engineering Theses & Dissertations

The Dual Contouring algorithm (DC) is a grid-based process used to generate surface meshes from volumetric data. However, DC is unable to guarantee 2-manifold and watertight meshes due to the fact that it produces only one vertex for each grid cube. We present a modified Dual Contouring algorithm that is capable of overcoming this limitation. The proposed method decomposes an ambiguous grid cube into a set of tetrahedral cells and uses novel polygon generation rules that produce 2-manifold and watertight surface meshes with good-quality triangles. These meshes, being watertight and 2-manifold, are geometrically correct, and therefore can be used to …

Electrotransfer Of Plasmid Dna Radiosensitizes B16f10 Tumors Through Activation Of Immune Response, Monika Savarin, Urska Kamensek, Maja Cemazar, Richard Heller, Gregor Sersa

Bioelectrics Publications

Background. Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors.

Materials and methods. The murine melanoma Bl6F10 tumors, growing on the back of C57BI/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, …

Tissue Specific Microenvironments: A Key Tool For Tissue Engineering And Regenerative Medicine, Patrick C. Sachs, Peter A. Mollica, Robert D. Bruno

School of Medical Diagnostics & Translational Sciences Faculty Publications

The accumulated evidence points to the microenvironment as the primary mediator of cellular fate determination. Comprised of parenchymal cells, stromal cells, structural extracellular matrix proteins, and signaling molecules, the microenvironment is a complex and synergistic edifice that varies tissue to tissue. Furthermore, it has become increasingly clear that the microenvironment plays crucial roles in the establishment and progression of diseases such as cardiovascular disease, neurodegeneration, cancer, and ageing. Here we review the historical perspectives on the microenvironment, and how it has directed current explorations in tissue engineering. By thoroughly understanding the role of the microenvironment, we can begin to correctly …

Modeling Beta-Traces For Beta-Barrels From Cryo-Em Density Maps, Dong Si, Jing He

Computer Science Faculty Publications

Cryo-electron microscopy (cryo-EM) has produced density maps of various resolutions. Although ά-helices can be detected from density maps at 5-8 angstrom resolutions, β-strands are challenging to detect at such density maps due to close-spacing of β-strands. The variety of shapes of β-sheets adds the complexity of β-strands detection from density maps. We propose a new approach to model traces of β-strands for β-barrel density regions that are extracted from cryo-EM density maps. In the test containing eight β-barrels extracted from experimental cryo-EM density maps at 5.5 angstrom-8.25 angstrom resolution, StrandRoller detected about 74.26% of the amino acids in the β-strands …

Comparing An Atomic Model Or Structure To A Corresponding Cryo-Electron Microscopy Image At The Central Axis Of A Helix, Stephanie Zeil, Julio Kovacs, Willy Wriggers, Jing He

Computer Science Faculty Publications

Three-dimensional density maps of biological specimens from cryo-electron microscopy (cryo-EM) can be interpreted in the form of atomic models that are modeled into the density, or they can be compared to known atomic structures. When the central axis of a helix is detectable in a cryo-EM density map, it is possible to quantify the agreement between this central axis and a central axis calculated from the atomic model or structure. We propose a novel arc-length association method to compare the two axes reliably. This method was applied to 79 helices in simulated density maps and six case studies using cryo-EM …

Hyper-Activation Of Pp60(Src) Limits Nitric Oxide Signaling By Increasing Asymmetric Dimethylarginine Levels During Acute Lung Injury, Sanjiv Kumar, Xutong Sun, Satish Kumar Noonepalle, Qing Lu, Evgeny Zemskov, Ting Wang, Saurabh Aggarwal, Christine Gross, Shruti Sharma, Ankit A. Sesai, John D. Catravas

Bioelectrics Publications

The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60^Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in …

Nanosecond Pulsed Electric Field Induced Changes In Cell Surface Charge Density, Diganta Dutta, Xavier-Lewis Palmer, Anthony Asmar, Michael Stacey, Shizhi Qian

Bioelectrics Publications

This study reports that the surface charge density changes in Jurkat cells with the application of single 60 nanosecond pulse electric fields, using atomic force microscopy. Using an atomic force microscope tip and Jurkat cells on silica in a 0.01 M KCl ionic concentration, we were able to measure the interfacial forces, while also predicting surface charge densities of both Jurkat cell and silica surfaces. The most important finding is that the pulsing conditions varyingly reduced the cells' surface charge density. This offers a novel way in which to examine cellular effects of pulsed electric fields that may lead to …