Biotechnology Commons^™

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 …

Vascular Endothelial Growth Factor-A Gene Electrotransfer Promotes Angiogenesis In A Porcine Model Of Cardiac Ischemia, Anna A. Bulysheva, Barbara Hargrave, Nina Burcus, Cathryn G. Lundberg, Len Murray, Richard Heller

Bioelectrics Publications

This study aimed to assess safety and therapeutic potential of gene electrotransfer (GET) as a method for delivery of plasmid encoding vascular endothelial growth factor A (VEGF-A) to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. GET following plasmid encoding VEGF-A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of the saline vehicle. Animals were monitored for 7 weeks and the hearts were evaluated for angiogenesis, myocardial infarct size and left ventricular contractility. …

Microbubble Generation By Piezoelectric Transducers For Biomedical Studies, Mohammed Alkhazal

Electrical & Computer Engineering Theses & Dissertations

Bubbles induced by blast waves or shocks are speculated as the major cause of damage in biological cells in mild traumatic brain injuries (TBI). Microbubble collapse was found to induce noticeable cell detachment from the cell substrate, changes in focal adhesion, and biomechanics. To better understand the bubble mechanism, a system needs to be constructed which allows clear differentiation on the impact of bubbles from that of shocks. Such a generator needs to be low profile in order to place under a microscope. A piezoelectric transducer system was designed to meet the need. The system uses either a flat or …

Experimental Assessment Of Mouse Sociability Using An Automated Image Processing Approach, Frency Varghese, Jessica A. Burket, Andrew D. Benson, Stephen I. Deutsch, Christian W. Zemlin

Electrical & Computer Engineering Faculty Publications

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III). The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called “stimulus mouse”). In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a …

The Cytotoxic Synergy Of Nanosecond Electric Pulses And Low Temperature Leads To Apoptosis, Claudia Muratori, Andrei G. Pakhomov, Elena C. Gianulis, Sarah D. Jensen, Olga N. Pakhomova

Bioelectrics Publications

Electroporation by nanosecond electric pulses (nsEP) is an emerging modality for tumor ablation. Here we show the efficient induction of apoptosis even by a non-toxic nsEP exposure when it is followed by a 30-min chilling on ice. This chilling itself had no impact on the survival of U-937 or HPAF-II cells, but caused more than 75% lethality in nsEP-treated cells (300 ns, 1.8-7 kV/cm, 50-700 pulses). The cell death was largely delayed by 5-23 hr and was accompanied by a 5-fold activation of caspase 3/7 (compared to nsEP without chilling) and more than 60% cleavage of poly-ADP ribose polymerase (compared …

Inactivation Of Acanthamoeba Spp. And Other Ocular Pathogens By Application Of Cold Atmospheric Gas Plasma, Wayne Heaselgrave, Gilbert Shama, Peter W. Andrew, Michael G. Kong

Bioelectrics Publications

Currently there are estimated to be approximately 3.7 million contact lens wearers in the United Kingdom and 39.2 million in North America. Contact lens wear is a major risk factor for developing an infection of the cornea known as keratitis due to poor lens hygiene practices. While there is an international standard for testing disinfection methods against bacteria and fungi (ISO 14729), no such guidelines exist for the protozoan Acanthamoeba, which causes a potentially blinding keratitis most commonly seen in contact lens wearers, and as a result, many commercially available disinfecting solutions show incomplete disinfection after 6 and 24 …

Isquest: Finding Insertion Sequences In Prokaryotic Sequence Fragment Data, Abhishek Biswas, David T. Gauthier, Desh Ranjan, Mohammad Zubair

Computer Science Faculty Publications

Motivation: Insertion sequences (ISs) are transposable elements present in most bacterial and archaeal genomes that play an important role in genomic evolution. The increasing availability of sequenced prokaryotic genomes offers the opportunity to study ISs comprehensively, but development of efficient and accurate tools is required for discovery and annotation. Additionally, prokaryotic genomes are frequently deposited as incomplete, or draft stage because of the substantial cost and effort required to finish genome assembly projects. Development of methods to identify IS directly from raw sequence reads or draft genomes are therefore desirable. Software tools such as Optimized Annotation System for Insertion Sequences …

Introduction To Fifth Special Issue On Electroporation-Based Technologies And Treatments, Damijan Miklavčič, Lluis M. Mir, P. Thomas Vernier

Bioelectrics Publications

This special issue of the Journal of Membrane Biology contains reports on recent developments in the field of electroporation by participants in the International Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments held in November 2014 in Ljubljana. This was the eighth session of what is now an annual event, first organized in 2003.

Sperm Dna Oxidative Damage And Dna Adducts, Hueiwang Anna Jeng, Chih-Hong Pan, Mu-Rong Chao, Wen-Yi Lin

Community & Environmental Health Faculty Publications

The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n =112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling …

Increased Tissue Temperature Improves Electro-Transfer Mediated Gene Delivery To Skin, Anna Bulysheva, Amy Donate, Chelsea Edelblute, Derrick Jung, Karl Schoenbach, Richard Heller

Bioelectrics Publications

[Introduction] Developing an optimal gene electro-transfer system for delivery of plasmid DNA to the skin in vivo has been challenging with expression often confined in the epithelium requiring high voltage electric fields, which can cause cellular and tissue damage. Minimizing such damage, while optimizing gene expression profiles, are highly desirable for therapeutic applications of gene delivery to the skin. Cell membrane fluidity is temperature dependent, thus moderate temperature elevation can increase membrane fluidity. Utilizing this concept, we developed a novel gene electro-transfer electrode for in vivo applications with ability to heat tissue with a laser integrated into a four pin …

Concepts Of Cancer And A Novel Cancer Therapy: Treating Tumors As An Aggressive Organ, Stephen J. Beebe

Bioelectrics Publications

No abstract provided.

Evaluations Of A Mechanistic Hypothesis For The Influence Of Extracellular Ions On Electroporation Due To High-Intensity, Nanosecond Pulsing, V. Sridhara, R. P. Joshi

Electrical & Computer Engineering Faculty Publications

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of …

Electrotransfer Of Single-Stranded Or Double-Stranded Dna Induces Complete Regression Of Palpable B16.F10 Mouse Melanomas, Loree Heller, Vesba Todorovic, Maja Cemazar

Bioelectrics Publications

Enhanced tumor delivery of plasmid DNA with electric pulses in vivo has been confirmed in many preclinical models. Intratumor electrotransfer of plasmids encoding therapeutic molecules has reached Phase II clinical trials. In multiple preclinical studies, a reduction in tumor growth, increased survival or complete tumor regression have been observed in control groups in which vector or backbone plasmid DNA electrotransfer was performed. This study explores factors that could produce this antitumor effect. The specific electrotransfer pulse protocol employed significantly potentiated the regression. Tumor regression was observed after delivery of single-stranded or double-stranded DNA with or without CpG motifs in both …

Next-Peak: A Normal-Exponential Two-Peak Model For Peak-Calling In Chip-Seq Data, Nak-Kyeong Kim, Rasika V. Jayatillake, John L. Spouge

Mathematics & Statistics Faculty Publications

Background: Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) can locate transcription factor binding sites on genomic scale. Although many models and programs are available to call peaks, none has dominated its competition in comparison studies.

Results: We propose a rigorous statistical model, the normal-exponential two-peak (NEXT-peak) model, which parallels the physical processes generating the empirical data, and which can naturally incorporate mappability information. The model therefore estimates total strength of binding (even if some binding locations do not map uniquely into a reference genome, effectively censoring them); it also assigns an error to an estimated binding location. The comparison study …

Response To "Sodium Current Inhibition By Nanosecond Pulsed Electric Field (Nspef) - Fact Or Artifact?" By Verkerk Et Al, Andrei G. Pakhomov

Bioelectrics Publications

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics.

Real-Time In Vivo Imaging Of Size-Dependent Transport And Toxicity Of Gold Nanoparticles In Zebrafish Embryos Using Single Nanoparticle Plasmonic Spectroscopy, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multicoloured optical molecular probes and sensors for real-time in vivo imaging. To effectively study biological functions in vivo, it is essential that the NP probes are biocompatible and can be delivered into living organisms non-invasively. In this study, we have synthesized, purified and characterized stable (non-aggregated) gold (Au) NPs (86.2 +/- 10.8 nm). We have developed dark-field single NP plasmonic microscopy and spectroscopy to study their transport into early developing zebrafish embryos (cleavage stage) and their effects on embryonic development …

Inactivation Of Bacterial Opportunistic Skin Pathogens By Nonthermal Dc-Operated Afterglow Atmospheric Plasma, L. C. Heller, C. M. Edelblute, A. M. Mattson, X. Hao, J. F. Kolb

Bioelectrics Publications

AIMS: Multidrug-resistant opportunistic pathogens are clinically significant and require the development of new antimicrobial methods. In this study, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus cells were exposed to atmospheric plasma on agar plates and in vitro on porcine skin for the purpose of testing bacterial inactivation.

METHODS AND RESULTS: Microbial inactivation at varying exposure durations was tested using a nonthermal plasma jet generated with a DC voltage from ambient air. The observed reduction in colony forming units was quantified as log₁₀ reductions.

CONCLUSIONS: Direct plasma exposure significantly inactivated seeded bacterial cells by approx. 6 log_{10 …}

Microfluidic Impedance Spectroscopy As A Tool For Quantitative Biology And Biotechnology, Ahmet C. Sabuncu, Jie Zhuang, Juergen F. Kolb, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to …

Evaluation Of Delivery Conditions For Cutaneous Plasmid Electrotransfer Using A Multielectrode Array, Bernadette Ferraro, Loree C. Heller, Yolmari L. Cruz, Siqi Guo, Amy Donate, Richard Heller

Bioelectrics Publications

Electroporation (EP) is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues. Delivery of plasmid DNA by EP to a large surface area is not practical because the distance between the electrode pairs, and therefore the applied voltage, must be increased to effectively permeabilize the cell membrane. The design of the multielectrode array (MEA) incorporates multiple electrode pairs at a fixed distance to allow for delivery of plasmid DNA to the skin, potentially reducing the sensation associated with in vivo EP. In this report, we evaluate the effects of field …

Electro-Gene Transfer To Skin Using A Noninvasive Multielectrode Array, Siqi Guo, Amy Donate, Gaurav Basu, Cathryn Lundberg, Loree Heller, Richard Heller

Bioelectrics Publications

Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccinations and several metabolic disorders. The critical factors for DNA delivery to the skin by electroporation (EP) are effective expression levels and minimal or no tissue damage. Here, we evaluated the non-invasive multielectrode array (MEA) for gene electrotransfer. For these studies we utilized a guinea pig model, which has been shown to have a similar thickness and structure to human skin. Our results demonstrate significantly increased gene expression 2 to 3 logs above injection …

Human Endogenous Retrovirus Transcription Profiles Of The Kidney And Kidney-Derived Cell Lines, Sonja Haupt, Michele Tisdale, Michelle Vincendeau, Mary Anne Clements, David T. Gauthier, Raymond Lance, O. John Demmes, Adriana Tuqueti-Neves, Elfriede Noessner, Christine Leib-Mösch, Alex D. Greenwood

Biological Sciences Faculty Publications

The human genome comprises approximately 8-9% of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to …

Efficacy Of The Pentavalent Rotavirus Vaccine, Rotateq (Rv5), Between Doses Of A 3-Dose Series And With Less Than 3 Doses (Incomplete Regimen), Penelope D. Dennehy, Timo Vesikari, David O. Matson, Robbin F. Itzler, Michael J. Dallas, Michelle G. Goveia, Mark J. Dinubile, Penny M. Heaton, Max Ciarlet

Community & Environmental Health Faculty Publications

Post-hoc analyses of the Rotavirus Efficacy and Safety Trial (RES T) were conducted to determine whether the pentavalent rotavirus vaccine (RV5) confers early protection against rotavirus gastroenteritis (RVGE) before completion of the 3-dose regimen. To evaluate the efficacy of RV5 between doses in reducing the rates of RVGE-related hospitalizations and emergency department (ED) visits in infants who ultimately received all 3 doses of RV5/placebo, events occurring from 2 weeks after the first and second doses to receipt of the subsequent dose (Analysis A) and events occurring from 2 weeks after the first and second doses to 2 weeks after the …

Electrically Mediated Delivery Of Plasmid Dna To The Skin, Using A Multielectrode Array, Richard Heller, Yolmari Criz, Loree C. Heller, Richard A. Gilbert, Mark J. Jaroszeski

Bioelectrics Publications

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take full advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is a strong candidate to meet this delivery criterion. Electroporation of the skin is a simple, direct, in vivo method to deliver genes for therapy. Previously, delivery to the skin was performed by means of applicators with relatively large distances between electrodes, resulting in significant muscle stimulation and pain. These applicators also had limitations in controlling the directionality of the applied field. …

Plasmid Injection And Application Of Electric Pulses Alter Endogenous Mrna And Protein Expression In B16.F10 Mouse Melanomas, L. C. Heller, Y. L. Cruz, B. Ferraro, H. Yang, R. Heller

Bioelectrics Publications

The application of electric pulses to tissues causes cell membrane destabilization, allowing exogenous molecules to enter the cells. This delivery technique can be used for plasmid gene therapy. Reporter gene expression after plasmid delivery with eight representative published protocols was compared in B16.F10 mouse melanoma tumors. This expression varied significantly based on the pulse parameters utilized for delivery. To observe the possible influence of plasmid injection and/or pulse application on endogenous gene expression, levels of stress-related mRNAs 4 and 24 h after delivery were determined by PCR array. Increases in mRNA levels for several inflammatory chemokines and cytokines were observed …

Electroporation-Mediated Delivery Of A Naked Dna Plasmid Expressing Vegf To The Porcine Heart Enhances Protein Expression, W. G. Marshall Jr., B. A. Boone, J. D. Burgos, S. I. Gografe, M. K. Baldwin, M. L. Danielson, M. J. Larson, D. R. Caretto, Y. Cruz, B. Ferraro, L. C. Heller, K. E. Ugen, M. J. Jaroszeski, R. Heller

Bioelectrics Publications

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly …

Apoptosis Initiation And Angiogenesis Inhibition: Melanoma Targets For Nanosecond Pulsed Electric Fields, Xinhua Chen, Juergen F. Kolb, R. James Swanson, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

Many effective anti-cancer strategies target apoptosis and angiogenesis mechanisms. Applications of non-ionizing, nanosecond pulsed electric fields (nsPEFs) induce apoptosis in vitro and eliminate cancer in vivo; however in vivo mechanisms require closer analysis. These studies investigate nsPEF-induced apoptosis and anti-angiogenesis examined by fluorescent microscopy, immunoblots, and morphology. Six hours after treatment with one hundred 300 ns pulses at 40 kV/cm, cells transiently expressed active caspases indicating that caspase-mediated mechanisms. Three hours after treatment transient peaks in Histone 2AX phosphorylation coincided with terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and pyknotic nuclei, suggesting caspase-independent mechanisms on nuclei/DNA. Large …

Quantitative Pcr Assay For Mycobacterium Pseudoshottsii And Mycobacterium Shottsii And Application To Environmental Samples And Fishes From The Chesapeake Bay, D. T. Gauthier, K. S. Reece, J. Xiao, M. W. Rhodes, H. I. Kator, R. J. Latour, C. F. Bonzek, J. M. Hoenig, W. K. Vogelbein

Biological Sciences Faculty Publications

Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium …

Increased Perfusion And Angiogenesis In A Hindlimb Ischemia Model With Plasmid Fgf-2 Delivered By Noninvasive Electroporation, B. Ferraro, Y. L. Cruz, M. Baldwin, D. Coppola, R. Heller

Bioelectrics Publications

Gene therapy approaches delivering fibroblast growth factor-2 (FGF-2) have shown promise as a potential treatment for increasing blood flow to ischemic limbs. Currently, effective noninvasive techniques to deliver plasmids encoding genes of therapeutic interest, such as FGF-2, are limited. We sought to determine if intradermal injection of plasmid DNA encoding FGF-2 (pFGF) followed by noninvasive cutaneous electroporation (pFGFE+) could increase blood flow and angiogenesis in a rat model of hindlimb ischemia. pFGFE+ or control treatments were administered on postoperative day 0. Compared to injection of pFGF alone (pFGFE-), delivery of pFGFE+ significantly increased FGF-2 expression for 10 days. Further, the …

Electric Pulses To Prepare Feeder Cells For Sustaining And Culturing Of Undifferentiated Embryonic Stem Cells, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Current challenges in embryonic-stem-cell (ESC) research include inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gammaradiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed-electric-fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10- nanosecond-electric-pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1s pulse interval, …

A New Pulsed Electric Field Therapy For Melanoma Disrupts The Tumor's Blood Supply And Causes Complete Remission Without Recurrence, Richard Nuccitelli, Xinhua Chen, Andrei G. Pakhomov, Wallace H. Baldwin, Saleh Sheikh, Jennifer L. Pomicter, Wei Ren, Chris Osgood, R. James Swanson, Juergen F. Kolb, Stephen J. Beebe, Karl H. Schoenbach

Bioelectrics Publications

We have discovered a new, ultrafast therapy for treating skin cancer that is extremely effective with a total electric field exposure time of only 180 mu sec. The application of 300 high-voltage (40 kV/cm), ultrashort (300 nsec) electrical pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. None of these melanomas recurred during a 4-month period after the initial melanoma had disappeared. These pulses generate small, long-lasting, rectifying nanopores in the plasma membrane of exposed cells, resulting in increased membrane permeability to …