Biotechnology Commons^™

Electroporation For The Delivery Of Dna-Based Vaccines And Immunotherapeutics: Current Clinical Developments, Angela M. Bodles-Brakhop, Richard Heller, Ruxandra Draghia-Akli

Bioelectrics Publications

Electroporation (EP) has been used in basic research for the past 25 years to aid in the transfer of DNA into cells in vitro. EP in vivo enhances transfer of DNA vaccines and therapeutic plasmids to the skin, muscle, tumors, and other tissues resulting in high levels of expression, often with serological and clinical benefits. the recent interest in nonviral gene transfer as treatment options for a vast array of conditions has resulted in the refinement and optimization of EP technology. current research has revealed that EP can be successfully used in many species, including humans. clinical trials are …

Intradermal Delivery Of Plasmid Vegf(165) By Electroporation Promotes Wound Healing, Bernadette Ferraro, Yolmari Cruz, Domenico Coppola, Richard Heller

Bioelectrics Publications

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF₁₆₅ (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal …

Comparison Of Electrically Mediated And Liposome-Complexed Plasmid Dna Delivery To The Skin, Loree C. Heller, Mark J. Jaroszeski, Domenico Coppola, Richard Heller

Bioelectrics Publications

BACKGROUND: Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

METHODS: Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

RESULTS: Experiments …

Optimization Of Cutaneous Electrically Mediated Plasmid Dna Delivery Using Novel Electrode, L. C. Heller, M. J. Jaroszeski, D. Coppola, A. N. Mccray, J. Hickey, R. Heller

Bioelectrics Publications

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo. A critical component of this technique is the electrode configuration. Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode. The highest transgene expression and efficiency of individual cell transformation with minimal damage was produced with eight 150 ms pulses at field strength of …

Potential For Stimulating Host Anti-Tumor Immune Response Via Rnai-Mediated Local Foxp3 Knockdown, N. Klaiber

Chemistry & Biochemistry Faculty Publications

Neoplastic growths represent a unique challenge for the host immune system. As they are indeed derived from self, many of the same mechanisms operating to prevent autoimmunity also provide an umbrella beneath which malignant cells are free to proliferate.1 Central among these immune regulatory boundaries are an influential subset of lymphocytes known as T regs. Hypothesized to exist decades ago, yet eluding definitive characterization until relatively recently, T regs have been demonstrated to play a crucial role in the proper functioning of the immune system as a whole. They may also, however, represent one of the primary obstacles to successful …

Sensory Genes And Mate Choice: Evidence That Duplications, Mutations, And Adaptive Evolution Alter Variation In Mating Cue Genes And Their Receptors, Lisa Horth

Biological Sciences Faculty Publications

Fascinating new data, revealed through gene sequencing, comparative genomics, and genetic engineering, precisely establish which genes are involved in mate choice and mating activity—behaviors that are surprisingly understudied from a genetic perspective. Discussed here are some of the recently identified visual and chemosensory genes that are involved in mate choice and mating behavior. These genes’ products are involved in the production, transmission, and receipt of crucial sensory mate-choice cues that affect fitness. This review exposes newfound evidence that alternative splicing, gene-expression pattern changes, and molecular genetic variation in sensory genes are crucial for both intra- and interspecific mate choice and …

Evaluation Of Toxicity Following Electrically Mediated Interleukin-12 Gene Delivery In A B16 Mouse Melanoma Model, Loree Heller, Kathleen Merkler, Jeffrey Westover, Yolmari Cruz, Domenico Coppola, Kaaron Benson, Adil Daud, Richard Heller

Bioelectrics Publications

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model.

EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically.

RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated …

Plasma Membrane Voltage Changes During Nanosecond Pulsed Electric Field Exposure, W. Frey, R. O. Price, P. F. Blackmore, R. P. Joshi, R. Nuccitelli, S. J. Beebe, K. H. Schoenbach, J. F. Kolb

Bioelectrics Publications

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of similar to 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat …

Nanosecond Pulsed Electric Fields: A New Stimulus To Activate Intracellular Signaling, Stephen J. Beebe, Karl H. Schoenbach

Bioelectrics Publications

When new technologies are introduced into the scientific community, controversy is expected and both excitement and disappointment enrich the lives of those who initiate the new ideas. It becomes the mission of the “inventors” to embrace the burden of proof to establish their ideas and convince the skeptics and disbelievers who will undoubtedly temper their enthusiasm and test their patience. While open mindedness is generally a scientific motto, those who review patents, manuscripts, and grants do not always readily practice it, even when the evidence is convincingly presented; old ideas and concepts often die hard. So it has been and …

Selective Field Effects On Intracellular Vacuoles And Vesicle Membranes With Nanosecond Electric Pulses, Ephrem Tekle, Hammou Oubrahim, Sergey M. Dzekunov, Juergen F. Kolb, Karl H. Schoenbach

Bioelectrics Publications

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of …

High-Temperature Fluorescent In Situ Hybridization For Detecting Escherichia Coli In Seawater Samples, Using Rrna-Targeted Oligonucleotide Probes And Flow Cytometry, Ying Zhong Tang, Karina Yew Hoong Gin, Tok Hoon Lim

OES Faculty Publications

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E. coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with …

Effect Of Humic Substance Photodegradation On Bacterial Growth And Respiration In Lake Water, Alexandre M. Anesio, William Granéli, George R. Aiken, David J. Kieber, Kenneth Mopper

Chemistry & Biochemistry Faculty Publications

This study addresses how humic substance (HS) chemical composition and photoreactivity affect bacterial growth, respiration, and growth efficiency (BGE) in lake water. Aqueous solutions of HSs from diverse aquatic environments representing different dissolved organic matter sources (autochthonous and allochthonous) were exposed to artificial solar UV radiation. These solutions were added to lake water passed through a 0.7-μm pore-size filter (containing grazer-free lake bacteria) followed by dark incubation for 5, 43, and 65 h. For the 5-h incubation, several irradiated HSs inhibited bacterial carbon production (BCP) and this inhibition was highly correlated with H₂O₂ photoproduction. The H_{2 …}

On Thacker's Biomedia (2004), Nicholas Ruiz Iii

Reconstruction: Studies in Contemporary Culture

[First paragraph]

Only we could have done it. The Code (DNA and its analogs, etc.) that we perhaps only narrate, never forgetting all of the fictions that narration portends, today consciously edits the text that evolution has edited unconsciously for eons. Thacker considers how humans -- perhaps little more than interactive Code, manipulate the Code -- with the technology of the Code. No other faction of Code has yet to make such heady ‘gains.’ Where posthumanity was always a literary endeavor, a literary bypass, out of instrumental science and into fictive states, it now is a technical endeavor, encompassing a …

Computational Protein Biomarker Prediction: A Case Study For Prostate Cancer, Michael Wagner, Dayanand N. Naik, Alex Pothen, Srinivas Kasukurti, Raghu Ram Devineni, Bao-Ling Adam, O. John Semmes, George L. Wright Jr.

Mathematics & Statistics Faculty Publications

Background: Recent technological advances in mass spectrometry pose challenges in computational mathematics and statistics to process the mass spectral data into predictive models with clinical and biological significance. We discuss several classification-based approaches to finding protein biomarker candidates using protein profiles obtained via mass spectrometry, and we assess their statistical significance. Our overall goal is to implicate peaks that have a high likelihood of being biologically linked to a given disease state, and thus to narrow the search for biomarker candidates.

Results: Thorough cross-validation studies and randomization tests are performed on a prostate cancer dataset with over 300 patients, obtained …

The Effects Of Intense Submicrosecond Electrical Pulses On Cells, Jingdong Deng, Karl H. Schoenbach, E. Stephen Buescher, Pamela S. Hair, Paula M. Fox, Stephen J. Beebe

Bioelectrics Publications

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the submicrosecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 μs– 60 ns, electric field intensities 3–150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of …

Real-Time Study Of Multidrug Resistance Mechanism In Pseudomonas Aeruginosa Using Nanoparticle Optics And Single Live Cell Imaging, Sophia Vasou Kyriacou

Chemistry & Biochemistry Theses & Dissertations

This thesis centers on the study of the xenobiotic efflux system in Pseudomonas aeruginosa, which is a ubiquitous bacterium. It resists many structurally and functionally diverse substrates due to expression of Mex-extrusion pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM systems. Despite extensive research, the structure and mechanism of multidrug resistance is unclear (1-9). For example, (i) how do MexA, MexB and OprM proteins assemble to extrude antibiotics? (ii) What is the antibiotic susceptibility of MexA, MexB, and OprM proteins? (iii) How do substrates cross the outer membrane of P. aeruginosa? (iv) Where are antibiotics accumulated inside the cell? This thesis …

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Electric Field Enhanced Plasmid Delivery To Liver Hepatocellular Carcinomas, Richard Gilbert, Mark J. Jaroszeski, Loree Heller, Richard Heller

Bioelectrics Publications

Electric field enhanced molecular delivery for cancer research and treatment is a new technology that has demonstrated its effectiveness in clinical trials using bleomycin or cisplatin (Heller, R., Gilbert, R., Jaroszeski, M. J. Clinical applications of electrochemotherapy, Advanced Drug Delivery Reviews 35,119-129, 1999), as chemotherapeutic agents. The technology is being investigated in research applications for applicability as a method to enhance gene expression in a target tumor. Success is predicated on an appropriate effective electric field mediated delivery protocol that triggers significant appropriate gene expression duration and levels. An electric field mediated delivery protocol includes a set of conditions …

Electrically Mediated Delivery Of Vector Plasmid Dna Elicits An Antitumor Effect, L. Heller, D. Coppola

Bioelectrics Publications

In vivo electroporation is an efficient means of increasing plasmid DNA delivery to normal tissues, such as skin and muscle, as well as directly to tumors. In the experiments described here, plasmid DNA was delivered by in vivo electroporation to B16 mouse melanomas using two very different pulsing protocols. Reporter expression increased 21- or 42-fold, respectively with electroporation over injection alone. The growth of experimental melanomas with an approximate diameter of 4 mm on the day of treatment was monitored after electroporation delivery of reporter plasmid DNA. Remarkably, short-term complete regressions using one of these pulsing protocols occurred in up …

Effect Of Electrically Mediated Intratumor And Intramuscular Delivery Of A Plasmid Encoding Ifn Α On Visible B16 Mouse Melanomas, Loree C. Heller, Stephanie F. Ingram, M. Lee Lucas, Richard A. Gilbert, Richard Heller

Bioelectrics Publications

Interferon α may be used as a single agent therapy for metastatic malignant melanoma or as an adjuvant to chemotherapy. Delivery of interferon α by gene therapy offers an alternative to recombinant protein therapy. Electrically mediated delivery enhances plasmid expression in a number of tissues, for instance skin, liver, muscle and tumors including melanomas. Here we compare the effect of delivery of a plasmid encoding mouse interferon α on growth of visible B16 mouse melanomas following electrically mediated delivery to muscle or directly to the tumor. Intratumoral delivery of interferon α plasmid not only slows melanoma growth, but induces complete, …

Il-12 Plasmid Delivery By In Vivo Electroporation For The Successful Treatment Of Established Subcutaneous B16.F10 Melanoma, M. Lee Lucus, Loree Heller, Domenico Coppola, Richard Heller

Bioelectrics Publications

Interleukin-12 (IL-12) has been used in numerous immunotherapy protocols against melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid DNA encoding IL-12 as an antitumor agent against B16.F10 melanoma. We treated mice bearing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injections of a plasmid encoding IL-12, followed by in vivo electroporation. For i.t. treatments, we used an applicator containing six …

Electrically Mediated Plasmid Dna Delivery To Hepatocellular Carcinomas In Vivo, L. Heller, M. J. Jaroszeski, D. Coppola, C. Pottinger, R. Gilbert, Richard Heller

Bioelectrics Publications

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Exploring Participation Processes For Technology Development: Case Studies Of Biotechnology Research And Development Projects In Thailand, Omjai Yuktavetya

Engineering Management & Systems Engineering Theses & Dissertations

There is increased awareness in the organizational and management literature that participation can make contributions in technology development. One of the problems in developing biotechnology in Thailand is that only a small portion of publicly funded research and development projects directly result in commercial success. Among the reasons cited (TDRI 1992a) is a lack of collaboration between various stakeholders; therefore, an effective technology policy to support their participation is needed. This research explores current participation processes in biotechnology research and development projects in Thailand in which there were different perspectives among various stakeholders. The quantitative and qualitative methodologies developed here …

A Computer-Simulated Restriction Fragment Length Polymorphism Analysis Of Bacterial Small-Subunit Rrna Genes: Efficacy Of Selected Tetrameric Restriction Enzymes For Studies Of Microbial Diversity In Nature, Craig L. Moyer, James M. Tiedje, Fred C. Dobbs, David M. Karl

OES Faculty Publications

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective nas to identify the optimal combination of TREs for RnP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were …

Phylogenetic Diversity Of The Bacterial Community From A Microbial Mat At An Active, Hydrothermal Vent System, Loihi Seamount, Hawaii, Craig L. Moyer, Fred C. Dobbs, David M. Karl

OES Faculty Publications

The phylogenetic diversity of small-subunit rRNA genes associated,vith the domain Bacteria was examined (by using previously defined operational taxonomic units [C. L. Moyer, F. C. Dobbs, and D. M. Karl, Appl. Environ. Microbiol. 60:871-879, 1994]; those for Pele's Vents Bacteria are hereafter abbreviated PVB OTUs) with samples from a microbial mat at an active, deep-sea hydrothermal vent system. A cluster of phylogenetically related PVB OTUs (OTUs 2, 3, 6, and 8) was closely affiliated with Thiovulum sp. contained within the epsilon subclass of the class Proteobacteria and accounted for 60.5% of the small-subunit rRNA bacterial clone library from Pele's Vents. …

Molecular Cloning And Rare Cleavage Mapping Of Human 2p, 6q, 8q, 12q, And 18q Telomeres, Roberto A. Macina, Ken Morii, Xue-Lan Hu, Dimitri G. Negorev, Chrysanthe Spais, Lisa A. Ruthig, Harold C. Riethman

School of Medical Diagnostics & Translational Sciences Publications

Large terminal fragments of human chromosomes 2p, 6q, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from 11 unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned Fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

Role Of Heat Shock Protein 70 Kda Cognate In Limiting Thermal Inactivation And Refolding Of Heat-Denatured Nuclear Type I Topoisomerase, Kuo-Kuang Wen

Theses and Dissertations in Biomedical Sciences

Previous studies (Ciavarra et al., 1994) demonstrated that the constitutive 70 kDa heat shock protein (hsc70) protected purified topoisomerase I from thermal injury. In addition, hsc70 was capable of regenerating catalytic activity of heat-denatured topoisomerase I. A whole cell lysate was also active in this reaction assay. The present study demonstrates that heat-denatured topoisomerase I is reactivated by a cytosolic fraction and that this activity is dependent on the presence of cytosolic hsc70. The efficacy of hsc70-mediated refolding of heat-denatured topoisomerase I is greatly enhanced by a cytosolic cofactor(s). In all these refolding reactions, exogenous ATP is not required. Size …

Estimation Of Diversity And Community Structure Through Restriction-Fragment-Length-Polymorphism Distribution Analysis Of Bacterial 16s Ribosomal-Rna Genes From A Microbial Mat At An Active, Hydrothermal Vent System, Loihi Seamount, Hawaii, Craig L. Moyer, Fred C. Dobbs, David M. Karl

OES Faculty Publications

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete …

A Comparison Of No And N20 Production By The Autophic Nitrifier Nitrosomonas Europaea And The Heterotrophic Nitrifier Alcaligenes Faecalis, Iris C. Anderson, Mark Poth, Julie Homstead, David J. Burdige

OES Faculty Publications

Soil microorganisms are important sources of the nitrogen trace gases NO and N₂O for the atmosphere. Present evidence suggests that autotrophic nitrifiers such as Nitrosomonas europaea are the primary producers of NO and N₂O in aerobic soils, whereas denitrifiers such as Pseudomonas spp. or Alcaligenes spp. are responsible for most of the NO and N₂O emissions from anaerobic soils. It has been shown that Alcaligenes faecalis, a bacterium common in both soil and water, is capable of concomitant heterotrophic nitrification and denitrification. This study was undertaken to determine whether heterotrophic nitrification might be …

Preservation Of Atp In Hypersaline Environments, Bruce J. Tuovila, Fred C. Dobbs, Paul A. Larock, B. Z. Siegel

OES Faculty Publications

High concentrations of particulate ATP were found in the anoxic brines of the Orca Basin and East Flower Garden, Gulf of Mexico. Other measurements indicative of growth and respiration suggested that the microbial community in the brines was inactive, but somehow the ATP associated with the cells persisted. Conceivably, when cells growing just above the interface sank into the brine, the increased osmotic stress could elicit an osmoregulatory response resulting in increased ATP. It was also possible that hydrolytic enzymes were inactivated, resulting in the preservation of ATP. Experiments in which a culture of marine bacteria was suspended in menstrua …