Biotechnology Commons^™

Optimizing Sperm Collection Procedures In Zebrafish, Madelyn B. Wasden, Rachel L. Roberts, April Delaurier

Journal of the South Carolina Academy of Science

Zebrafish are a highly-valued model organism used for developmental biology research. Zebrafish can be used for genetic manipulation and hence, many mutant and transgenic lines exist. It is impractical to maintain lines of adult zebrafish, due to resource constraints and the need to continuously produce new generations. Therefore, a practical way to preserve zebrafish lines is to freeze sperm and retrieve lines using in vitro fertilization of fresh eggs. Most existing in vitro protocols used by research labs have a wide variety of fertilization rates (ranging from 0% to >90%). Due to this variability, lines may be at risk of …

Mitochondrial Reactive Oxygen Species In Lipotoxic Hearts Induces Post-Translational Modifications Of Akap121, Drp1 And Opa1 That Promote Mitochondrial Fission, Kensuke Tsushima, Heiko Bugger, Adam R. Wende, Jamie Soto, Gregory A. Jenson, Austin R. Tor, Rose Mcglauflin, Helena C. Kenny, Yuan Zhang, Rhonda Souvenir, Xiao X. Hu, Crystal L. Sloan, Renata O. Pereira, Vitor A. Lira, Kenneth W. Spitzer, Terry L. Sharp, Kooresh I. Shoghi, Genevieve C. Sparagna, Eva A. Rog-Zielinska, Peter Kohl, Oleh Khalimonchuk, Jean E. Schaffer, E. Dale Abel

Department of Biochemistry: Faculty Publications

Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood.

Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo.

Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation …

Characterization Of Different Molecular Markers For Identification Of Salmonella Enterica Serovar Typhi In Pakistani Population, Faizan Muttiullah, Fida Muhammad Khan, Fakhar-I- Abbas, Sabiha Shamim

Journal of Bioresource Management

Typhoid is caused by Salmonella enterica serovar Typhi that is usually diagnosed by using serologic and immuno-chromatographic techniques in developing counties including Pakistan, which is thought to be an unreliable diagnostic method. For accurate diagnosis we used molecular techniques to amplify 204 bp StyR-36 and 498 bp flagellin gene for the identification of Salmonella enterica serovar Typhi. This study was done on 58 individuals diagnosed positive of typhoid via serologic tests and 50 healthy individuals as a control group. Success rate of amplification for flagellin gene was 77.58% while that for StyR-36 gene was 68.97% showing that flagellin gene primer …

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 …

Validation Of The Pre-B Cell Receptor As A Therapeutic Target In B Cell Precursor Acute Lymphoblastic Leukemia, Michael Frank Erasmus

Biomedical Sciences ETDs

This dissertation is built upon the fundamental idea that the pre-B cell receptor (pre-BCR) is important to leukemia cell survival and a logical therapeutic target in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The pre-BCR is expressed early at a specific stage during B cell development where it plays a central role in survival of healthy B lymphocytes. This receptor is composed of the membrane heavy chain (mIgμ) associated with surrogate light chain components, 5 and VpreB. Through the use of advanced imaging modalities, in particular two-color single particle tracking (SPT), we showed that pre-BCRs formed transient, homotypic interactions. These …

Stem Cell Derived Osteoprogenitors And Their Role In Bone Repair Using Morphogenic Activators, George Washington Brownridge Iii, Sylvanna Islas, Angelina Miller, Warren Hoeffler

Student Research Posters

Bone constantly cycles through a dynamic process of breakdown and remodeling. Osteoblasts are the specialized mesenchymal stem cells that have a major role in bone formation and the remodeling process whereas their counterpart osteoclasts, handle bone resorption. Embryonic stem cells can be partially differentiated into Progenitor cells, and we worked with #18, a candidate for being an osteoprogenitor that has the potential to respond to morphogenic activators. In the case of bone remodeling, TGF-β 2, BMP-2 and an abundance of CA++ have been shown to be potential activators of differentiation into osteoblasts. Eight different trials were conducted with the cells …

Comparative Genomic Analysis Of Two Serotype 1/2b Listeria Monocytogenes Isolates From Analogous Environmental Niches Demonstrates The Influence Of Hypervariable Hotspots In Defining Pathogenesis, Aidan Casey, Kieran Jordan, Aidan Coffey, Edward M. Fox, Olivia Mcauliffe

Department of Biological Sciences Publications

The vast majority of clinical human listeriosis cases are caused by serotype 1/2a, 1/2b, 1/2c, and 4b isolates of Listeria monocytogenes. The ability of L. monocytogenes to establish a systemic listeriosis infection within a host organism relies on a combination of genes that are involved in cell recognition, internalization, evasion of host defenses, and in vitro survival and growth. Recently, whole genome sequencing and comparative genomic analysis have proven to be powerful tools for the identification of these virulence-associated genes in L. monocytogenes. In this study, two serotype 1/2b strains of L. monocytogenes with analogous isolation sources, but …

Loss Of Exogenous Androgen Dependence By Prostate Tumor Cells Is Associated With Elevated Glucuronidation Potential, Brenna M. Zimmer, Michelle E. Howell, Qin Wei, Linlin Ma, Trevor Romsdahl, Eileen G. Loughman, Jennifer E. Markham, Javier Seravalli, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD⁺-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate, and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen dependent versus castration resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration resistant tumor cells expressed higher …

Hiv Vaccines: Progress, Limitations And A Crispr/Cas9 Vaccine, Omar A. Garcia Martinez

Biology: Student Scholarship & Creative Works

ABSTRACT: The HIV-1 pandemic continues to thrive due to ineffective HIV-1 vaccines. Historically, the world’s most infectious diseases, such as polio and smallpox, have been eradicated or have come close to eradication due to the advent of effective vaccines. Highly active antiretroviral therapy is able to delay the onset of AIDS but can neither rid the body of HIV-1 proviral DNA nor prevent further transmission. A prophylactic vaccine that prevents the various mechanisms HIV-1 has to evade and attack our immune system is needed to end the HIV-1 pandemic. Recent advances in engineered nuclease systems, like the CRISPR/Cas9 system, have …

Design And Testing Of Novel Anthrax Vaccines Utilizing A Tobacco Mosaic Virus Expression System, Ryan C. Mccomb

KGI Theses and Dissertations

Anthrax is a potentially fatal disease caused by the bacteria Bacillus anthracis. Infection and disease occur after spores gain entry into the body, germinate into vegetative bacteria, and produce toxin. Bacillus anthracis spores have been engineered as bioweapons and have been used repeatedly in warfare and terrorism to inflict casualties in military and civilian populations. Currently, only one vaccine has been approved for prevention of anthrax in the United States. This vaccine is an undefined product that is difficult to produce, requires a long vaccination schedule, and is reactogenic. Efforts to make an improved anthrax vaccine are being pursued. …

A Tail Of Two Phages: Genomic And Functional Analysis Of Listeria Monocytogenes Phages Vb_Lmos_188 And Vb_Lmos_293 Reveal The Receptor-Binding Proteins Involved In Host Specificity, Aidan Casey, Kieran Jordan, Horst Neve, Aidan Coffey, Olivia Mcauliffe

Department of Biological Sciences Publications

The physical characteristics of bacteriophages establish them as viable candidates for downstream development of pathogen detection assays and biocontrol measures. To utilize phages for such purposes, a detailed knowledge of their host interaction mechanisms is a prerequisite. There is currently a wealth of knowledge available concerning Gram-negative phage-host interaction, but little by comparison for Gram-positive phages and Listeria phages in particular. In this research, the lytic spectrum of two recently isolated Listeria monocytogenes phages (vB_LmoS_188 and vB_LmoS_293) was determined, and the genomic basis for their observed serotype 4b/4e host-specificity was investigated using comparative genomics. The late tail genes of these …

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 …

Enhanced Expression Of Codon Optimized Mycobacterium Avium Subsp. Paratuberculosis Antigens In Lactobacillus Salivarius, Christopher D. Johnston, John P. Bannatine, Rodney Govender, Lorraine Endersen, Daniel Pletzer, Helge Weingart, Aidan Coffey, Jim O'Mahony, Roy D. Sleator

Department of Biological Sciences Publications

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants …

Self-Assembled Gold Nanoplexes For Cancer-Targeted Sirna Delivery, Yongliang Shi

Master's Theses

Through layer-by-layer method, the authors have constructed three Au nanoplexes: AuPEI/RNA/PEI, AuPEI/RNA/PEI-mPEG, and AuPEI/NA/PEI-PEG-FA. All the nanoplexes are characterized by UV-vis spectrometry, DLS, and zeta potential. The surface density of the first layer PEI and second layer RNA were also determined. Moreover, the Au nanoplexes can protect siRNA from RNase degradation and are stable in cell culture medium.

siGLuc-ppp, ssRNA₈₀, and siF17 were delivered by the assembled Au nanoplexes, and the results were analyzed by GLuc assay, TB assay, and Luciferase assay. In terms of RNA delivery, the Au nanoplexes AuPEI/RNA/PEI exhibited excellent gene silencing efficiency (or causing …

The Role Of Sirtuin 1 And Ceramide In T10C12 Conjugated Linoleic Acid Induced Delipidation In 3t3-L1 Adipocytes, Wei Wang

Department of Animal Science: Dissertations, Theses, and Student Research

Project 1: Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) reduces triglyceride (TG) levels in adipocytes through multiple pathways, with AMP-activated protein kinase (AMPK) generally facilitating, and peroxisome proliferator-activated receptor γ (PPARγ) generally opposing these reductions. Sirtuin 1 (SIRT1), a histone/protein deacetylase that affects energy homeostasis, often functions coordinately with AMPK, and is capable of binding to PPARγ, thereby inhibiting its activity. This study investigated the role of SIRT1 in the response of 3T3-L1 adipocytes to t10c12 CLA by testing the following hypotheses: 1) SIRT1 is functionally required for robust TG reduction; …

Nudging The Fda, W. Nicholson Price Ii, I. Glenn Cohen

Law Faculty Scholarship

[Excerpt] "The FDA’s regulation of drugs is frequently the subject of policy debate, with arguments falling into two camps. On the one hand, a libertarian view of patients and the health care system holds high the value of consumer choice. Patients should get all the information and the drugs they want; the FDA should do what it can to enforce some basic standards but should otherwise get out of the way. On the other hand, a paternalist view values the FDA’s role as an expert agency standing between patients and a set of potentially dangerous drugs and potentially unscrupulous or …

Biochemical Assay Optimization And Computational Screening Efforts To Identify Potential Luxs Inhibitors, Keeshia Q. Wang

Master's Theses

Quorum sensing (QS) is a process of coordination of bacterial gene expression in response to cell population. System two QS is regulated by the small signaling molecule autoinducer-2 (AI-2) and is implicated in the infectious behaviors of various bacterial species. AI-2 is biosynthesized from S-ribosylhomocysteine (SRH) by the enzyme LuxS and induces interspecies cell-to-cell communication. Inhibition of LuxS would therefore inhibit interspecies QS. Herein, a search for novel molecular species that will competitively bind with SRH in the LuxS binding site is performed in silico. Computational screening results are then validated in vitro using an optimized LuxS inhibition …

A Miniaturised Spectrometer Device For The Detection Of Nitrogen Dioxide In An Urban Environment, Brian Devine

Doctoral

Monitoring of air pollutants, such as Nitrogen Dioxide (NO2), that are toxic or environmentally damaging is a key metric for environmental protection agencies worldwide. There is a constant need to develop new technologies and methodologies that provide real-time, low cost pollution measurements over a broad range of sampling sites, particularly in urban and industrial areas. Typically, detection of pollutants in urban environments is performed using a variety of techniques, many of which are expensive, require complex setups and are in fixed locations. The novel system presented in this thesis is designed for portable, low cost and in-situ detection of pollutants …

The Effects Of Hydrostatic Pressure On Early Endothelial Tubulogenic Processes, Ryan M. Underwood

Theses and Dissertations--Biomedical Engineering

The effects of mechanical forces on endothelial cell function and behavior are well documented, but have not been fully characterized. Specifically, fluid pressure has been shown to elicit physical and chemical responses known to be involved in the initiation and progression of endothelial cell-mediated vascularization. Central to the process of vascularization is the formation of tube-like structures. This process—tubulogenesis—is essential to both the physiological and pathological growth of tissues. Given the known effects of pressure on endothelial cells and its ubiquitous presence in the vasculature, we investigated pressure as a magnitude-dependent parameter for the regulation of endothelial tubulogenic activity. To …

Biomass Assisted Synthesis Of Antibacterial Gold Nanoparticles And Commentary On Its Future Potential And Applications In Medicine, Chad Willis

Mahurin Honors College Capstone Experience/Thesis Projects

Xylose is a natural monosaccharide found in biomass such as straw, pecan shells, cottonseed hulls, and corncobs. Using this monosaccharide, we report the green synthesis and characterization of biocompatible, biodegradable xylose encapsulated gold nanoparticles (Xyl-GNPs) with potential antibacterial activity. GNPs were synthesized using the bioreduction property of xylose on the chloroaurate anions in an aqueous solution at room temperature and at atmospheric pressure. The characterization of synthesized GNPs was examined by UV-vis spectroscopy; transmission electron microscopy (TEM), energy-dispersive x-ray spectroscopy (EDS) and fourier transform infrared spectroscopy (FTIR). Results indicate that the particles were stable; near spherical in shape with an …

Quantitative Reagent-Free Detection Of Fibrinogen Levels In Human Blood Plasma Using Raman Spectroscopy, Kelvin Poon, Fiona Lyng, Peter Knief, Orla L. Howe, Aidan Meade, James Curtin, Hugh Byrne, Joseph Vaughan

Articles

Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and …

Emerging Dynamics Of Human Campylobacteriosis In Southern Ireland, Susan Bullman, Daniel Corcoran, James O'Leary, Derry O'Hare, Brigid Lucey, Roy D. Sleator

Department of Biological Sciences Publications

Infections with Campylobacter spp. pose a significant health burden worldwide. The significance of Campylobacter jejuni/Campylobacter coli infection is well appreciated but the contribution of non-C. jejuni/C. coli spp. to human gastroenteritis is largely unknown. In this study, we employed a two-tiered molecular study on 7194 patient faecal samples received by the Microbiology Department in Cork University Hospital during 2009. The first step, using EntericBio® (Serosep), a multiplex PCR system, detected Campylobacter to the genus level. The second step, utilizing Campylobacter species-specific PCR identified to the species level. A total of 340 samples were confirmed as Campylobacter genus positive, 329 of …

Campylobacter Ureolyticus: An Emerging Gastrointestinal Pathogen?, Susan Bullman, Daniel Corcoran, James O'Leary, Brigid Lucey, Deirdre Byrne, Roy D. Sleator

Department of Biological Sciences Publications

A total of 7194 faecal samples collected over a 1-year period from patients presenting with diarrhoea were screened for Campylobacter spp. using EntericBios, a multiplex-PCR system. Of 349 Campylobacter-positive samples, 23.8% were shown to be Campylobacter ureolyticus, using a combination of 16S rRNA gene analysis and highly specific primers targeting the HSP60 gene of this organism. This is, to the best of our knowledge, the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis and may suggest a role for this organism as an emerging enteric pathogen.

Exhausted Or Unlicensed: Can Field-Of-Use Restrictions In Biotech License Agreements Still Prevent Off-Label Use Promotion After Quanta Computer?, Kristal M. Wicks

The University of New Hampshire Law Review

[Excerpt] “In the biotechnology (biotech) industry, companies must be increasingly aware of their intellectual property and how their licensing strategies can impact their rights. When licensing patented technology, it is common practice for biotech companies to include restricted field-of-use provisions in their license agreements. Such provisions permit a licensee to only use licensed technology in a defined field and restrict use or development in another field. This licensing strategy plays an important role within the biotech industry because it allows companies to more effectively control their intellectual property and to more efficiently research and develop pharmaceutical products.

A problem that …

Development Of Near Real-Time Assessment System For Cancer Cells., Nahrizul Adib Kadri

Student Works (2010-2019)

Dielectrophoresis (DEP) is an electrical phenomenon that occurs when a polarisable particle is placed in non-uniform electrical fields, generating DEP force (FDEP) that is dependent upon the electrophysiological make-up of the particle in use. In cell cytometry studies, DEP offers the advantage of conducting various cell manipulations in vitro without the need of specific surface markers. Its drawbacks, namely the time-consuming processes involved and the inability to conduct large batch cell assays, had limited its usage. This thesis presents the work that offered solution to the said obstacles, by developing a system capable of conducting DEP experiments within a very …

Estimation Of The Product Yield And Collection Efficiency Of Peripheral Blood Stem Cell Harvesting Using Peripheral Blood Cd34[+] Cells Quantification., Habsah Aziz

Student Works (2010-2019)

Peripheral blood stem cells (PBSC) have been used for transplantation since the early 1990s. CD34+ has been used as a marker of hematopoietic stem cells to determine the optimal timing for PBSC harvesting. However, a wide ranges of CD34+ cells cut-off values from 8/ul to 66/ul have been used to initiate PBSC harvesting in autologous patients. We performed here a study to determine the peripheral blood CD34+ cells cut-off value for the prediction of stem cell yield of 2 x106 CD34+ cells/kg body weight patients, to study the correlation between peripheral blood CD34+ cells and stem cell yield, to study …

The Effect Of Endothelin-1 On The Expression Of Cdk Inhibitors P21 & P27 In Bovine Corneal Endothelial Cells, Lakshmi Reddy Bollu

Masters Theses & Specialist Projects

Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell …

Respecting, Rather Than Reacting To, Race In Biomedical Research: A Response To Professors Caulfield And Mwaria, Michael J. Malinowski

Journal Articles

This Commentary is part of a colloquy on race-based genetics research.

United States Regulation Of Stem Cell Research: Recasting Government's Role And Questions To Be Resolved, Owen C. B. Hughes, Alan L. Jakimo, Michael J. Malinowski

Journal Articles

This article directly addresses the stem cell controversy, but also the broader history and norms regarding the roles of federal and state government in U.S. science research funding.

Desulfovibrio Desulfuricans G20 Tetraheme Cytochrome Structure At 1.5 A˚ And Cytochrome Interaction With Metal Complexes, Mrunalini Pattarkine, J J. Tanner, C A. Bottoms, Y H. Lee, Judy D. Wall

Faculty Works

The structure of the type I tetraheme cytochrome c3 from Desulfovibrio desulfuricans G20 was determined to 1.5 A˚ by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated …