Biotechnology Commons^™

Transposon-Mediated Stable Suppression Of Gene Expression In The Developing Chick Retina, Masaru Nakamoto, Chizu Nakamoto

Biology Faculty Publications

The embryonic chick has long been a favorite model system for in vivo studies of vertebrate development. However, a major technical limitation of the chick embryo has been the lack of efficient loss-of-function approaches for analyses of gene functions. Here, we describe a methodology in which a transgene encoding artificial microRNA sequences is introduced into embryonic chick retinal cells by in ovo electroporation and integrated into the genome using the Tol2 transposon system. We show that this methodology can induce potent and stable suppression of gene expression. This technique therefore provides a rapid and robust loss-of-function approach for studies of …

Molecularly Imprinted Polymers Synthesized As Adsorbents For Ergot Alkaloids: Characterization And In Vitro And Ex Vivo Assessment Of Effects On Ergot Alkaloid Bioavailability, Manoj B. Kudupoje

Theses and Dissertations--Animal and Food Sciences

Alkaloid toxicities negatively impact livestock health and production and are of serious economic concern to animal industries. To date, few strategies have been developed to evaluate alkaloid levels in feed or to counteract alkaloid toxicities. The present research evaluated the applicability of imprinting technology to synthesize polymers that have potential to interact with ergot alkaloids and therefore reduce their bioavailability in the GIT. The studies also evaluated applicability of synthesized polymers for use in the ruminal environment using an in vitro ruminal fermentation model, and for the ability to ameliorate vasoconstriction using ex vivo myographic evaluations.

In the first experiment, …

Development Of Immunoassay On Paper-Based Microfluidic Devices For Determination Of Ractopamine, Alpha-Fetoprotein, Human Thyroid Stimulating Hormone, Pattarachaya Preechakasedkit

Chulalongkorn University Theses and Dissertations (Chula ETD)

In this dissertation, immunoassays on paper-based microfluidic devices have been developed using different types of label for various applications in agriculture safety and medical diagnosis, which can be divided into three parts. In the first part, the conjugation method of ractopamine (RAC) and protein carrier is developed for fabricating paper-based lateral flow strip test of RAC using gold nanoparticles (AuNPs) as a label. The developed conjugation method provided a simple and one-step method with the use of less chemicals. The strip test for RAC determination in animal feed offers a linearity of 0.075-0.750 ng g-1 and a limit of detection …

Hydrogen Sulfide Removal By Nitrate Reducing And Sulfide Oxidizing Bacteria In Anoxic Bioreactors, Wannapawn Watsuntorn

Chulalongkorn University Theses and Dissertations (Chula ETD)

The strain MAL 1HM19, a nitrate reducing and sulfur oxidizing bacteria (NR-SOB) was successfully isolated from Mae Um Long Luang hot spring from Mae Hong Son province (Thailand) using hydrogen sulfide (H2S) and nitrate (NO3-) as an electron donor and acceptor, respectively. Among the isolates of NR-SOB from different sources, the strain MAL 1HM19 was the most promising novel strain of NR-SOB because of its ability to yield ~100% H2S removal, under anoxic conditions, within 5.5 h at an initial H2S concentration of 650 ppmv. The identification of strain MAL 1HM19 based on the 16S rDNA nucleotide sequence suggests that …

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, …

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 …

Seasonal Switchgrass Ecotype Contributions To Soil Organic Carbon, Deep Soil Microbial Community Composition And Rhizodeposit Uptake During An Extreme Drought, Catherine E. Stewart, Damaris Roosendaal, Karolien Denef, Elizabeth Pruessner, Louise H. Comas, Gautam Sarath, Virginia L. Jin, Marty R. Schmer, Madhavan Soundararajan

Department of Biochemistry: Faculty Publications

The importance of rhizodeposit C and associated microbial communities in deep soil C stabilization is relatively unknown. Phenotypic variability in plant root biomass could impact C cycling through belowground plant allocation, rooting architecture, and microbial community abundance and composition. We used a pulse-chase ¹³C labeling experiment with compound-specific stable-isotope probing to investigate the importance of rhizodeposit C to deep soil microbial biomass under two switchgrass ecotypes (Panicum virgatum L., Kanlow and Summer) with contrasting root morphology. We quantified root phenology, soil microbial biomass (phospholipid fatty acids, PLFA), and microbial rhizodeposit uptake (¹³C-PLFAs) to 150 cm over …

The Assembly Factor Pet117 Couples Heme A Synthase Activity To Cytochrome Oxidase Assembly, Nicholas G. Taylor, Samantha Swenson, Nicholas J. Harris, Edward M. Germany, Jennifer L. Fox, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for …

Oxidative Stress, Metabolomics Profiling, And Mechanism Of Local Anesthetic Induced Cell Death In Yeast, Cory Honsinger Thomas Boone, Ryan A. Grove, Dana Adamcova, Javier Seravalli, Jiri Adamec

Department of Biochemistry: Faculty Publications

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC₅₀ dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by …

Secs (Sinusoidal Endothelial Cells), Liver Microenvironment, And Fibrosis, Vaishaali Natarajan, Edward N. Harris, Srivatsan Kidambi

Department of Biochemistry: Faculty Publications

Liver fibrosis is awound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs …

Carbon And Acyl Chain Flux During Stress-Induced Triglyceride Accumulation By Stable Isotopic Labeling Of The Polar Microalga Coccomyxa Subellipsoidea C169, James W. Allen, Concetta C. Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO₂ balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-¹³C]glucose, ¹³CO₂, or D₂O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid …

Development Of Gene-Based Microsatellite Markers In Acer Saccharum Marsh., Monica Harmon

Dissertations, Master's Theses and Master's Reports

Sugar maple (Acer saccharum Marsh) is an important ecological and economic resource. Despite its significance, there are few molecular genetic resources available to date. This study used previously developed EST libraries generated for sugar maple and other hardwood species to develop and characterize EST-SSRs in sugar maple and test their transferability to other Acer species. Out of 58 markers tested, 22 showed amplification in the expected size range. From these, 16 polymorphic markers were selected to test transferability and 13 of these showed amplification in at least one other Acer species.

Toxoplasma Dj-1 Regulates Organelle Secretion By A Direct Interaction With Calcium-Dependent Protein Kinase 1, Matthew A. Child, Megan Garland, Ian Foe, Peter Madzelan, Moritz Treeck, Wouter A. Van Der Linden, Kristina Oresic Bender, Evanthie Weerapana, Mark A. Wilson, John C. Boothroyd, Michael L. Reese

Department of Biochemistry: Faculty Publications

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-A resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, …

Structural And Mechanistic Insights Into Hemoglobincatalyzed Hydrogen Sulfide Oxidation And The Fate Of Polysulfide Products, Victor Vitvitsky, Pramod K. Yadav, Sojin An, Javier Seravalli, Uhn-Soo Cho, Ruma V. Banerjee

Department of Biochemistry: Faculty Publications

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H₂S but, lacking organelles, cannot dispose of H₂S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H₂S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human …

Short Carboxylic Acid-Carboxylate Hydrogen Bonds Can Have Fully Localized Protons, Jiusheng Lin, Edwin Pozharski, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a defining feature of low barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homolog YajL was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp23 that satisfies standard donor-acceptor distance criteria for …

Quantification Of Cell Signaling Networks Using Kinase Activity Chemosensors, Jon R. Beck, Edward N. Harris, Cliff I. Stains

Department of Biochemistry: Faculty Publications

The ability to directly determine endogenous kinase activity in tissue homogenates provides valuable insights into signaling aberrations that underlie disease phenotypes. When activity data is collected across a panel of kinases, a unique “signaling fingerprint” is generated that allows for discrimination between diseased and normal tissue. Here we describe the use of peptide-based kinase activity sensors to fingerprint the signaling changes associated with disease states. This approach leverages the phosphorylation-sensitive sulfonamido-oxine (Sox) fluorophore to provide a direct readout of kinase enzymatic activity in unfractionated tissue homogenates from animal models or clinical samples. To demonstrate the application of this technology, we …

Re-Framing Biotechnology Regulation, Alison Peck

Law Faculty Scholarship

Biotechnology is about to spill the banks of federal regulation. New genetic engineering techniques like CRISPR-Cas9 promise revolutionary breakthroughs in medicine, agriculture, and public health-but those techniques would not be regulated under the terms of the Coordinated Framework for Regulation of Biotechnology. This revolutionary moment in biotechnology offers an opportunity to correct the flaws in the framework, which was hastily patched together at the advent of the technology. The framework has never captured all relevant technologies, has never satisfied the public that risk is being effectively managed, and has never been accessible to small companies and publicly-funded labs that increasingly …

Bioactive Compounds From Ventilago Denticulata Willd., Wannapha Molee

Chulalongkorn University Theses and Dissertations (Chula ETD)

New naphthalene derivatives (1 and 2) and a new isomer of ventilagolin (3), together with three known anthraquinones, including chrysophanol (4), physcin or emodin 3-methyl ether (5), and emodin (6), were isolated from vines of Ventilago denticulata. The isolated compounds exhibited cytotoxic activity against cancer cell lines (HuCCA-1, A549, HepG-2, MOLT-3) with IC50 values of 1.15-40.54 µg/mL. Compounds 1-3 exhibited weak antibacterial activity (MIC 200.0-400.0 µg/mL), while emodin (6) displayed moderate antibacterial activity with MIC 25.0 µg/mL. Compounds 1-3, 5, and 6 exhibited nitric oxide radical scavenging activities with IC50 232.0-604.3 µM and compounds 1 and 2 exhibited DPPH radical …

Antifungal Genome Mining And Genetics In Filamentous Actinomycete Bacteria Isolated From Local Soils, Jacob Alex, Kevin Lou, Bilal Makhdoom

Undergraduate Research Symposium Posters

Actinomycetes are gram positive, filamentous bacteria that produce useful antibiotics, antitumor agents, and agricultural products. A series of enrichments were undertaken to isolate actinomycetes from local soils, varying enrichment media, antibacterials, and soil treatments (including heat and CaCO3). Isolates were characterized by 16S rDNA sequencing, phenotypic and morphological observations, and antibiotic production. The genetic tractability of select isolates was analyzed using a panel of integrating vectors derived from ɸC31, ɸBT1, and OzzyJ phage using intergeneric conjugation. Further, a semi-degenerate multiplex PCR assay to detect ɸBT1 genomic integrants was designed and tested for the first time. Finally, PCR screens were used …

Chaperonin Containing Tcp1 (Cct) As A Target For Cancer Therapy, Ana Carr

Electronic Theses and Dissertations

Treatments for aggressive cancers like triple negative breast cancer (TNBC) and small-cell lung cancer (SCLC) have not improved and remain associated with debilitating side effects. There is an unmet medical need for better, druggable targets and improved therapeutics. To this end, we investigated the role of Chaperonin-Containing TCP1 (CCT), an evolutionarily conserved protein-folding complex composed of eight subunits (CCT1-8), in oncogenesis. Our laboratory was the first to report that the CCT2 subunit is highly expressed in breast cancer and could be therapeutically targeted. To determine whether CCT is a marker of disease progression in other cancers, we analyzed CCT2 gene …