Biotechnology Commons^™

Gene And Protein Sequence Optimization For High-Level Production Of Fully Active And Aglycosylated Lysostaphin In Pichia Pastoris, Hongliang Zhao, Kristina Blazanovic, Yoonjoo Choi, Chris Bailey-Kellogg, Karl E. Griswold

Dartmouth Scholarship

Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can …

Genome-Wide Rnai Ionomics Screen Reveals New Genes And Regulation Of Human Trace Element Metabolism, Mikalai I. Malinouski, Nesrin M. Hasan, Yan Zhang, Javier Seravalli, Jie Lin, Andrei Avanesov, Svetlana Lutsenko, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Trace elements are essential for human metabolism and dysregulation of their homoeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires …

Arabidopsis Accelerated Cell Death 11, Acd11, Is A Ceramide-1-Phosphate Transfer Protein And Intermediary Regulator Of Phytoceramide Levels, Dhirendra K. Simanshu, Xiuhong Zhai, David Munch, Daniel Hofius, Jennifer E. Markham, Jacek Bielawski, Alicja Bielawska, Lucy Malinina, Julian G. Molotkovsky, John W. Mundy, Dinshaw J. Patel, Rhoderick E. Brown

Department of Biochemistry: Faculty Publications

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1- phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center …

Arizona Clinical Research Workforce Survey, Cris E. Wells, Julie A. Robbins, Gaye Luna

Online Journal for Workforce Education and Development

Objective: We characterize the current clinical research workforce in Arizona with the goal of identifying possible educational training gaps for the future workforce. Background: The science community, government entities, educational institutions, and community programs continue to advocate for additional funding and resources to educate the bioscience workforce, of which clinical research is a part. However, there is little evidence that additional training and/or education is needed within the clinical research profession. Method: We utilized an electronic survey tool that was designed to solicit workforce data from clinical research professionals actively working or seeking employment in the field of clinical research …

Measuring Radiation Exposure In Human Blood Using Gene Expression, Krystal Naranjo, Melissa Bentley, Harsha Konery, Matthew Coleman

STAR Program Research Presentations

Mammalian cells are known to express genes that are associated with repairing damaged DNA. The transcript CDKN1A is one of several cell cycle regulator genes expressed in response to cell damage by ionizing radiation (IR). In this study, male and female lymphocytes; previously exposed ex vivo to IR, were used to demonstrate linear gene expression responses that may vary between genders. We used qRT-PCR to generate response curves for CDKN1A. No differences were identified for the endogenous control gene GAPDH. CDKN1A expression demonstrated average fold changes well above three fold for three of the four healthy patient donors at 24 …

The Effects Of Electrolyte Chemical Composition, Concentration And Ph On The Electrochemical Redox Reactions, Sea Choi, Xiao-Ying Yu

STAR Program Research Presentations

Electrolyte composition, concentration, pH, ionic strength, and temperature have long been known to play a role in the interface of the electrode-electrolyte. However, detailed studies are not sufficient. Cyclic voltammetry (CV) is a widely used electrochemical technique for obtaining qualitative information in electroanalysis. CV is often the first experiment performed in an electrode surface in an electrochemical study. It measures the number of electrons transferred and intermediates formed during oxidation-reduction (redox) reactions by observing its current, intensity and position of peak potential with applied voltage. This is accomplished by using a three-electrode system including working electrode (WE), counter electrode (CE) …

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 …

Effect Of Origanum Chemotypes On Broiler Intestinal Bacteria, Liliana Betancourt, Fernando Rodriguez, Vienvilay Phandanouvong, Claudia Ariza-Nieto, Michael Hume, David Nisbet, German Afanador-Tellez, Alexandra Martynova-Van Kley, Armen Nalian

Faculty Publications

Essential oils have been proposed as alternatives to antibiotic use in food animal production. This study evaluated 3 chemotypes of the Origanum genus, containing varying amounts of secondary metabolites carvacrol, thymol, and sabinene, in the broiler chicken diet. Aerial parts of Origanum vulgare L. (OL), O. vulgare L. ssp. hirtum (OH), and O. majorana (OM) were collected from a greenhouse located in the high altitude Sabana de Bogotá (Savanna of Bogotá) and O. vulgare L. ssp. hirtum (OG) produced and ground in Greece. Oregano essential oils (OEO) from these plants were obtained by steam distillation and analyzed by gas chromatography …

Development Of Loop-Mediated Isothermal Amplification Combined With Colorimetry Using Nanogold Probe For Chlamydia Trachomatis Detection, Ilada Choopara

Chulalongkorn University Theses and Dissertations (Chula ETD)

Chlamydia trachomatis จัดเป็นแบคทีเรียที่ก่อโรคติดต่อทางเพศสัมพันธ์ที่พบมากที่สุดทั่วโลกทั้งในเพศหญิงและชาย ถึงแม้ว่าโรคติดเชื้อนี้จะไม่ทําให้ผู้ติดเชื้อถึงตายและไม่แสดงอาการเริ่มต้นหรือแสดงเพียงเล็กน้อยนั้น การติดเชื้อนี้ในระยะยาวมีส่วนทําให้เป็นหมันหรือมีบุตรยาก และส่งเสริมการเกิดมะเร็งปากมดลูกจากไวรัสเอชพีวีได้ จึงเป็นที่น่าเสียดายที่การตรวจสุขภาพประจําปียังไม่มีการตรวจเชื้อนี้รวมอยู่ด้วย และยังไม่มีวิธีการตรวจเชื้อนี้ในคลินิกหรือสถานีอนามัยต่างๆ ที่การตรวจเชื้อนี้มีความสําคัญ ปัจจุบันวิธีมาตรฐานที่ใช้ตรวจคือการวิเคราะห์อาการโดยแพทย์ หรือการวิเคราะห์ลําดับเบสของยีน ompA (วิธีนี้ประกอบด้วยขั้นตอนการสกัดดีเอ็นเอด้วยชุดสกัดสําเร็จรูป การทําปฏิกิริยาลูกโซ่โพลีเมอเรส การรันเจลอิเล็กโตรโฟริซิส และการหาลําดับเบส) ดังนั้น งานวิจัยนี้จึงได้พัฒนาวิธีที่ไม่ซับซ้อนและประหยัดค่าใช้จ่ายขึ้นจากเทคนิคลูปเมดิเอเตดไอโซเทอร์มอลแอมพลิฟิเคชัน (LAMP) ร่วมกับการวัดสีโดยใช้โพรบติดอนุภาคทองระดับนาโน (AuNP) พบว่าวิธีที่พัฒนาขึ้นให้ผลความจําเพาะและความไวเทียบเท่าวิธีมาตรฐาน และใช้เวลาเพียง 24.5 นาที ขณะที่วิธีมาตรฐานใช้เวลามากกว่า 24 ชั่วโมง เมื่อรวมกับวิธีสกัดดีเอ็นเออย่างหยาบที่เหมาะสมกับชนิดของตัวอย่างพบว่าใช้เวลารวมทั้งสิ้นเพียง 29.5 นาที และมีค่าใช้จ่ายเพียง $3.6 ขณะที่วิธีมาตรฐานราคา $13.8 นอกจากนี้เมื่อทําการสุ่มตรวจในกลุ่มคนไข้และกลุ่มคนปกติ จํานวน 130 คน พบว่าวิธีที่พัฒนาขึ้นให้ผลตรงกับวิธีมาตรฐาน

Evidence For Hysteretic Substrate Channeling In The Proline Dehydrogenaseand ∆1-Pyrroline-5-Carboxylate Dehydrogenase Coupled Reaction Of Proline Utilizationa(Puta), Michael A Moxley, Nikhilesh Sanyal, Navasona Krishnan, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism.

Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step.

Conclusion: Substrate channeling in PutA exhibits hysteretic behavior.

Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation.

Evaluation Of Three Herbicide Resistance Genes For Use In Genetic Transformations And For Potential Crop Protection In Algae Production, Andrew J. Bruggeman, Daniel Kuehler, Donald P. Weeks

Department of Biochemistry: Faculty Publications

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.79-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, …

Agricultural Biotechnology-An Opportunity To Feed A World Of Ten Billion, Nina V. Fedoroff, Drew L. Kershen

Faculty Articles

No abstract provided.

Pharmacokinetic And Biodistribution Assessment Of A Near Infrared-Labeled Psma-Specific Small Molecule In Tumor-Bearing Mice, Joy L. Kovar, Lael L. Cheung, Melanie A. Simpson, D. Michael Olive

Department of Biochemistry: Faculty Publications

Prostate cancer is themost frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27. IRDye 800CW was conjugated to YC-27 or an anti-PSMA antibody used for reference. Human 22Rv1, PC3M-LN4, and/or LNCaP prostate tumor cells were exposed to the labeled compounds. In vivo targeting and clearance properties were determined in tumor-bearing mice. Organs and tumors were excised and imaged to assess probe localization. YC-27 exhibited a dose …

Preliminary Joint X-Ray And Neutron Protein Crystallographic Studies Of Ecdhfr Complexed With Folate And Nadp+, Qun Wan, Audrey Y. Kovalevsky, Mark A. Wilson, Brad C. Bennett, Paul Langan, Chris Dealwis

Department of Biochemistry: Faculty Publications

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP⁺ of 4 x 1.3 x 0.7 mm (3.6 mm³) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 A resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 A resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP⁺ ternary-complex structure in order to examine …

Ycf1-Mediated Cadmium Resistance In Yeast Is Dependent On Copper Metabolism And Antioxidant Enzymes, Wenzhong Wei, Nathan Smith, Xiaobin Wu, Heejeong Kim, Javier Seravalli, Oleh Khalimonchuk, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Aims: Acquisition and detoxification of metal ions are vital biological processes. Given the requirement of metallochaperones in cellular copper distribution and metallation of cuproproteins, this study investigates whether the metallochaperones also deliver metal ions for transporters functioning in metal detoxification. Results: Resistance to excess cadmium and copper of the yeast Saccharomyces cerevisiae, which is conferred by PCA1 and CaCRP1 metal efflux P-type ATPases, respectively, does not rely on known metallochaperones, Atx1p, Ccs1p, and Cox17p. Copper deficiency induced by the expression of CaCRP1 encoding a copper exporter occurs in the absence of Atx1p. Intriguingly, CCS1 encoding the copper chaperone for superoxide …

Integrating Mitosis, Toxicity, And Transgene Expression In A Telecommunications Packet-Switched Network Model Of Lipoplex-Mediated Gene Delivery, Timothy M. Martin, Beata Wysocki, Jared P. Beyersdorf, Tadeusz A. Wysocki, Angela K. Pannier

Department of Agricultural and Biological Systems Engineering: Faculty Publications

Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms, including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of …

Characterization Of Bacteriophage Specific To Non-Agglutinable Vibrio Cholerae, Dulyapat Benyajirapatch

Chulalongkorn University Theses and Dissertations (Chula ETD)

Vibrio spp. the main bacteria found in marine shrimp can cause diseases either in shrimp or human. Using antibiotics is a first option to reduce this problem. Unfortunately, this treatment has no sufficient potential due to antibiotic resistance can occur after long term of use. Therefore, bacteriophage has become a better alternative to control Vibrio spp. in shrimp. In this study, 100 isolates of Vibrio spp. were screened and isolated from Pacific white shrimp Litopaeneus vannamei and cultured water from shrimp hatcheries and ponds. The suspected Vibrio spp. were determined and classified into 5 species including V. cholerae, V. parahaemolyticus, …

Production And Characterization Of Monoclonal Antibodies Against Listeria Monocytogenes, Korrakot Prommajan

Chulalongkorn University Theses and Dissertations (Chula ETD)

Listeria monocytogenes contaminated in foods is a bacteria that can cause Listeriosis in human and animals. Detection of this bacteria in food products is required in order to reduce the opportunity of infection and increases consumers’ trust on taking food safely. Diagnostic based on immunological method with monoclonal antibody (MAb) specific to pathogen is accurate and precise. The objective of this research was to produce MAbs against L. monocytogenes NCTC 11994 and EGD-e strains by immunization mice with heat-inactivated, sonicated- and SDS-mercaptoethanol treated forms. After cell fusion 21 of hybridoma were obtained. MAbs reacted crossly with some Listeria spp. and …

Purification And Characterization Of A Novel Selenocysteine Lyase From Enterococcus Faecalis, Samantha Nelson

Electronic Theses and Dissertations

A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a …

The Anti-Toxin Properties Of Grape Seed Phenolic Compounds, Patrick Cherubin

Electronic Theses and Dissertations

Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. …