Biotechnology Commons^™

Session C-5: Biotech Basics, Elizabeth Martinez, Emily Hood Ferrin

Professional Learning Day

The field of biotechnology is rapidly expanding. What is biotech? What are the applications? How is biotech studied? These topics will be explored as participants engage in hands-on lessons developed at an appropriate level for middle school students. Activities may be used to introduce students to the world of biotech.

Genesis And Growth Of Extracellular Vesicle-Derived Microcalcification In Atherosclerotic Plaques, Joshua D. Hutcheson, Claudia Goettsch, Sergio Bertazzo, Natalia Maldonado, Jessica L. Ruiz, Wilson Goh, Katsumi Yabusaki, Tyler Faits, Carlijn Bouten, Gregory Franck, Thibaut Quillard, Peter Libby, Masanori Aikawa, Sheldon Weinbaum, Elena Aikawa

Publications and Research

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the …

Methods For Detecting Microbial Methane Production And Consumption By Gas Chromatography, Jared T. Aldridge, Jennie L. Catlett, Megan L. Smith, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic …

Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials For Small Molecule Delivery, Min Dai, Ja Frezzo, E Sharma, R Chen, N Singh, C Yuvienco, E Caglar, S Xiao, A Saxena, Jk Montclare

Publications and Research

We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrate a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP …

Comparing Apples And Oranges?: Next Generation Sequencing And Its Impact On Microbiome Analysis, Adam G. Clooney, Fiona Fouhy, Roy D. Sleator, Aisling O'Driscoll, Stanton Catherine, Paul D. Cotter, Marcus J. Claesson

Department of Biological Sciences Publications

Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to …

Stabilin-1 And Stabilin-2 Are Specific Receptors For The Cellular Internalization Of Phosphorothioate-Modified Antisense Oligonucleotides (Asos) In The Liver, Colton M. Miller, Aaron J. Donnerr, Emma E. Blank, Andrew W. Egger, Brianna M. Kellar, Michael E. Østergaard, Punit P. Seth, Edward N. Harris

Department of Biochemistry: Faculty Publications

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro^TM, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform …

Dna Sequencing Activity, Sarah O'Leary-Driscoll

Sequencing & Genome Mining

This activity is meant to introduce students to basic DNA sequencing techniques. Using a hands-on approach helps them understand the fundamentals of what is happening in the lab, and the questions that accompany the activity can lead to more in depth discussions about sequencing, and the development of the next generation sequencing techniques.

Hydrodynamic Loading In Concomitance With Exogenous Cytokine Stimulation Modulates Differentiation Of Bovine Mesenchymal Stem Cells Towards Osteochondral Lineages, Stephen M. Goldman, Gilda A. Barabino

Publications and Research

Background: Mesenchymal stem cells (MSCs) are viewed as a having significant potential for tissue engineering and regenerative medicine therapies. Clinical implementation of MSCs, however, demands that their preparation be stable and reproducible. Given that environmental and bioprocessing parameters such as substrate stiffness, seeding densities, culture medium composition, and mechanical loading can result in undirected differentiation of the MSC population, the objective of this study was to systematically investigate how hydrodynamic loading influences the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) towards the osteochondral lineages both in the presence and absence of exogenous, inductive factors.

Methods: Expanded bovine MSCs were …

Screening Of Synthetic Chemical Agents “Chromenes” With Potent Anti-Breast Cancer Activity, Aysha Hamad Zaher Saeed Al Meqbali

Theses

Cancer is the second leading cause of death worldwide. Conventional therapies cause serious side effects and, at best, merely extend the patient’s lifespan by a few years. Cancer control may therefore benefit from the potential that resides in alternative therapies. There is thus an increasing demand to utilize alternative concepts or approaches to the prevention of cancer. The principal aim of this research is to screen and identify new synthetic compounds “chromenes”, with high efficiency, for breast cancer therapy. In this study, we have screened four newly designed and synthesized chromenes (C1, C15, C28 and C29) for their potential anticancer …

Utilization Of The Clinical Laboratory For The Implementation Of Concussion Biomarkers In Collegiate Football And The Necessity Of Personalized And Predictive Athlete Specific Reference Intervals, Stefanie Podlog (Nee Schulte), Natalie N. Rasmussen, Joseph W. Mcbeth, Patrick Q. Richards, Eric Yochem, David J. Petron, Frederick G. Strathmann

Athletic Training Collection

Background: A continued interest in concussion biomarkers makes the eventual implementation of identified biomarkers into routine concussion assessment an eventual reality. We sought to develop and test an interdisciplinary approach that could be used to integrate blood-based biomarkers into the established concussion management program for a collegiate football team.

Methods: We used a CLIA-certified laboratory for all testing and chose biomarkers where clinically validated testing was available as would be required for results used in clinical decision making. We summarized the existing methods and results for concussion assessment across an entire season to identify and demonstrate the challenges with the …

Genome-Wide Transcriptomic And Proteomic Analyses Of Bollworm-Infested Developing Cotton Bolls Revealed The Genes And Pathways Involved In The Insect Pest Defence Mechanism, Saravanan Kumar, Kanakachari Mogilicherla, Dhandapani Gurusamy, Krishan Kumar, Prabhakaran Narayanasamy, Padmalatha Kethireddy Venkata, Amolkumar Solanke, Savita Gamanagatti, Vamadevaiah Hiremath, Ishwarappa S. Katageri, Sadhu Leelavathi, Polumetla Ananda Kumar, Vanga Siva Reddy

Entomology Faculty Publications

Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome-wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi-quantitative real-time PCR. Comparative analyses have revealed that 39% of the transcriptome and 35% of the proteome were differentially regulated during bollworm infestation. Around 36% of significantly regulated transcripts and 45% …

Biological Lignocellulose Solubilization: Comparative Evaluation Of Biocatalysts And Enhancement Via Cotreatment, Julie M. D. Paye, Anna Guseva, Sarah K. Hammer, Erica Gjersing

Dartmouth Scholarship

Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions.

Nitrogenase Femoco Investigated By Spatially Resolved Anomalous Dispersion Refinement, Thomas Spatzal, Julia Schlesier, Eva-Maria Burger, Daniel Sippel, Limei Zhang, Susana L.A. Andrade, Douglas C. Rees, Oliver Einsle

Department of Biochemistry: Faculty Publications

The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S 1⁄4 3/2 state the common iron oxidation state assignments must be reconsidered. By a spatially resolved refinement of the anomalous scattering contributions of the 7 Fe atoms of FeMoco, we conclude that three irons (Fe1/3/7) are more reduced than the other four (Fe2/4/5/6). Our data are in agreement with the …

Vo-Ohpic Treatment Reduces Cardiac Remodeling In Doxorubicin-Induced Cardiomyopathy, Taylor Johnson

Electronic Theses and Dissertations

Doxorubicin (Doxo) is one of multiple anthracycline drugs used to effectively treat various forms of cancer. Unfortunately, Doxo treatment, as a side effect, induces cardiomyopathy and subsequent heart failure. We have previously demonstrated that transplanted embryonic stem (ES) cells and their conditioned medium (CM) modulate the PTEN pathway and reduce apoptosis, fibrosis and hypertrophy in a Doxo induced cardiomyopathy (DIC) model. However, mechanisms of inhibited apoptosis mediated through PTEN pathway are completely unknown. Therefore, we used VO-OHpic (VO), a potent PTEN inhibitor to understand the mechanism of apoptosis as well as its effect on cardiac remodeling in DIC. Animals were …

The Role Of Type-I Interferon In Limiting Spread And Killing Of An Oncolytic Rna Virus In Prostate Cells, Kritika Kedarinath

Electronic Theses and Dissertations

Prostate cancer is the second most prevalent cancer amongst men and there is an urgent need to address viable therapeutic options for its treatment. Development of viruses which target and kill cancer cells has gained momentum due to the first FDA approved oncolytic virus for treating human cancer patients. Our previous work with the RNA virus, Parainfluenza Virus 5 (PIV5), has led to the generation of mutants that are potential candidates for oncolytic viruses: 1) the hyperfusogenic (P/V/F) mutant has a mutated P/V and fusion gene which activates anti-viral responses and causes massive cell-cell fusion respectively, and 2) the Leader …

Malondialdehyde (Mda) And Glutathione Peroxidase (Gpx) Are Elevated In Crohns Disease-Associated With Mycobacterium Avium Subspecies Paratuberculosis (Map), Ahmad Qasem

Electronic Theses and Dissertations

Inflamed tissue in Crohn’s disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The level of MAP antibodies in bovine sera was determined by IDEXX kit whereas detection of MAP DNA was performed by IS900-based nPCR. A total of 42 cattle (21 infected with MAP and 21 …

Manufacturing Barriers To Biologics Competition And Innovation, W. Nicholson Price Ii, Arti K. Rai

Faculty Scholarship

As finding breakthrough small-molecule drugs gets harder, drug companies are increasingly turning to “large molecule” biologics. Although biologics represent many of the most promising new therapies for previously intractable diseases, they are extremely expensive. Moreover, the pathway for generic-type competition set up by Congress in 2010 is unlikely to yield significant cost savings.

In this Article, we provide a fresh diagnosis of, and prescription for, this major public policy problem. We argue that the key cause is pervasive trade secrecy in the complex area of biologics manufacturing. Under the current regime, this trade secrecy, combined with certain features of FDA …

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 …

Role Of Apoptotic Hepatocytes In Hcv Dissemination: Regulation By Acetaldehyde, Murali Ganesan, Sathish Kumar Natarajan, Jinjin Zhang, Justin L. Mott, Larisa I. Poluektova, Benita L. Mcvicker, Kusum K. Kharbanda, Dean J. Tuma, Natalie A. Osna

Department of Biochemistry: Faculty Publications

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, …

Inhibition Of Protein Ubiquitination By Paraquat And 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways, Juliana Navarro-Yepes, Anandhan Annadurai, Erin Bradley, Iryna Bohovych, Bo Yarabe, Annemieke De Jong, Huib Ovaa, You Zhou, Oleh Khalimonchuk, Betzabet Quintanilla-Vega, Rodrigo Franco

Department of Biochemistry: Faculty Publications

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP⁺) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells …

Tumor Suppressive Role Of Sestrin2 During Colitis And Colon Carcinogenesis, Seung-Hyun Ro, Xiang Xue, Sadeesh K. Ramakrishnan, Chun-Seok Cho, Sim Namkoong, Insook Jang, Ian A. Semple, Allison Ho, Hwan-Woo Park, Yatrik M. Shah, Jun Hee Lee

Department of Biochemistry: Faculty Publications

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery …

Potassium And The K+/H+ Exchanger Kha1p Promote Binding Of Copper To Apofet3p Multi-Copper Ferroxidase, Xiaobin Wu, Heejeong Kim, Javier Seravalli, Joseph J. Barycki, P. John Hart, David W. Gohara, Enrico Di Cera, Won Hee Jung, Daniel J. Kosman, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K⁺) compartmentalization to the trans-Golgi network via Kha1p, a K⁺/H⁺ exchanger. K⁺ in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K⁺ is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K⁺ acts as a chemical factor in Fet3p maturation, a …

Analysis Of Oligomerization Properties Of Heme A Synthase Provides Insights Into Its Function In Eukaryotes, Samantha Swenson, Andrew Cannon, Nicholas J. Harris, Nicholas G. Taylor, Jennifer L. Fox, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Heme a is an essential cofactor for function of cytochrome c oxidase in the mitochondrial electron transport chain. Several evolutionarily conserved enzymes have been implicated in the biosynthesis of heme a, including the heme a synthase Cox15. However, the structure of Cox15 is unknown, its enzymatic mechanism and the role of active site residues remain debated, and recent discoveries suggest additional chaperone-like roles for this enzyme. Here, we investigated Cox15 in the model eukaryote Saccharomyces cerevisiae via several approaches to examine its oligomeric states and determine the effects of active site and human pathogenic mutations. Our results indicate that Cox15 …

Global Reach, Local Markets: The Challenges Of Leading Global Innovation, Sarah Higgins

Honors Theses

This Global Studies honors thesis addresses how managers and leaders of global firms manage innovation across multiple markets. Current research on multinational corporations provides an understanding of different kinds of innovation and the ways to attend to multiple markets. However, there is less documentation of how these innovation strategies are actually implemented on the ground and the tensions that these efforts might produce. Therefore, my research focuses in particular on the challenges and tensions faced by leaders of global firms as they implement transnational innovation strategies. This study is based upon in-depth interviews with 20 participants who held positions in …

การผลิตโปรตีเอสจากวัสดุเหลือทิ้งทางการเกษตรโดย Bacillus Pumilus, พัชราภรณ์ การงาน

Chulalongkorn University Theses and Dissertations (Chula ETD)

โปรตีเอสเป็นเอนไซม์ที่เร่งปฏิกิริยาการย่อยสลายโปรตีนให้เป็นกรดอะมิโน และเปปไทด์สายสั้นๆ ซึ่งสามารถนำไปประยุกต์ใช้ในการบำบัดน้ำเสียที่มีโปรตีนนมสูงจากร้านกาแฟได้ ซึ่งแบคทีเรียในสกุล Bacillus เป็นแบคทีเรียที่ผลิตโปรติเอสได้ดี โดย Bacillus pumilus มีความสามารถในการผลิตโปรติเอสปริมาณสูง ซึ่งคัดแยกได้จากบ่อน้ำพุร้อนทางภาคเหนือของประเทศไทย วิทยานิพนธ์นี้สนใจการผลิตโปรตีเอสจาก B. pumilus และศึกษาปัจจัยของแหล่งคาร์บอน และไนโตรเจน รวมถึงภาวะที่เหมาะสมต่อการผลิตโปรติเอส การผลิตโปรติเอสจาก B. pumilus มีค่ากิจกรรมของเอนไซม์โปรติเอสสูงที่สุด เท่ากับ 2.545 ยูนิตต่อมิลลิลิตร โดยเลี้ยงในอาหารสำหรับผลิตโปรติเอสประกอบด้วย 1 เปอร์เซ็นต์ โมลาส 1 เปอร์เซ็นต์ รำข้าว ภาวะในการเลี้ยงที่เหมาะสม คือ ค่าความเป็นกรด-ด่างเริ่มต้นในการเลี้ยงเท่ากับ 8.0 บ่มที่อุณหภูมิ 30 องศาเซลเซียส ความเร็วรอบ 250 รอบต่อนาที เป็นเวลา 24 ชั่วโมง ราคาอาหารเป็นหนึ่งในปัจจัยสำคัญสำหรับการผลิตโปรติเอส ดังนั้นการใช้วัสดุเหลือทิ้งทางการเกษตรเป็นแหล่งอาหารเลี้ยงเชื้อ เป็นการลดต้นทุนในการผลิตโปรตีเอส จึงเหมาะกับการนำไปประยุกต์ใช้ในระดับอุตสาหกรรม และการประยุกต์ใช้ในการบำบัดน้ำเสีย

Alcoholic Vs Non-Alcoholic Fatty Liver In Rats: Distinct Differences In Endocytosis And Vesicle Trafficking Despite Similar Pathology, Karuna Rasineni, Daniel D. Penrice, Sathish Kumar Natarajan, Mark A. Mcniven, Benita L. Mcvicker, Kusum K. Kharbanda, Carol A. Casey, Edward N. Harris

Department of Biochemistry: Faculty Publications

Background: Non-alcoholic and alcoholic fatty liver disease (NAFLD and AFLD, respectively) are major health problems, as patients with either condition can progress to hepatitis, fibrosis, and cirrhosis. Although histologically similar, key differences likely exist in these two models. For example, altered content of several vesicle trafficking proteins have been identified in AFLD, but their content in NAFLD is unknown. In this study, we compared select parameters in NAFLD and AFLD in a rat model.

Methods: We fed either Lieber- DeCarli liquid control or alcohol-containing (35 % as calories) diet (AFLD model) or lean or high-fat (12 or 60 % derived …

Atomic Resolution Experimental Phase Information Reveals Extensive Disorder And Bound 2-Methyl-2,4-Pentanediol In Ca2+-Calmodulin, Jiusheng Lin, Henry Van Den Bedem, Axel T. Brunger, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca²⁺-CaM, but no assignment of these features has been made. In addition, Ca²⁺-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 A resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca²⁺-CaM. …

An Evaluation Of New And Established Methods To Determine T-Dna Copy Number And Homozygosity In Transgenic Plants, Katarzyna Glowacka, Johannes Kromdijk, Lauriebeth Leonelli, Krishna K. Niyogi, Tom E. Clemente, Stephen P. Long

Department of Biochemistry: Faculty Publications

Stable transformation of plants is a powerful tool for hypothesis testing.Arapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations.Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher …

Microbiological Control And Mechanisms Of Action Of High Voltage Atmospheric Cold Plasma, Lu Han

Doctoral

Dielectric barrier discharge-atmospheric cold plasma (DBD-ACP) is a promising non-thermal technology effective against a wide range of microorganisms. These studies were performed using a custom built DBD-ACP system. The inactivation efficacy was found to be governed by a series of critical control parameters, including treatment time, mode of exposure (Direct/Indirect exposure), applied voltage, applied gas content but was also very dependent on the characteristics of treatment targets. In this study, these parameters were investigated using in-package design along with a post-treatment storage procedure to align with industrial processing times as well as mitigation of post-process contamination. A range of food …