OU - Dissertations

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Open Access
A NEW STRATEGY FOR PHOTO-TRIGGERED RELEASE OF DRUGS BY VISIBLE/NEAR IR LIGHT: PHOTO-UNCLICK CHEMISTRY
(2012) Bio, Moses Kwabena; YOU, YOUNGJAE||Glatzhofer, Daniel
Spatio-temporally controlled release of therapeutic or diagnostic agents by the use of light has gained much attention in recent times. Controlled release of bioactive molecules and drugs is a critical issue for many biological applications. In cell biology, caged compounds (photo-releasable compounds) have been used to study the molecular processes in biological systems. In drug delivery, controlled release of active form of drugs from inactive forms (prodrugs and nano-drug delivery carriers) is critical to achieve local expression of pharmacological action of the drug, especially toxic drugs such as anti-cancer drugs. Current strategies use UV light or short-visible light to release the active compounds. However, UV or short visible light can be used only at a cellular level. Its application at a tissue/animal level (in vivo) has been hampered by its limited tissue penetration. The other concern is the cellular damage by UV light itself.
Open Access
Accessing Natural Products from Silent Biosynthetic Pathways Using Chemical Epigenetics
(2011) Henrikson, Jon C.; Cichewicz, Robert H
Natural products have played a major role in the history of medicine. Despite this history, many view the area of natural products discovery to be tapped dry, thus the focus of drug development companies has shifted towards other avenues to discover new drug leads (i.e. combinatorial chemistry). In recent years, there has been an expansion of antimicrobial-resistant organisms accompanied by a lack of new drug entities coming to market. These trends illustrate a need to find new approaches other than the current status quo.
Open Access
ACTIVATION OF FUNGAL SILENT BIOSYTHETIC PATHWAYS BY EPIGENETIC MODIFICATION
(2011) Wang, Xiaoru; CICHEWICZ, Robert H
Natural products have played an important role as drug leads for different diseases. They provide unique structural cores with diverse biological activities. Because of the overuse of antibiotics many pathogens have developed antibiotic-resistance; there is an urgent and continuing need for new antibiotics.
Open Access
The Advancement of Narrow Open Tubular Liquid Chromatography
(2021-12-17) Xiang, Piliang; Yang, Zhibo; Nanny, Mark; Shao, Yihan; Wu, Si
Open tubular columns are primarily used in gas chromatography, and they were regarded as the best choice to achieve high separation efficiency in gas chromatography. Substances diffuse much slower in liquids than in gases. An open tubular liquid chromatography column must have a small inner diameter to achieve high separation efficiency. We are one of the pioneers that explored narrow open tubular liquid chromatography with 2 μm inner diameter capillaries. Based on previous high efficiency separation of 11 fluorescently derivatized amino acids, we improved the column preparation and greatly enhanced the separation efficiency of the narrow open tubular column. This thesis focuses on optimizing the narrow open tubular column for high efficiency separation, high speed separation, high throughput separation and coupling it with mass spectrometer for single cell bottom-up proteomics.
Open Access
Advancement of Natural Products: Optimization of Instrumentation and Examples of their Application to the Isolation of New Compounds
(2019) Mattes, Allison; Cichewicz, Robert; Stevenson, Bradley; Glatzhofer, Daniel; Sharma, Indrajeet; Yang, Zhibo
The world of natural products has a long rich history dating back thousands of years and was the origin of medicine as we know it. Advancements in the field of natural products can be seen not only in the isolation of new molecules, but also in the advancement of new tools that are used to elucidate the structure of new compounds, identify lead compounds, as well as monitor the pharmokinetics of bioactive substances. The third chapter in this dissertation introduces a new tool that could be used in the later steps of drug development by being able to rapidly quantify a fluorinated compound in complex mixtures, the fourth chapter includes a comparison of an advanced spheroid bioassay with the traditional 2D assay that can be used to identify appropriate lead compounds, and the fifth chapter reveals techniques for applying natural products to a less discussed target: agricultural fungicides.
Open Access
Analysis of biogenic amines and their metabolites in brain tissue by high performance liquid chromatography with electrochemical detection.
(1977) Sasa, Suleiman Issa,
Open Access
Analysis of the effect of student cognizance of the Learning Cycle in general chemistry
(2012) Czapla, Matthew Jordan; Abraham, Michael R
While the benefits of the using the Learning Cycle have been well researched, one area that has received surprisingly little investigation is the effect that student cognizance of the learning cycle has on student performance in chemistry. The Learning Cycle, with its strong theoretical roots in scientific practice and learning theory, offers a logical opportunity to educate students in the nature of science and metacognition. In addition, by examining the class holistically, students will have the opportunity to better link the lab and lecture components of the course. We hypothesized that since a keen understanding of the nature of science, strong metacognitive ability, and a holistic view of Learning Cycle classes have all been shown to increase student comprehension in general chemistry, students who were taught to understand the Learning Cycle would perform better than students who were not. Statistical analysis of survey and grade data will be presented.
Open Access
ANTIBIOTIC COMBINATION THERAPY AGAINST MULTIDRUG-RESISTANT STAPHYLOCOCCUS EPIDERMIDIS BIOFILMS AND BROADENING ANTIBIOTIC SPECTRUM USING POLYETHYLENIMINE
(2020-07) Lam, Anh; Rice, Charles; Nanny, Mark; Wu, Si; Yang, Zhibo; Shao, Yihan
Antibiotic resistance (AR) is a serious growing threat around the globe. There has been no new antibiotic class developed in the past 30 years, while antibiotic-resistant superbugs are emerging everywhere and becoming more and more life-threatening. In 2019, AR pathogens took away about 35,000 lives and infected over 2.8 million people a year in the United States alone. Experts predict that, by 2050, AR will be the top leading cause of death, claiming 10 million lives a year. Motivated and dedicated to thousands of families who lose their loved ones each year to antibiotic-resistant infections, our research lab studies the defense mechanisms of superbugs. Instead of finding a new antibiotic, we study how to remove their resistance using a potentiator called BPEI (branched polyethylenimine). BPEI is a chemical compound that can disable the resistance factors of superbugs while traditional antibiotics (i.e. amoxicillin) can now actively target the vulnerable pathogens. It is called “combination therapy”. My initial specific contribution is study to fight one of the most commonly clinical isolates of Staph infections—multidrug-resistant Staphylococcus epidermidis. Previously known as a harmless commensal species on human skin, Staphylococcus epidermidis is now the first-ranking causative agent of hospital-related infections, with 24% mortality. It has become resistant to many antibiotics and thus acquired the name MRSE (Methicillin- Resistant Staphylococcus epidermidis). Additionally, MRSE bacteria can form dangerous biofilms – extra layers of self-made material – that protects them from antibiotics and helps them live on inanimate surfaces like medical devices for weeks to months. Persistent biofilms are also a leading cause of chronic wound infections. In the United States, a cost of $2 billion/year is estimated for S. epidermidis vascular-catheter-related bloodstream infections. This resistance is mainly governed by a protein called PBP2a, which the susceptible Staph bacteria do not have. The protein PBP2a has a very low affinity for traditional β-lactam antibiotics, thereby making first-choice antibiotics ineffective. Here, the use of BPEI becomes effective because exposure to BPEI molecules inhibits the function of PBP2a of MRSE, and therefore making them susceptible to existing antibiotics. Many experiments and analyses were conducted by using multiple biochemical techniques including microtiter plate assays, growth and time-killing curves, bacterial colony forming units, visible and fluorescence spectroscopies, electron microscopies, Fourier-transform infrared spectroscopy, and mass spectrometry. Not only being effective against MRSE, BPEI can also broaden antibiotic spectrum against other bacterial species like MRSA, Pseudomonas aeruginosa, E. coli and their biofilms. Exact concentrations of each combination treatment were found for each bacterial strain. New mechanisms of action of BPEI against different bacteria and its effects on human inflammatory responses were also elucidated and reported in this dissertation. Better understanding of how superbugs react to each treatment helps us to design more and more powerful potentiators. The efficacy of BPEI in combination therapy has shown to be effective against more than 20 clinical isolates (patients’ swab from OUHSC) of drug-resistant pathogens. This enables BPEI to function as a broad-spectrum antibiotic potentiator which expands the opportunities to improve drug design (synthesis of BPEI analogs), antibiotic development, and therapeutic approaches (i.e. anti-inflammatory) against pathogenic germs.
Open Access
Application of kinetic doping of silica sol-gel thin films to internal coating of capillary tubes and doping of branched polyethylenimine
(2020-05) Jensen, Jessica; Yip, Wai Tak; Rice, Charles; Bumm, Lloyd; Glatzhofer, Daniel; Schroeder, Susan
The applications of kinetic doping in silica sol-gel thin films are a mostly under-developed area. Previously, our lab has demonstrated a high loading capacity for cationic dye and protein guest molecules in silica thin films coated on flat-surface coverslips. Expanding on this work, we hypothesized that doping of films internally on capillary tubes was possible, with the aim of developing protocols for loading enzymes on these substrates. Such devices could be very useful for biocatalysis in microfluidic devices. Additionally, we theorized that branched polyethylenimine (BPEI) could be loaded into a silica thin film with kinetic doping. An organic molecule with a cationic charge at neutral pH, BPEI was a good candidate for kinetic doping and could not be loaded via traditional methods. Loading of BPEI could produce films and coatings that are useful in heavy metal remediation or inhibition of biofilm formation. In chapter three, kinetic doping is applied to loading internally coated capillary tubes. Parameters for internally doping capillary tubes were developed with rhodamine 6G, producing internally coated thin films with approximately 90 nm thickness. Horseradish peroxidase (HRP) was loaded into the thin films, with a 47000X increase in concentration over the loading solution. Activity of the loaded HRP was determined to be 0.019 ± 0.003 U/mg, and it was shown to have a stronger resistance to denaturation by methanol than surface-adsorbed HRP. In chapter four, kinetic doping was utilized to functionalize silica thin films with 25000 MW BPEI. To our knowledge, this is the first time that a highly basic guest such as BPEI has been doped into silica thin films. Parameters for the kinetic doping of 1800 and 750000 MW BPEI into silica sol-gel thin films were developed in chapter 5. Solvent accessible amines were quantified: 25000 and 750000 MW BPEI doped films were found to have similar amounts of amines while 1800 MW BPEI doped films had significantly less. SEM images of the films revealed drastic morphology differences between the films. Two applications of these films were tested. The 25000 MW films were tested for copper (II) sequestration to assess their potential for heavy metal sequestration, and showed high loading capacity of 10 ± 6 mmol/g. They proved to be reusable, with only a 6% reduction in the amount of copper (II) ions sequestered by the third use. The films were also stable against leaching over the course of one week in solution, with less than 1% of the original BPEI lost under various storage conditions. The efficacy of the 1800, 25000, and 750000 MW films against S. epidermis biofilms were tested with a crystal violet assay, and all films proved to be effective in inhibiting biofilm formation (p-value < 0.05). The best dopant, 25000 MW BPEI, caused an 89% reduction in biofilm growth and surpassed the performance of the clinical antibiotic gentamycin (p-value < 0.003). Most of the results in chapters three and five are pending publication at this time. Most of the results in chapter four have been previously published in 2019 in ACS Omega (Jensen, J. M.; Yip, W. T., Amine Functionalization of Silica Sol–Gel Thin Films via Kinetic Doping: A Novel, Green Approach. ACS Omega 2019, 4 (20), 18545-18554.).
Open Access
Applications of Button Sample Holders for Mid-Infrared Spectroscopy Measurements
(2021-12-17) Singh, Jaspreet; White, Robert; Nanny, Mark; Rice, Charles; Wu, Si; Yip, Wai-Tak
Infrared (IR) spectroscopy is a rapid and non-destructive method for chemical analysis that is based on interactions between electromagnetic radiation and matter. Despite its widespread popularity in commercial and research environments, there have been no major developments in IR sampling methods since the 1960’s. This dissertation describes applications of a new mid-IR spectroscopy sampling method that involves the use of a unique “button” sample holder. Buttons are fabricated from stainless-steel and comprise a backing to which a wire mesh is attached. This type of sample holder can accommodate thin layers of neat solid or liquid samples, eliminating the need for time-consuming and expensive sample dilution protocols. Because infrared radiation reflects from button surfaces over a wide range of angles, diffuse reflection optics are needed to collect the radiation after it interacts with the sample. The small wire diameters characteristic of the meshes used to construct buttons allow thin sample layers to be analyzed. Path lengths on the order of a few microns can be achieved, permitting analysis of high absorptivity neat samples, such as coals, that would otherwise need to be diluted in a non-absorbing sample matrix. The unique mechanism by which radiation interacts with samples produces spectra with greatly enhanced dynamic range when compared to conventional methods. The unconventional buttons sample holder can be employed for quantitative analyses of both solids and liquids, yielding linear calibration curves. A modified button containing a sample reservoir can be utilized as a variable path length liquid cell, with path lengths determined by the sample volume. Although path lengths are not directly proportional to sample volume, quantitative liquid sample analyses can be achieved when the effective path length is constant, which requires that constant sample volumes are added to the reservoir. The robust button design permits abrasive sampling by simply rubbing the rough button mesh over sample surfaces to remove small quantities of material. This approach was used to obtain infrared spectra of commercial pharmaceutical tablets quickly and easily. The sensitivity and reproducibility of this methodology was sufficient for product discrimination by principal component analysis (PCA). The utility of the button sample holder was extended by constructing several unique accessories. An eight-sample carousel was built to allow multiple samples to be analyzed with minimal effort. A low-temperature thermoelectric cooling accessory was constructed to rapidly cool and heat button samples. By using this device, infrared spectra at sub-ambient temperatures were obtained. This apparatus was also useful for measuring infrared spectra of highly volatile samples. By using a temperature controller, unique temperature ramp profiles were applied to solid samples to discriminate between reversible and irreversible sample changes. A button sample holder was incorporated into a variable temperature diffuse reflectance infrared Fourier transform (VT-DRIFTS) apparatus to investigate molecular level processes occurring during zeolite dehydrations. Subtle, temperature-dependent changes in cation-exchanged Linde Type A (LTA) and NaY zeolites were identified and characterized. By using spectral subtractions, sodium-LTA dehydration was found to occur by a step-wise mechanism involving temperature-dependent changes in water-cation interactions. A VT-DRIFTS study of calcium-exchanged LTA identified a complex sequence of concurrent processes, which included the formation of carbonate species. For NaY zeolite, VT-DRIFTS difference spectroscopy was used to selectively monitor changes in the hydroxyl groups contained in water and attached to zeolite framework acid sites.
Open Access
Applications of Hyphenated Analytical Techniques in Pharmaceutical Industry
(2011) Cheng, Chang; Liu, Shaorong
Analytical chemistry plays a critical role in pharmaceutical research and development. A typical analysis includes separating and identifying active pharmaceutical ingredients and impurities, determining the quantity of counterions, residual solvents, moisture, heavy metals in drug substances and drug products, and analyzing drugs and their metabolites in biological fluids. Analytical method development and validation are arguably the most important procedures in analytical research and development, because the qualities of drug substance and drug product are controlled by using analytical methods. This dissertation focuses on the application of various analytical techniques towards pharmaceutical industry.
Open Access
BACTERIAL FLAGELLA TEMPLATED SYNTHESIS OF HYBRID NANOMATERIALS BY BIOMIMETIC MINERALIZATION IN MILD CONDITIONS
(2011) Li, Dong; Mao, Chuanbin
Biological template directed nanomaterials have unique properties. Under genetic control, they can specifically interact with other macromolecules or inorganics. Moreover, the biologically based materials are easily assembled into nano-, micro- and macro-scales in a hierarchical manner because of their inherent self-assembly characteristics via molecular recognition. By integrating molecular biology, chemistry and materials science, we can control peptide-material interactions with a genetic approach. This approach provides unprecedented opportunities to design and synthesize novel nanomaterials. Bacterial flagella are composed of thousands of flagellin (FliC) proteins through self-assembly. By means of genetic engineering called peptide display, a foreign peptide can be inserted on FliC and finally displayed on the surface of bacterial flagella. At the same time, it is surface-exposed. The bio-engineered flagella can be used as a display tool for extracellular secretions, live vaccines, protein-ligand interactions, peptide display libraries, and so forth. Recently, flagella-templated synthesis and assembly of inorganic nanomaterials have exhibited a variety of promising applications as nanotubes or nanowires.
Open Access
BACTERIOPHAGE NANOFIBERS ENABLED NANOSTRUCTURES AND TARGET-RECOGNITION FOR BIO-APPLICATIONS
(2015) Xu, Hong; Mao, Chuanbin; Liu, Shaorong; Bumm, Lloyd; Yang, Zhibo; Yip, Wai Tak
Nanostructure is an object system that has dimensions on the nanoscale, i.e. between 0.1 and 100 nanometers. It includes nanotextured surface, nanotube, nanorod, nanofilament, nanoparticle, etc. The field of nanostructure science and technology is an explosively growing area in the past decades. Its tremendous potential for innovating the material science (referred as nanotechnology) and promising applications in the conjugation with current medicine (referred as nanomedicine) to preserve and improve human health is already clear, though the understanding of nanostructure functionalities is just beginning to reveal. Nanostructures can be considered as novel rising objects in the scientific community. However, to biologists, nanostructures are familiar since even the simplest organism contains astoundingly sophisticated elements functionalizing at the nanoscale which are still difficult even under the most advanced technologies, such as protein folding, formation of protein aggregates, pairing of nucleotides, and precise assembly of microbes. Filamentous bacteriophage, the subject of this dissertation, is a representative instance of naturally biological nanostructures. Using standard molecular cloning technique, foreign peptides can be fused into the phage protein on the surface, termed as Phage Display technique. Although phage display has primarily been used for studying protein-protein interactions, it has two main research directions in the area of nanotechnology by using two different aspects of phage. The filamentous shape of the phage in the nano range provides the bio-template function of phages used within nanotechnology and material science while phage display can also provide an easy way to discover new targeting motifs, drugs or epitopes for medical applications. Both methods are becoming more popular, but the possibility of identifying targeting peptides against live cells and intact tissues including tumors greatly increases the potential application of using phage display in the biomedical field. My research followed the two main research directions of phage display in nanotechnology. I also focused on the uses and advances of phage display in the biomedical field. Chapter 2 is the application of phage as a bio-template for bone regeneration: Oriented nucleation of hydroxylapatite crystals on self-assembled bacteriophage bundles. This work demonstrates the hydroxyapatite crystals can be nucleated on self-assembled bacteriophages in a preferred c-axis orientation. The resultant bacteriophage may serve as the building block to substitute collagen and form bone implant. It has a great potential in the field of tissue engineering. It also proved that the driving force of phage self-assembly is beta sheet secondary structure and beta sheet between phage bundles can induce the orientated nucleation of hydroxyapatite crystals. The beta sheet formation can be used as a general method to precisely control assemblies between engineerable biomaterials. In natural bone, HAP crystals are nucleated and grown with their c-axis preferentially parallel to collagen fibrils. In the work of Chapter 2, we also found that HAP crystals were also nucleated on EQ bundles with their c-axis preferentially parallel to phage bundles, which greatly mimicked the base level of hierarchical structure of natural bone. We further found that calcium ions were attracted to the glutamic acids and aspartic acids of peptide E and Q with a pattern matching with calcium ions on the (001) plane of HAP crystals. This specific matching then induced the HAP nucleation on the beta sheet and the growth along the c-axis. This work provided the proof for a new way of fabricating bone biomaterials by biomimicking and bioinspiration. Chapter 3 is the phage as a bio-template for the synthesis of phage-gold nanocomposites. Bacteriophage-gold nanocomposite was constructed in the work of Chapter 3 to use as a model to study the optimum condition for best TEM imaging of bioinorganic nanohybrids. In this work, wild type M13 phage was combined with gold nanoparticles to form phage-gold nanocomposite. Most Au-NPs were aligned into large belts, forming a net-like structure. TEM imaging of negatively stained bioinorganic nanohybrids is a simple way to characterize these nanostructures that may have biomedical functions. By studying the effects of different factors on the negative staining of phage-gold nanocomposites, we found that the optimum staining conditions for these bioinorganic nanohybrids are using 0.5% UA solution with pH 4.5, staining for 10 seconds, and rapidly drying the stains. This work demonstrates that each staining factor has an influence to the final appearance of stained specimens and optimum staining condition needs to be customized to the individual type of bioinorganic nanohybrids. The effects of each staining factor and a general guideline for selecting staining condition were provided in this work. Therefore, this research provides useful information for the characterization of bioinorganic nanomaterials by TEM imaging. It will help the research in the field of nanomaterials and bionanotechnology. Chapter 4 is discovering a novel targeting motif to mesenchymal stem cells by phage display to improve the efficiency and specificity of gene or drug delivery. In this work, we did cell panning on primary mesenchymal stem cells and identified peptide HMGMTK as MSC binding peptide. Further bioinformatics analysis suggests that peptide HMGMTK has high similarity to the signaling regions of chemokine CX3CL1. This may suggest a possible mechanism that could explain the MSC homing effect of the selected peptide. We then constructed a novel and effective mesenchymal stem cell gene delivery vector by liposome/protamine/DNA complex incorporating with identified MSC-targeting motif and Nuclear localized signal (NLS) motif. Through several evaluations, we proved that this resultant complex is multifunctional, effective, but low-cytotoxic MSC gene delivery vector. The transfection mediated by the resultant complex LPD+PA+NLS showed higher efficiency and specificity compared to those achieved by LPD only, LPD with control peptide, or LPD with PA only. Meanwhile, MTT results supported the low cytotoxicity of LPD+PA+NLS complex. Based on all the results, we can conclude that a novel rMSC binding peptide HMGMTK was identified and could be used to build up a promising gene carrier for facilitating liposome-mediated MSC gene delivery.
Open Access
Bacteriophage-based biomaterials for manipulating derivation and differentiation of human induced pluripotent stem cells
(2020-05) Zhou, Ningyun; Rajan, Rakhi; Hewes, Randall; Wu, Si; Burgett, Anthony
Induced pluripotent stem cells (iPSCs), which are derived from somatic cells, can differentiate into any cell type. They are promising tools in medical applications including regenerative medicine, personalized cell therapy, disease modeling, and drug discovery. The current stem cell research faces at least the following two major challenges: how to improve the reprogramming efficiency in iPSCs derivation; and how to control the differentiation of stem cells into certain cell types. The works in this dissertation attempt to find solutions to tackle the above two challenges. To enhance the reprogramming efficiency of somatic cells into iPSCs, human dermal fibroblasts (HDFs)-internalizing peptides were selected using Phage Display Peptide Library. After the selection, 3 HDF-binding peptides with high occurrences were selected for further screening. Finally, the HDF-binding peptide with the strongest affinity and high specificity was chemically conjugated to the surface of a nanoparticle plasmid carrier to improve the endocytosis efficiency and further help with the reprogramming process. To induce directional differentiation of iPSCs or iPSC-derived stem cells, a novel 2D virus-based substrate with unique self-assembled hierarchical nano- and micro-topographies was developed. This substrate can direct the bidirectional differentiation of iPSC-derived neural progenitor cells (NPCs) into neurons and astrocytes without the use of costly growth factors, which also provide a new approach for studying the interaction between neurons and astrocytes.
Open Access
Binding and transport through ferric siderophore receptors, FepA and FhuA.
(2006) Annamalai, Rajasekaran.; Klebba, Phillip F.,
Under iron deficient conditions, Gram-negative bacteria like E. coli secrete siderophores to chelate iron in the extracellular medium. Ligand-gated porins (LGPs) transport the ferric siderophore complexes across the outer membrane (OM) of the bacterium. The C-terminal domains of LGPs form channels across the OM, which are occluded by their N domains. The LGPs, FepA and FhuA transport ferric enterobactin (FeEnt) and ferrichrome (Fc) respectively into the periplasm of E. coli.
Open Access
A BINDING ISOTHERM BETWEEN FERROCENE MEDIATORS AND GLUCOSE OXIDASE FOR USE IN BIOANODE DEVELOPMENT AND INITIAL STUDIES OF AN ELECTROCATALYTIC DEOXYDEHYDRATION REACTION
(2019) Bamper, Daniel; Glatzhofer, Daniel; Nicholas, Kenneth; Crossley, Steven; Halterman, Ronald; Yip, Wai Tak
Enzymatic bioelectrodes are powered by renewable biomass sources. Glucose-powered bioelectrodes are prepared from ferrocene-functionalized linear poly(ethylenimine) (Fc-LPEI) cross-linked with ethylene glycol diglycidyl ether (EGDGE). Glucose bioanodes are normally only powered by the oxidation of glucose, however it can be envisioned that the bioanode could be used to power an electrochemical reduction. In considering a disproportionation-like system, a reaction that reduces glucose could be powered by an anodic oxidation of glucose. A potential candidate for the electrochemical reduction is deoxydehydration (DODH), as glucose contains vicinal hydroxyl groups. In this work, a Langmuir binding isotherm was detected electrochemically using cyclic voltammetry when Glucose Oxidase is treated as an inhibitor for (ferrocenylmethyl)trimethylammonium chloride (FcTAMCl) oxidation/reduction. A binding constant, Ka, was calculated from cyclic voltammetric data. This binding isotherm was observed in other ferrocene molecules, including ferrocencarboxylic acid, (((methoxy)ethoxy)ethoxy)methylferrocene, and bis(trimethylammoniummethyl)ferrocene, and were on the magnitude of 2.0 x 106 through 6.4 x 107. The magnitude of the binding isotherm varies between each derivative, with the glycol ether derivative exhibiting the strongest isotherm. Based on this Ka, a new glycol ether containing ferrocene-functionalized LPEI (FcG2LPEI) was prepared. The amperometric performance of this new material in the presence of 100 mM glucose is compared against similarly substituted ferrocenymethyl-functionalized LPEI (FcC1LPEI) that was developed by the Glatzhofer group. The amperometric performance of the FcG2LPEI is better than the amperometric performance of the FcC1LPEI, suggesting that this binding isotherm may be indicative of potential mediator performance in the polymer-supported state. Rhenium-based deoxydehydration catalysts were prepared and are demonstrated to be effective DODH catalysts for vicinal diols, using a variety of molecular and elemental reductants. The electrochemical behavior of (Me3TACN)ReO3PF6 wa investigated in aprotic media in the absence and presence of a variety of acids. The strength of the acid appears to affect the reduction potential of the complex. Attempts to prepare the Re(V) glycolate made from ReOCCl3(PPh3)2, Me3TACN, and styrene glycol were successful in deuterated dichloromethane, but not successful in deuterated acetonitrile. Attempts to prepare the Re(V) glycolate using bulk electrolysis were not successful.
Open Access
The binding of deoxyguanosine and adenosine to chemically modified polycytidylic acid.
(1979) Boyd, Phillip A.,
Open Access
Bioactive Molecules Detection and Identification in Complex Biological Systems through Activity-Based Metabolomics and Proteomics
(2020-07) Ma, Hongyan; Cichewicz, Robert; Yang, Zhibo; Karr, Elizabeth; Richter-Addo, George; White, Robert
The detection and identification of bioactive molecules (e.g. therapeutic small-molecule lead compounds, functional proteins) from complex mixtures is often a critical step in various biochemical and biomedical research fields. Conventional approaches typically rely on the purification of the molecules of interest that is guided by a specific activity/function followed by structure elucidation, chemical/physical property characterization, and bioactivity evaluation. The bioassay-guided purification/characterization process is often conducted in a labor-intensive and low-throughput manner consisting of repeated experimental steps. The lack of tools to directly pinpoint the bioactive molecules from complex crude biological samples is currently a painful challenge in the discovery of new bioactive molecules from biological samples. Emerging LC-MS-based metabolomic and proteomic approaches enabled rapid and high-throughput identification of the proteins and bioactive small molecules from complex biological samples (e.g. natural product extracts/fractions, cell lysates). Nevertheless, it remains challenging to incorporate both the activity and chemical identifications of the bioactive molecules directly from the complex mixtures. To address this problem, we have developed two new approaches: 1) Lickety-split Ligand Affinity based Molecular Angling System (LLAMAS), and 2) Activity-Correlated Quantitative Protein Profiling Platform (ACPP), for direct detection and identification of bioactive small molecules and proteins, respectively, from complex biological samples by combining the activity-based assays with quantitative metabolomics and proteomics studies. In natural product discovery, bioactive compounds are usually purified via bioassay-guided fractionations. Loss of the activity or failure in the purification of the minor or unstable bioactive compounds during the commonly multi-step workflows (e.g. multiple rounds of biological tests and chemical fractionations) is a common and costly limitation in the isolation of bioactive natural products. A major reason behind the problem lies in that most common bioassay models (e.g. phenotypic and genotypic) are not accessible to the direct detection and identification of individual bioactive chemical components from complex fractions and crude extracts rich in di-verse secondary metabolites. To address this, the Lickety-split Ligand Affinity based Molecular Angling System (LLAMAS), an ultrafiltration-LC-PDA-MS/MS-based DNA binding assay, coupled with modern dereplication tools (e.g. GNPS, and DNP), was established for quick and easy identification and dereplication of DNA binding agents in complex samples. This assay was developed using eight known small-molecule DNA binders (1-8) with different properties (e.g. DNA binding mechanism, solubility, and LC-MS performance). It was further validated by the successful detection of two DNA binders that were spiked into a highly complex soil extract and the dereplication of three known DNA intercalators including actinomycin D (9), V (10) and X0β (11) from the crude extract of Streptomyces antibioticus. To improve the throughput, the approach was further optimized to a 96-well plate-based assay—LLAMAS 2.0. Seven DNA binding candidates, including berberine (12), palmatine (13), coptisine (14), fangchinoline (15), tetrandrine (16), daurisoline (17), and dauricine (18) were detected and identified from three herbal extracts through high-throughput screening of 394 plant extracts. The results demonstrated that LLAMAS enables efficient identification and dereplication of DNA binding molecules directly from complex metabolite extracts in the early stage of natural product discovery pipelines to avoid repeated purification of known active compounds. Functional protein (e.g. catalytic enzymes) identification and its activity validation is often a time-consuming and labor-intensive process due to the inclusion of protein overexpression and purification steps. Proteomics approaches have been applied for the discovery of novel enzyme candidates from microbial secretomes by comparing protein expression profiles with the enzyme activity of different secretome cocktails obtained under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident enzymatic activity assignment, which remains to be elucidated. To address this challenge, an Activity-Correlated Protein Profiling Platform (ACPP) was developed, that can systematically correlates protein-level enzymatic activity patterns with protein elution profiles obtained from the “native” LC prefractionation and quantitative proteomics analysis. Enzymatic activity patterns in sequential fractions measured in different micro-scale bioassays were correlated with protein elution pro-files (protein relative intensities in each fraction) using the Pearson Correlation algorithm to generate R-scores. The activity was then confidently assigned based on R-score and record of predicted functions in free-accessible databases, such as NCBI and UniProt. The ACPP has been successfully applied to the identification of two types of active biomass-degrading enzymes (e.g. starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major active starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major active cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.
Open Access
THE BIOCHEMICAL CHARACTERIZATION OF TWO PHOSPHORELAY SIGNALING PROTEINS: YPD1 HOMOLOGS FROM SACCHAROMYCES CEREVISIAE AND CRYPTOCOCCUS NEOFORMANS
(2016-08) Kennedy, Emily; West, Ann; Karr, Elizabeth; Cichewicz, Robert; Bourne, Christina; Zgurskaya, Elena
Histidine-to-aspartate multi-step phosphorelay systems are used extensively by eukaryotes and bacteria to sense and respond to changes in their external environment and to control the regulation of crucial biological functions such as cell cycle, mating, and virulence. These pathways generally consists of a hybrid histidine kinase (HHK), a single histidine phosphotransfer protein (HPt), and one or more response regulator proteins (RR). The histidine phosphotransfer protein, Ypd1, is found at the branch point of these signal transduction pathways in several fungal organisms, and acts as an intermediate that is responsible for relaying messages between the HHK, which senses the signal, and the RR, which initiates a cellular response. This protein, or a homolog, is often the sole HPt in fungal organisms. In several fungal species it has been shown to be essential for viability. These organisms include the common baker’s yeast Saccharomyces cerevisiae (Sc) and the human fungal pathogen Cryptococcus neoformans (Cn). HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H+4 position, relative to the phosphorylatable histidine is found in a significant number of annotated HPts. Substitution of residues with larger side chains at this position typically disrupts signal transduction by altering the structure and/or enzymatic activity of the phosphotransfer-competent complex formed between HPts and their cognate partners. Previous work using yeast two-hybrid assays has shown that the residue in the H+4 position on the surface of ScYpd1 in the G68 position has an only slightly diminished activity in its ability to accept a phosphoryl group from the receiver domain of ScSln1, but a highly diminished activity in its ability to transfer its phosphoryl group to the receiver domain of ScSsk1. Here we report a comprehensive analysis of a set of point mutants at position 68 of Ypd1 to gain insights into the evolutionary constraints leading to the exceptionally high conservation of glycine at position H+4 in HPts. We combine bioinformatics, phosphoryl transfer assays, X-ray crystallography, fluorescence-based binding assay and molecular dynamics techniques to help explain the delicate balance of interatomic interactions required for phosphotransfer to occur between an HPt and its cognate RR. The conclusions that can be drawn clarify the importance of a small and/or hydrophilic residue in the H+4 position. Ypd1 from C. neoformans is distinct from Ypd1 from S. cerevisiae. Based on sequence alignments, CnYpd1 includes an extended N-terminal region that is not present in ScYpd1 of approximately 60 residues. This extended N-terminal region shares no close sequence homology with any other known protein domain. Organisms with stand-alone HPt proteins that include an extended N-terminal region are rare, and are found strictly in fungal species that have been shown to be pathogenic to plants or humans, or that live in extreme environments. Results from this study indicate that CnYpd1 exhibits unique characteristics in comparison to other histidine phosphotransfer proteins, such as an extended N-terminal amino acid sequence, which we find contributes to structural integrity, a longer phosphorylated life-time and the ability to bind calcium ions.
Open Access
BIOCHEMISTRY OF HEMIN UPTAKE IN LISTERIA MONOCYTOGENES AND THE FUCTION OF TONB IN ESCHERICHIA COLI
(2009) Jiang, Xiaoxu; Klebba, Philip E
Iron is an essential metal element for the metabolism pathway of most microorganisms. For pathogens, iron is also an important factor for virulence. But in the host body, the availability of free iron is very low. Iron mainly associates with heme in hemoglobin or is tightly bound to iron-associated proteins like transferrin, lactoferrin and ferritin. Both Gram-negative and Gram-positive pathogens have iron transporters in their cell envelopes to acquire iron from the environment, and one of the target iron compound for these transport systems is heme in the red blood cells.