Protein Behavior Directed by Heparin Charge and Chain Length

Glycosaminoglycans (GAGs), highly charged biological polyelectrolytes, are of growing importance as biomaterials and pharmaceutical drugs due to their immense range of physiological functions. They bind to many proteins; however, the degree of structural selectivity in GAG-protein interactions is largely unknown .Our studies have focused on the importance of heparin (a model GAG) charge and chain length in protein binding in order to explore its potential applications in biofunctional tissue scaffold materials, as polysaccharide drugs in anticoagulation, and as inhibitory agents in protein aggregation. We used electrospray ionization mass spectrometry, capillary electrophoresis, size exclusion chromatography, dynamic/static light scattering and electrostatic protein modeling

Porphyrin as a Spectroscopic Probe of Net Electric Fields in Heme Proteins

Heme proteins have diverse functions as well as varied structures but share the same organic, conjugated cofactor. Similarly varied approaches have been taken to deduce how heme can take on different roles based on its protein environment. A unique approach is to view the protein matrix as a constellation of point charges that generates a defined, reproducible, net internal electric field that has influence over the electronic properties of the heme cofactor. This work considers how porphyrins, the basic chromophore building block of heme, can be used as a native spectroscopic sensor of internal electric field at the active site of heme proteins. First, a number of approaches to model the electrostatic nature of protein structure are described. One approach based on Coulomb’s law is used to estimate the net electric field in myoglobin, easily placing the internal electric field on the order of MV/cm. A closer inspection of myoglobin structure reveals that slight changes in position or strategic mutations can cause appreciable change in the field magnitude and direction. Then, the idea of a porphyrin probe is further developed, followed by a theoretical and spectral characterization of porphyrins substituted into heme proteins for use in emission spectroscopy as non-emissive heme must be replaced by other porphyrin analogs with higher quantum yield. Once the porphyrin–protein system has been established as the guest–host system of interest, the hole-burning Stark spectroscopy method was used to quantitatively measure the magnitude and direction of the internal electric field vector generated by the protein. The collected Stark spectra had a more established classical analysis method for analysis, but a major aspect of this work is a quantum-mechanical analysis method that has been advanced for more practical and widespread usage. This novel quantum-mechanical approach to the method has promise for greater accuracy for internal electric field determination as well as the ability to resolve the field into spatial components in order to determine not just field magnitude but also direction. The results from the new analysis of experimental data for myoglobin of the in-plane components of the field places both at 1.7 MV/cm. Finally, two ab initio excited-state methods, CIS and TDDFT, were used to calculate electronic state energies and transition dipole moment values in support of this new quantum-mechanical analysis method. The two methods are described thoroughly with presentation of benefits and drawbacks to each method

Computational and biochemical characterizations of anhydrobiosis-related intrinsically disordered proteins.

Anhydrobiosis is the remarkable phenomenon of “life without water”. It is a common technique found in plant seeds, and a rare technique utilized by some animals to temporarily stop the clock of life and enter a stasis for up to several millennia by removing all of their cellular water. If this phenomenon can be replicated, then biological and medical materials could be stored at ambient temperatures for centuries, which would address research challenges as well as enhance the availability of medicine in areas of the world where refrigeration, freezing, and cold-chain infrastructure are not developed or infeasible. Furthermore, modifying crop tissues could make them resistant to droughts, addressing one of the greatest threats to food stability around the world. This work utilizes a combination of computational techniques and novel approaches to performing biochemistry without water to elucidate the mechanisms of function of specialized proteins that are responsible for anhydrobiosis in animals, particularly the anhydrobiotic cysts of the brine shrimp Artemia franciscana. A detailed evaluation of the chemical properties of anhydrobiosis-related, intrinsically disordered proteins indicates that there are multiple protein-based strategies to achieve anhydrobiosis, but that late embryogenesis abundant (LEA) proteins are the most well understood. However, the mechanisms of LEA protein function have never been demonstrated, resulting in a wide variety of hypotheses regarding their ability to confer desiccation tolerance. This work demonstrates that a group 1 LEA protein, AfLEA1.1, and a group 6 LEA protein, AfrLEA6, undergo liquid-liquid phase separations during desiccation and thereby transiently form novel protective membraneless organelles which partition specific proteins and nucleic acids. These desiccation-induced cellular compartments are a novel mechanism to explain how LEA proteins confer desiccation tolerance, and the drivers of this behavior have been linked to the consensus sequences that define these LEA proteins. Therefore, the separation of aqueous proteins into a specialize compartment during drying is unlikely to only be a function of AfLEA1.1 and AfrLEA6, but actually the mechanism by which group 1 and group 3 LEA proteins function in plant seeds and anhydrobiotic animals. These results indicate that when water is unavailable, anhydrobiotic organisms substitute it with their own solvents

Novelty processing and smart delivery of Ganoderma Lucidum spores

In recent decades the traditional Chinese medicinal mushroom Ganoderma lucidum (GL), a fungal specie widely consumed homoeopathically in the Eastern Hemisphere, has been studied particularly with respect to antitumour and immunoenhancing effects. Research into the various claims however remains limited owing to the lack of quality and consistency across investigations. As such, efficacy and feasibility of scaleup has not been evaluated in a way that allows widespread consumption or approved treatment. This project tackles three aspects of drug development from Ganoderma lucidum: Biocompound extraction, healthcare evaluation via in-vitro testing, and encapsulation for smart delivery. These avenues are brought together for the first time to evaluate the prospects of developing GL for effective and safe healthcare. This research investigates the parameters that would influence the extractability of a biocompound from the spores of Ganoderma lucidum (GLS), via two conventional methods: Hot Water Extraction (HWE) and Ultrasound-Assisted Extraction (UAE). They are evaluated with respect to their crude water-soluble polysaccharide yield (GLPS). Solvent polarity and process duration were statistically significant factors affecting extract yield, with both extraction methods showing considerable gains over similar setups in literature, recovering over 6% crude GLPS using shorter durations and lower temperatures than other published investigations. This investigation highlighted the importance of solvent viscosity on specific DGlucan extraction in the GLPS yield. Bioactive effects of the extract were evaluated via cytotoxicity toward Human Osteosarcoma (HOS) cells in-vitro, achieving over 40% cell growth inhibition. Cytotoxicity however was only achieved when water-insoluble fractions were administered – suggesting cytotoxicity was a result of the unextracted crude triterpenoids (GLTP) containing Ganoderic Acids. Therefore, HOS-inhibitory capabilities are then compared to a GLPS extract containing Ganoderic Acids (in this work termed “PSGA”), extracted using HWE subject to supervised machine learning optimisation. As well as determining that this yield was maximised at the longest HWE duration and smallest solvent volume, it was observed to inhibit HOS growth by nearly 58% after 24 hours. Low doses and shorter incubation were most effective - suggesting concepts such as resistance (clonal selectivity) and delayed apoptosis, but further work will verify the reported effects of PSGA dosage and exposure time on cancer proliferation. Lastly, research effort is devoted to creating an alginate matrix for the controllable delivery of GLS using Electrohydrodynamic Atomisation (EHDA). Significant effects of the system’s process parameters on particle morphology are observed, in particular EHDA voltage. The carrier’s size, shape and surface features are correlated with its release profile. Importantly, GLS content (something traditionally compromised to maintain particle integrity) was maximised at 50 wt% whilst maintaining a controlled and spherical shape and size – making this study novel and extremely important. It is established that GLS-Alginate particles could offer controlled release over a 2-week administration in pH-neutral conditions; an environment not yet established as “stable” for alginate, yet reflective of physiological passage. Thus, for the first time sodium alginate is proven to be a real contender in controlling the delivery of GLS biomolecules. The reconciliation of these essential stages of drug development highlights some crucial points of focus as GL continues to undergo rigorous development in the realm of drug discovery

Program and Proceedings: The Nebraska Academy of Sciences 1880-2010

PROGRAM FRIDAY, APRIL 23, 2010 REGISTRATION FOR ACADEMY, Lobby of Lecture wing, Olin Hall Aeronautics and Space Science, Session A, Olin 249 Aeronautics and Space Science, Session B, Olin 224 Chemistry and Physics, Section A, Chemistry, Olin A Collegiate Academy, Biology Session A, Olin B Collegiate Academy, Chemistry and Physics, Session A, Olin 324 Biological and Medical Sciences, Session A, Olin 112 Biological and Medical Sciences, Session B, Smith Callen Conference Center Chemistry and Physics, Section B, Physics, Planetarium History and Philosophy of Science, Olin 325 Junior Academy, Judges Check-In, Olin 219 Junior Academy, Senior High REGISTRATION, Olin Hall Lobby NWU Health and Sciences Graduate School Fair, Olin and Smith Curtiss Halls Junior Academy, Senior High Competition, Olin 124, Olin 131 Aeronautics and Space Science, Poster Session, Olin 249 Teaching of Science and Math, Olin 325 MAIBEN MEMORIAL LECTURE, OLIN B Dr. Mark Greip, Vice-Chair, Department of Chemistry, University of Nebraska-Lincoln LUNCH, PATIO ROOM, STORY STUDENT CENTER (pay and carry tray through cafeteria line, or pay at NAS registration desk) Aeronautics Group, Conestoga Room Anthropology, Olin 111 Biological and Medical Sciences, Session C, Olin 112 Biological and Medical Sciences, Session D, Smith Callen Conference Center Chemistry and Physics, Section A, Chemistry, Olin A Chemistry and Physics, Section B, Physics, Planetarium Collegiate Academy, Biology Session A, Olin B Collegiate Academy, Biology Session B, Olin 249 Collegiate Academy, Chemistry and Physics, Session A, Olin 324 Junior Academy, Judges Check-In, Olin 219 Junior Academy, Junior High REGISTRATION, Olin Hall Lobby Junior Academy, Senior High Competition, (Final), Olin 110 Earth Science, Olin 224 Junior Academy, Junior High Competition, Olin 124, Olin 131 NJAS Board/Teacher Meeting, Olin 219 Junior Academy, General Awards Presentations, Smith Callen Conference Center BUSINESS MEETING, OLIN B SOCIAL HOUR for Members, Spouses, and Guests First United Methodist Church, 2723 N 50th Street, Lincoln, NE ANNUAL BANQUET and Presentation of Awards and Scholarships First United Methodist Church, 2723 N 50th Street, Lincoln, N

Washington University Senior Undergraduate Research Digest (WUURD), Spring 2018

From the Washington University Office of Undergraduate Research Digest (WUURD), Vol. 13, 05-01-2018. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research and Associate Dean in the College of Arts & Scien