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Yes / The use of biological macromolecules like quince seed mucilage (QSM), as the common curative practice has a long history in traditional folk medicine to cure wounds and burns. However, this gel cannot be applied on exudative wounds because of the high water content and non-absorption of infection of open wounds. It also limits cell-to-cell interactions and leads to the slow wound healing process. In this study to overcome these problems, a novel QSM-based hybrid scaffold modified by PCL/PEG copolymer was designed and characterized. The properties of this scaffold (PCL/QSM/PEG) were also compared with four scaffolds of PCL/PEG, PCL/Chitosan/PEG, chitosan, and QSM, to assess the role of QSM and the combined effect of polymers in improving the function of skin tissue-engineered scaffolds. It was found, the physicochemical properties play a crucial role in regulating cell behaviors so that, PCL/QSM/PEG as a smart/stimuli-responsive bio-matrix promotes not only human-adipose stem cells (h-ASCs) adhesion but also supports fibroblasts growth, via providing a porous-network. PCL/QSM/PEG could also induce keratinocytes at a desirable level for wound healing, by increasing the mechanobiological signals. Immunocytochemistry analysis confirmed keratinocytes differentiation pattern and their normal phenotype on PCL/QSM/PEG. Our study demonstrates, QSM as a differentiation/growth-promoting biological factor can be a proper candidate for design of wound dressings and skin tissue-engineered substrates containing cell.

<p>Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3‑butanediol, a product formed in an alternative pathway to mixed acid fermentation.</p> <p>While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism. </p> <p>Here, the ALS and AHAS enzymes from <i>Klebsiella pneumoniae</i> were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site‑saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS <i>in vivo</i>.</p>

A method for measurement of crystallization induction time periods using a spectrophotometer is described. The turbidity of lysozyme solutions at sodium chloride concentrations of 0%, 3%, 6%, 9% (w/v) in 0.1 M NaAc (pH 4.0) and a lysozyme concentration of 10mg/ml was monitored at 350nm and showed a dramatic increase after nucleation occurred. Heterogeneous sources of nucleation were removed via filtration with 100,000 and 500,000 MWCO filters. Larger pore size, 0.2μm, filters were not able to remove these particles. The non-filtered and 0.2μm filtered had the same turbidity profiles except that the 0.2μm filtered had an induction time of 152min compared with the 119min induction time for the non-filtered solution. The 500,000 MWCO filter had a significant reduction in the formation of prenucleation aggregates and the induction time increased from 119min for non-filtered to 178min for the 500,000 MWCO filtered. Also, when the 100,000 MWCO filter was used there was no nucleation or crystallization over the 240min time frame. The increase in turbidity was attributed to an increase in particle size and not to bacterial growth because the solutions were found to be free of bacteria or fungi by microbiological analysis. Results from hanging drop vapor diffusion crystallization on the same solutions used in the turbidity study indicate that the sources removed by filtration lead to nucleation. Finally, sodium azide was added to the solutions at a concentrations of 0.17% (w/w) and was found to interfere with or delay lysozyme nucleation.

<p dir="ltr">UBE2C is upregulated in castration-resistant prostate cancer and shows strong correlation with high tumor grade. Currently, the scarcity of UBE2C inhibitors is alarming. This study is the first to report UBE2C post-translational modulation mediated by LIMK2 kinase. A proteome-wide screen previously conducted in the Shah lab has identified UBE2C as a direct substrate of LIMK2 using an innovative chemical genetic approach. LIMK2 regulates UBE2C in a variety of ways. First, LIMK2 directly associates with UBE2C in cells. Second, LIMK2 phosphorylates UBE2C at S123 and increases its stability at the protein level. Third, LIMK2 upregulates UBE2C mRNA and protein expression levels in cells. Contrary to its well-established function as an enzyme involved in the ubiquitin-proteosome pathway, UBE2C stabilizes LIMK2 protein expression in a reciprocal loop. This study is the first to show UBE2C stabilizing its substrate. Likewise, UBE2C increases LIMK2 mRNA and protein levels; however, the mechanism is to be elucidated. LIMK2-UBE2C loop is extremely oncogenic creating CRPC pathogenesis <i>in vivo</i>. Targeting LIMK2 is a suitable approach to effectively degrade both UBE2C and LIMK2 which leads to significant inhibition of tumor formation, cancer stem cell phenotype and epithelial to mesenchymal transition <i>in vivo</i>. Additionally, CDK1 for the longest time was thought to be the only protein of the cyclin dependent kinase family which binds to and is activated by cyclin B1 to regulate cell cycle progression. We first showed CDK5 activity in cell division and its importance in maintaining mitotic fidelity. We first established the activation of CDK5 by cyclin B1 <i>in vitro</i>. The phospho-mimetic CDK5 was observed to be less active when bound to cyclin B1 than its wild-type counterpart.</p>

<p> Understanding the function and structure of a protein is crucial for informing on rational drug design and for developing successful drug candidates. However, this understanding is often limited by the protein pipeline, i.e. the necessary steps to go from developing protein constructs to generating high-resolution structures of macromolecules. Because each step of the protein pipeline requires successful completion of the prior step, bottlenecks are often created and therefore this process can take up to several years to complete. Addressing current limitations in the protein pipeline can help to reduce the time required to successfully solve the structure of a protein. </p><p>The field of nonlinear optical (NLO) microscopy provides a potential solution to many issues surrounding the detection and characterization of protein crystals. Techniques such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) have already been shown to be effective methods for the detection of proteins with high selectivity and sensitivity. Efforts to improve high throughput capabilities of SHG microscopy for crystallization trials resulted in development of a custom microretarder array (μRA) for depth of field (DoF) extension, therefore eliminating the need for z-scanning and reducing the overall data acquisition time. Further work was done with a commercially available μRA to allow for polarization dependent TPE-UVF. By placing the μRA in the rear conjugate plane of the beam path, the patterned polarization was mapped onto the field of view and polarization information was extracted from images by Fourier analysis to aid in discrimination between crystalline and aggregate protein. </p><p>Additionally, improvements to X-ray diffraction (XRD), the current gold standard for macromolecular structure elucidation, can result in improved resolution for structure determination. X-ray induced damage to protein crystals is one of the greatest sources of loss in resolution. Previous work has been done to implement a multimodal nonlinear optical (NLO) microscope into the beamline at Argonne National Lab. This instrument aids in crystal positioning for XRD experiments by eliminating the need for X-ray rastering and reduces the overall X-ray dosage to the sample. Modifications to the system to continuously improve the capabilities of the instrument were done, focusing on redesign of the beam path to allow for epi detection of TPE-UVF and building a custom objective for improved throughput of 1064 nm light. Furthermore, a computational method using non-negative matrix factorization (NMF) was employed for isolation of unperturbed diffraction peaks and provided insight into the mechanism by which X-ray damage occurs. This work has the potential to improve the resolution of diffraction data and can be applied to other techniques where X-ray damage is of concern, such as electron microscopy.</p><div><br></div>

<p>Zein is a major storage protein of corn, with unique amphiphilic film forming properties. It is insoluble in water, but soluble in 70% ethanol and acetic acid, and has been declared ‘generally recognized as safe’ (GRAS) by the FDA. Due to new advances in food nanotechnology, zein is being investigated for various applications such as biodegradable packaging, oral delivery of proteins and peptides, scaffold for tissue engineering, as well as biodegradable sensor platforms. The time consuming and highly complicated methods for toxin and allergen analysis in the food industry necessitates the need for a rapid, selective, compact and easy-to-use method of detection for analytes. In the scope of this dissertation, we investigated the feasibility of functional zein nanocomposite films and formation of a zein nanocomposite sensor assembly for rapid and highly selective electrochemical measurements of food toxins and allergens. Fabrication of a zein based electrochemical amperometric sensor assembly was studied, first through the comparison of various zein film characteristics changes with the application of Laponite®, graphene oxide and carbon nanotube nanoparticles, followed by a proof-of-concept study by detecting the gluten allergen protein gliadin. </p> <p>To mechanistically study the functional zein nanocomposite films, Laponite®, a silica nanoparticle, was added in the presence of 70% ethanol solvent and oleic acid plasticizer. The films were studied using various characterization techniques like transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), water contact angle measurements etc. Through Si-N bond formation between Laponite® and zein, fabricated zein nanocomposite films showed increase in surface hydrophobicity, water vapor barrier properties, tensile strength and Young’s modulus. Graphene oxide (GO), a carbon nanoparticle, was also incorporated into zein through the solvent casting process. Uniform dispersion of GO nanoparticles within zein matrix were confirmed up to 1% GO loading, and covalent and hydrogen bonding mechanisms were proposed. Similar to zein-Laponite® (Z-LAP) nanocomposites, zein-GO (Z-GO) showed increase in hydrophobic tendencies, rougher surface and a 300% improvement in Young’s modulus and 180% improvement in tensile strength at only 3% GO loading. Both nanoparticles increased tensile strength, thermal stability and water vapor barrier property of the films, indicating a potential for food packaging as an alternative application for the nanocomposite films.</p> Finally, the research focused on the fabrication of an electrochemical amperometric sensor, capable of detecting the protein gliadin, which is responsible for the allergic reaction with people having celiac disease. Novel biodegradable coatings made from zein nanocomposites: zein-graphene oxide, zein-Laponite® and zein-multiwalled carbon nanotubes (Z-CNT) using drop casting technique were tested for fabricating the electrochemical sensors using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) techniques. As Z-CNT produced the strongest signals compared to other nanomaterials, the active tip of the electrochemical sensor was functionalized through a sequence of layer by layer deposition of Z-CNT nanocomposite, antibody and target analyte. Here, Z-CNT acts as a natural linker molecule with large number of functional groups, that causes immobilization of capture antibody and target, to ensure high sensor performance. Both CV curves and SWV curves indicated successful sequential immobilization of gliadin antibody onto the Z-CNT coated electrode. The Z-CNT biosensor was successfully able to give CV signals for gliadin toxins for as low as 0.5 ppm and was highly specific for gliadin in the presence of other interfering molecules, and remained stable over a 30-day period. The low-cost, thin, conductive zein films offered a promising alternative for protein immobilization platforms used in sensors and can be extended to other matrices in biosensors as well as other functional film applications

The objective of this research is to develop and apply a HILIC UHPLC stationary phase that allows for separation of intact glycoproteins. In Chapter 1 I give an overview of the problems of current glycosylation profiling with regards to biotherapeutics, and my strategy to separate the intact glycoprotein with HILIC. Chapter 2 describes the methods used to produce the nonporous packing material and stationary phase. In Chapter 3 I describe previous work in developing a HILIC polyacrylamide stationary phase, and further improvements I have made. Chapter 4 describes development of an assay in collaboration with Genentech of therapeutic mAb glycosylation. In Chapter 5, I show HILIC-MS of digested ribonuclease B as a beginning step to analyze glycosylated biomarkers.

<p>G-protein coupled receptors (GPCRs) are membrane proteins that constitute ~30% of the FDA-approved drug targets. Opioid receptors are a subtype of GPCRs with four different receptor types: delta, kappa, mu, and nociception opioid receptors. Opioids such as morphine have been used for thousands of years and are deemed the most effective method for treating pain. However, opioids can have detrimental effects if used illicitly or over an extended period of time. Intriguingly, most of the clinically used opioids act on the mu opioid receptor (µOR). Hence, efforts in recent decades have focused on other opioid receptors to treat pain and other disorders. The delta opioid receptor (δOR) is one of four opioid receptors expressed in the central and peripheral nervous system. The δOR has attracted much attention as a potential target for a multitude of diseases and disorders including substance and alcohol use disorders, ischemia, migraine, and neurodegenerative diseases. However, to date, no δOR agonists, or drugs that act directly at the δOR, have been successful as clinical candidates. Nonetheless, the therapeutic potential of the δOR necessitates the targeting its pharmacologically. In this dissertation, I highlight peptide-based modulation as well as the identification of novel agonists at the δOR. I report research findings in the context of biased agonism at δOR, which is a hypothesized cellular signaling mechanism with potential therapeutic benefits. The focus on this work is the molecular determinants of biased agonism, which were investigated using a combination of cellular and computational approaches.  </p>

<p>The pathological hallmarks of sickle cell disease originate from a single mutation of the beta hemoglobin gene resulting in a valine at position 6 instead of the canonical glutamic acid. This small change perpetuates many factors, manifesting into chronic embolic processes in the microvasculature, causing painful vaso-occlusive episodes and eventual organ failure. There have been numerous therapies developed to reduce the mortality of sickle cell ranging from agents to induce production of fetal hemoglobin to chronic blood transfusions. Although each of these options are effective at improving the quality of life for sickle cell patients, they only treat one aspect of the disease and, for some, become ineffective over time. In the hope of producing a better therapy, a better understanding of the pathogenesis of vaso-occlusive episodes is needed. While many models have been offered to account for these vaso-occlusive events, one recently proposed mechanism stems from the elevated tyrosine phosphorylation of the cytoplasmic domain of the major erythrocyte membrane protein, Band 3. Band 3 serves as a hub for many critical proteins in the red cell. It binds ankyrin, which associates the spectrin cortical cytoskeleton to the red cell membrane, deoxygenated hemoglobin, the kinases Wnk1 and OSR1, which regulate cation transport, and a glycolytic enzyme metabolon that regulates the production of ATP and glutathione. When Band 3 is tyrosine phosphorylated, each of these proteins dissociate, causing significant changes to red cell homeostasis. These changes include an accumulation of reactive oxygen species, vesiculation and release of prothrombotic microvesicles, leakage of cell free hemoglobin, and a decrease in cell volume. Normally, Band 3 exists in a predominantly unphosphorylated state, however, in sickle cell disease, Band 3 is abundantly tyrosine phosphorylated. Reduction in the tyrosine phosphorylation of Band 3 has been documented to prevent the release of microvesicles and hemoglobin from sickle cell red blood cells. Because these microvesicles and cell free hemoglobin contribute to the vaso-occlusive episodes in sickle cell patients, inhibiting the mechanism for their release offers a potential therapeutic option. But to accomplish this, the molecular cause for the elevated tyrosine phosphorylation in sickle cell disease must be identified. Since tyrosine phosphorylation is performed by a tyrosine kinase and removed by a tyrosine phosphatase, the elevation in phosphorylation must be due to changes in both of these processes. Unfortunately, the identity and nature of these kinases and phosphatases are poorly understood. In this dissertation, I identified the tyrosine kinases Syk, Lyn, and Src attributed to Band 3</p> <p>15</p> <p>phosphorylation that facilitates the release of microvesicles and hemoglobin in sickle cell red blood cells. Inhibition of Syk or one of the two Src family kinases is sufficient to prevent the destabilization of the red blood cell membrane. These kinases function in a hierarchy, where one of the three Src family kinase, Lyn phosphorylates Syk, activating it, and promoting the phosphorylation of Band 3 at tyrosines 8 and 21. Prevention of either phosphorylation event prevents the release of microvesicles and cell free hemoglobin. I also report the identification of PTP1B as the tyrosine phosphatase responsible for maintaining Band 3 in an unphosphorylated state. Interestingly, in sickle cell disease, this tyrosine phosphatase is proteolytically cleaved, resulting in a reduction in dephosphorylating potential. It has been reported previously that PTP1B is a substrate of the calcium dependent protease, calpain and that calpain inhibitors improve the cell morphology of sickle erythrocytes. Inhibition of this proteolytic process may offer an additional therapeutic option for the treatment of sickle cell disease.</p>