God The Creator

...The last book I wrote was on the first eleven chapters of Genesis. And so I\u27m going to focus what I say on those first eleven chapters

The Role of the Propeptide and its Residues in Activation and Secretion of Elastase, an M4 Metalloprotease Secreted by Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone

Standing On The Rock

...And so the question is, as we look at that, what is that phrase referring to here, when Paul describes the last days as being times when people are characterized by all these things? Is he talking about a final, terrible period of world apostasy just before the coming of Jesus Christ, or is he describing the age in which we live, which would describe the culture of America in our time? I\u27m not sure in every case that we can decide that clearly. And it may very well be here that he\u27s thinking about the last times, but it\u27s significant, certainly, that he\u27s writing these words to Timothy in order to instruct Timothy what to do in such days

DEVELOPMENT OF SMALL MOLECULE NEUROPROTECTANTS

Neurodegenerative diseases are a class of conditions that lead to progressive atrophy of different parts of the central nervous system (CNS). These diseases lead to devastating clinical outcomes to patients and give rise to an enormous socio-economical burden on society.1 One commonality among some of the most well-known neurodegenerative disorders, e.g. Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), is neuroinflammation.2-4 Neuroinflammation stems from interactions of the innate immune system with toxins and insults to the central nervous system. In the case of irremovable or chronic insults and toxins, this leads to chronic damaging inflammation that hastens neuronal degeneration and exacerbates disease pathology.5,6 Recently, inflammasomes of the innate immune system have been indicated in playing essential roles in the observed inflammatory responses. The most studied inflammasome is the nod-like receptor pyrin containing 3 (NLRP3) inflammasome.7–9 Recently our research group has successfully developed sulfonamide-based small molecule inhibitors of the NLRP3 inflammasome, such as JC-21 and JC-171, as potential therapeutics for AD and MS. Our studies established that JC-21 is a selective inhibitor of the NLRP3 inflammasome.10,11 Structural modifications led to the development of JC-171 with improved pharmacokinetic properties. More importantly, our studies demonstrated the in vivo activity of JC-171 to effectively ameliorate the experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.12 Our data also strongly suggested that inhibitors based on this chemical scaffold may directly target the NLRP3 inflammasome.10–12 In this dissertation, we conducted biophysical, biochemical, and modeling studies to further elucidate the mechanistic information of these compounds as inhibitors of the NLRP3 inflammasome. In order to conduct further mechanistic studies, the NLRP3 protein was produced via transfection of HEK 293 cells with a modified plasmid of full-length human NLRP3 protein.13 Furthermore, LC-MS studies were conducted to confirm the blood-brain barrier penetration (BBB) of JC-171. Our studies established that JC-171 directly binds to the NLRP3 protein. The results also suggested that JC-171 may bind to the NACHT domain of NLRP3 while in a site that is distinct from the ATP binding site. This notion is supported by the fact that our compounds do not interfere with the ATPase activity of NLRP3. Docking studies of JC-171 to the homology model of the NACHT domain of NLRP3 also supported this assertion by showing the interaction of JC-171 with residues that are not overlapping with the ATP binding pocket. BBB penetration studies in combination with LC-MS analysis confirmed that JC-171 shows better BBB penetration when compared to MCC950. Collectively, our results strongly support that our compounds function as NLRP3 inflammasome inhibitors by directly binding to the NLRP3 protein, a novel and distinct mechanism of action when compared to the known inhibitors that target the NLRP3 inflammasome pathway. These results strongly encourage further development of such inhibitors as potential therapeutics for neurodegenerative diseases

Classification and Quantification of Phenotypic Characteristics of Normal and Dysplastic Oral Cells

Introduction Oral cancer accounts for 2.5% of all cancer cases in the United States. The 5 year survival rate or stage 1 or localized oral cancer is 82%, and the survival rate of unstaged oral cancer is only 50%; therefore, early diagnosis is key. Current clinical practices for determining malignancy of tissue include physical examination and surgical biopsy, which can be plagued by high variability between observers and are invasive for the patient. Proflavine intercalates between base pairs of nucleic acids and has excitation and emission peaks at 455 (+/- 20) nm and 515 nm respectively; therefore, it can be used as a fluorophore to highlight cell structures in vitro. Methods Four cell lines, SCC-25, CAL-27, FaDu, and NCI cancer cells, were cultured in media, stained, and then imaged with a fluorescent microscope. Images were then analyzed to determine the phenotypic differences that existed between the cell types. Normal oral epithelial cells were also recovered and given the same treatment and analysis to provide a control group for comparison. Results The average brightness and size of the nucleus and cytoplasm was determined for each of the four cell types. Average entropy of each cell line was also determined. Discussion These results showed a significant difference in phenotypic characteristics between normal and cancerous cell lines. Using this information, we can begin to construct an algorithm that can be used to classify cells as normal or abnormal in automated and semi-automated screening tests

Entering the Closet: An Examination of Views About Sexual Orientation on Campus

This piece originally appeared in the CSPA Journal in 1994 and presented literature on LGBT students, particularly noting the lack of research at that time.  It also presents findings from a qualitative project talking to LGBT students at that time about their experience

Entering the Closet: An Examination of Views About Sexual Orientation on Campus

This piece originally appeared in the CSPA Journal in 1994 and presented literature on LGBT students, particularly noting the lack of research at that time. It also presents findings from a qualitative project talking to LGBT students at that time about their experience

Field Testing of Bridge Girder Webs Subjected to Horizontal Loads

In construction of plate girder bridges, when no ground supported falsework is used, metal brackets bolted to the web of the exterior girders are used to support construction loads. The loads include the weight of the falsework, the weight of the freshly poured concrete of the overhanging portion of the bridge deck, and the weight of the finishing machine. Figure 1 shows a cross section of the bridge with the bracket mounted on the exterior girder and formwork in place. The brackets transmit to the plate girder web a vertical shear force and a couple. The couple applied to the girder web causes both significantly high stresses and deflections which, in most cases, have not been considered in designing the girder. Since the deflection allows rotation of the bracket, the overhanging portion of the deck is lowered causing a corresponding lowering of the finishing machine. The result is an undesirable decrease in deck thickness over the girders. Figure 2 shows construction brackets mounted on a bridge girder before formwork was in place. At the present time in South Dakota, it is common practice to place the brackets at a distance of six inches or less from the nearest stiffener. Since stiffener spacing cannot be standardized economically across the entire span, a bracket to be placed without regard to stiffener spacing is needed. If brackets could be placed at standard intervals, the necessary formwork could be standardized and used on different bridges. Such standardization would contribute a great deal to economy in bridge construction. The newly recommended Load Factor Method of design which-would eliminate the lateral stiffeners in areas of low shear dictates a need for a bracket that can be used without regard to stiffener spacing. Therefore stresses and deflections relative to bracket depth and distance from the nearest stiffener are being studied to determine if certain brackets could be placed without restriction to distance from the nearest stiffener