The physicochemical features that affect cell-membrane permeability of bacteria are currently not well understood. Due to the lack of tools to predict penetration of the bacterial envelope, the discovery and development of novel antibiotics is at a standstill. For this reason, the increasing spread of resistant bacteria has become a major threat to public health because these pathogens remain unchallenged. To understand how intrinsic properties, such as porins and efflux pumps, of Gram-negative bacteria influence membrane permeability and consequently, antibiotic uptake, we have developed a systematic approach to evaluate the penetration of various compounds into several strains of Escherichia coli. We study Wild Type, a porin (ompR) knockout, and an efflux pump (tolC) knockout to measure the effects that these physicochemcial properties have on antibacterial uptake. Minimum inhibitory concentrations are calculated for eighteen different compounds when tested against these three strains of E. coli. Next, compound accumulation is assessed through LC-MS/MS analysis and killing kinetics are observed. Through this, a relationship between chemical structure and bacterial membrane permeability can be identified, thus a better understanding of the roles of porins and efflux pumps in the development of antimicrobial resistance is gained.

In the past 5 years, almost 100,000 children have been internationally adopted. Research suggests that many of these children have growth and developmental delays, but few large scale studies have been completed. In the current study data obtained from a restrospective chart review of 534 children followed in the International Adoption Clinic at Cincinnati Children’s Hospital Medical Center between 1999 and 2007 was analyzed. Prevalence of developmental delays and sensory processing issues initially and after 6 months in country, the relationship of delays/issues with previously identified risk factors, and the frequency of recommendations for early intervention (EI) and therapy services were all examined. Results demonstrated the following: 1) Development: most children fell > 1SD below the mean in at least one developmental domain (Vineland Developmental Motor Scales) initially, however improvements in developmental scores were noted between initial and follow up visits. The number of children with a delay in at least one domain dropped from 58% (initial visit) to 44% (follow up). Developmental scores at six months had the strongest correlation with age at time of adoption; children adopted at older ages had lower scores at follow up. 2) Sensory processing: Infant/toddler sensory profile scores for children aged 1-3 were compared at initial and follow up visits. The percentage of children with atypical scores in at least one area of sensory processing decreased from 68% initially to 48% by follow up. Similarly, children > 3 years of age demonstrated improvements in sensory processing; at follow up, only 15% had an atypical total score on the short sensory profile compared to 42% initially. 3) Referral: Referral data was available for 61% of children; of these approximately half received a recommendation for additional services. Most referrals were for EI services (31%); 22% received a referral to speech therapy, and 12% received referrals to PT and OT. Consistent with other work, a significant number of internationally adopted children demonstrate delays in developmental skills and sensory processing abilities. Although improvements in these areas were generally noted, developmental delays and sensory processing issues remained for some children 6 months after adoption. Over half of the children received recommendations for additional services; this number increased for those with more significant delays.

ABSTRACT SCREENING PROSTATE SPECIFIC ANTIGEN EFFECTS ON RACIAL DISPARATE MORTALITY: A PROPENSITY SCORE ANALYSIS By R. David McNally, Ph.D., M.S.H.A. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2011 Dissertation Chair: Jeffrey S. Legg, Ph.D., Associate Professor and Chair, Doctoral Program in Health Related Sciences Department of Radiation Sciences Prostate cancer is the most commonly diagnosed cancer among men in the United States. It is frequently cited that racial disparities in mortality between Caucasian and African American men with localized prostate cancer exist. In addition, the question of whether prostate cancer screening with the prostate specific antigen blood test (PSA) leads to reduced mortality remains unanswered. Outcomes theory and survival analysis have shown controversial inconsistencies in support of early detection methods for prostate cancer to the extent that experts in the medical community do not agree on best-practice guidelines suggestive of eliminating such disparities and reducing mortality. The purpose of this study was to explore the relationship between screening PSA tests and racial differences in mortality among Caucasian and African American men with application of a propensity scoring analysis on a large population-based data set. Prostate cancer patients diagnosed from January 1, 1986 through December 31, 2006 (n = 515,802 cases) from the SEER-17 data set linked to Medicare claims files were included. A separate analysis using a 5% randomized group of over 263,000 men without prostate cancer was also examined. The results demonstrated that no statistically significant differences in mortality between Caucasians and African Americans in the prostate cancer group existed (p=0.993). Further, the same result was found among men from the 5% randomized group without prostate cancer (p= 0.832), that no statistically significant difference exists for this study population when using a propensity scoring analysis and a conditional Cox regression model. From both analyses, no survival benefit was found for screened men versus non-screened men when using the PSA test for early detection. In addition, because age is a well-known predictor of death, a separate analysis was performed on age-matched men. The results for the age analysis also demonstrated no statistically significant differences in racial mortality or whether screening PSA reduced mortality after applying a propensity scoring analysis to a conditional Cox regression model. In conclusion, it is believed that using a propensity scoring method and Cox regression analysis improved the evaluation of this large population data set where censoring for survival time was important and where matched pairs were utilized. Further work in health services research using large population-based data sets should be pursued and incorporating Cox regression with a propensity analysis can be helpful.

The main purpose of this research was the development of an experimental model that allows for the assessment of pressure and thereby, the forces associated with interventions related to airway management. The foundation of this research was to develop, and assess the validity and reliability, of a method of quantifying the force experienced by a patient during airway management. Following IRB approval and the development of a unique simulation model that employs transducers situated in key anatomical locations to determine forces, a multivariate profile analysis with covariate of experience using a MANCOVA approach was conducted. The statistical design consisted of 102 subjects testing the dependent measure of pressure for the following techniques: Fiberoptic intubation, the Fastrach™ LMA, the # 3 C-Mac video laryngoscope, and the Trachlight®. Independent variables analyzed were practitioner types: emergency medicine physicians, certified registered nurse anesthetists, and anesthesiologists, all tested over five locations: Chicago, Las Vegas, Atlanta, Seattle, and Boston, with a co-variable of experience. Analysis demonstrated no difference in force attributed to the location, the airway provider or their interactions. This was contrasted by the finding that 81% of the variance in pressure scores was due to differences in airway techniques. The mannequin was also able to discern a subpopulation within techniques which lends to its validity. The mannequin preformed consistently regarding reproducible findings following the setup and dismantling over time and locations. This would seem to begin to form the bases of a valid and reliable tool for this and future research.

Congenital cytomegalovirus infection is the leading cause of sensorineural hearing loss in the U.S. CMV vaccines developed to date do not protect the majority of women of childbearing age from primary CMV infection. Insufficient vaccine-induced epithelial entry neutralizing activity may be the reason for poor performance of these vaccines. CMV entry into endothelial and epithelial but not fibroblast cells requires the virion envelope complex gH/gL/UL128-131. Since current vaccines do not target this complex, epithelial entry mediators UL128-131 are attractive subunit CMV vaccine candidates, since they should target mucosal immunity. The mucosal immune response, specifically salivary epithelial entry neutralizing activity, has not been previously described. This report demonstrates that salivas from CMV seropositive children under two, adolescents, and Towne vaccine recipients do not have epithelial or fibroblast neutralizing activity. Epithelial but not fibroblast neutralizing activity was identified in half of the salivas from CMV seropositive adults tested. This activity correlated with the level of serum neutralizing activity, suggesting that salivary neutralizing activity results from passively transferred serum IgG. Furthermore, this report describes three highly immune individuals with serum and saliva neutralizing titers two- to four-fold above average. These individuals also have UL130 antibodies detectable in western blot assays. This is the first report of antibodies by western blot in CMV seropositive sera to UL128, UL130, or UL131. To determine the feasibility of UL128-131 as vaccine candidates both peptide and DNA vaccines were tested in animal models. Rabbit anti-peptide sera from UL130 and UL131 vaccinated animals induced epithelial entry neutralizing activity similar to that found following natural infection. Mixing anti-peptide UL130 and UL131 sera neutralized CMV infection of epithelial cells at titers higher than natural infection. DNA vaccination with these proteins was not as successful but based on DNA vaccination of mice UL130 is the most immunogenic of the three proteins. These data support further development of UL130 as a CMV vaccine. Future vaccines, including the vaccine candidates described in this report, should strive to induce levels of immunity seen in the three highly immune individuals, specifically serum epithelial neutralizing titers >1:7,000 and saliva epithelial neutralizing titers >1:20.

Cytomegalovirus (CMV) infections remain a significant problem in congenitally infected infants and immunocompromised individuals. Modest antiviral activities of currently approved drugs coupled with dose-limiting toxicities restrict effectiveness and promote development of resistance. The potential for ribonucleotide reductase (RR) inhibitors hydroxyurea (HU), Didox, and Trimidox to synergize, through reduction of nucleotide pools, with the deoxynucleotide analog Ganciclovir (GCV) was examined. A yield reduction assay that utilizes luciferase expressed by a recombinant virus as a surrogate measure of viral infectious units was developed and used to determine effective dose ranges for each drug. RR inhibitors exhibited intrinsic anti-CMV activities on their own with IC50 values well below toxic levels. Moreover, RR inhibitors significantly synergized with GCV. These findings provide a rationale for exploration of RR inhibitors and deoxynucleotide analogs in anti-CMV combination therapy.

Staphylococcus aureus is a significant cause of morbidity and mortality world-wide. This opportunistic pathogen is capable of causing several severe diseases that are exacerbated by its diverse and widespread antibiotic resistance profile. Therefore it is necessary to identify novel therapeutic targets to effectively treat S. aureus disease. Lorenz et al first described the Immunodominant Surface Antigen B, IsaB, because it was 1 of 4 unique proteins immunogenic during septicemia and not colonization, suggesting that IsaB may be a virulence factor and a possible novel therapeutic target. Interestingly, IsaB has no homology to proteins of known function and appears to be found only in Staphylococci. We sought to characterize the function of IsaB in S. aureus. We began our studies by determining how isaB was regulated by known S. aureus regulators and environmental stimuli. It was observed that the transcriptional regulator SarA represses expression of isaB, while serum and acidic pH induce expression. We found that IsaB is an extracellular nucleic acid binding protein, able to bind to dsDNA, ssDNA, and RNA and leads significant accumulation of eDNA on the cell surface. We employed multiple virulence models to ascertain the role of IsaB in virulence. Excitingly, we found that IsaB significantly protects S. aureus from antimicrobial peptides and Neutrophil Extracellular Traps, both components of the innate immune system. Another virulence mechanism of S. aureus is the ability to form biofilms. While recent studies show a significant role for eDNA in S. aureus biofilms, we found that IsaB actually had a negative affect on biofilms under certain growth conditions. Finally, to group IsaB into a known functional class, we successfully expressed and purified mature IsaB for structural determination by Nuclear Magnetic Resonance, which is currently underway. Our studies show that IsaB is a novel virulence factor of S. aureus, able to bind eDNA and significantly protect from AMPs and NETs, and could therefore play a key role in immune evasion.

The peroxisome proliferator-activated receptors (PPARs) are the transcriptional regulators of glucose, lipids and cholesterol metabolisms. It has been established that PPAR-γ is the receptor for thiazolidinediones (TZDs) class of type II anti-diabetic drugs. These compounds act as agonists of PPAR-γ. They may delay the development of type II diabetes in individuals at high risk of developing the condition, and have been shown to have potentially beneficial effects on cardiovascular risk factors. PPAR-γ receptor activation by TZDs improves insulin sensitivity by promoting fatty acid uptake into adipose tissue, increasing production of adiponectin (responsible for glucose regulation and fatty acid metabolism) and reducing levels of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor-1(PAI-1) and interleukin-6 (IL-6). Our goal is to take advantage of the mode of binding of known PPAR-γ agonists, such as Rosiglitazone to PPAR-γ to rationally design novel agonists of PPAR-γ. Our long-term objective is to generate new and potent PPAR-γ agonists that could be used to treat diabetes. To achieve our goal the study was divided into five specific aims, including: Aim 1. Expression and purification of PPAR-γ ligand binding domain (LBD). Aim 2. Molecular modeling to design PPAR-γ receptor modulators. Aim 3. Synthesis of potential PPAR-γ receptor modulators. Aim 4. Functional studies to determine the binding affinity of PPAR-γ receptor modulators. Aim 5. Structural studies of PPAR-γ LBD in complex with PPAR-γ receptor modulators. We expressed the His-tagged PPAR-γ LBD protein in Rosetta DE3 cells, and used a one step affinity chromatography (Ni-NTA column) to obtain a significant yield of pure protein. Using the structural features and the known binding mode of Rosiglitazone to PPAR-γ LBD as a starting point, two classes of compounds (type-I and type-II compounds) were designed as potential PPAR-γ agonists. These novel compounds were rationalized to improve on the binding modes of Rosiglitazone via additional hydrogen-bonding and/or hydrophobic interactions to the protein. Five type-I and II compounds were synthesized and tested against PPAR-γ receptor for binding affinity, using fluorescent polarization assay. The IC-50 value of the most potent compound (compound B) was found to be ~ 7-fold lower than Rosiglitazone, significantly lower than the expected value. It seems that unlike Rosiglitazone which has free rotatable thiazolidinedione ring that can make optimal interactions with His323 and His449 (two critical residues that are important for binding affinity), the thiazolidinedione ring in our compounds are fixed in one position that may not lead to optimal contact with the protein. We are currently synthesizing analogs of our compounds with rotatable thiazolidinedione ring for further studies. X-ray crystallographic study has been initiated to determine the binding modes of our compounds with PPAR-γ LBD which would allow for structural modifications for improving existing interactions and/or formation of new favorable interactions that could lead to higher affinity and potency. We have also initiated testing of these compounds to determine their PPAR-γ agonistic effects.

The serotonin (5-HT) receptors, with seven subtypes and at least fifteen distinct members, mediate a wide range of physiological functions both in the central nervous system and in the periphery. All members of the 5-HT family except the 5-HT3 subtype belong to the family of aminergic G protein-coupled receptors (GPCRs). Over the years, various molecules have been reported which act selectively at 5-HT2 receptors. However, there are no ligands that exhibit complete selectivity for one subpopulation of 5-HT2 receptors. Insight into how drugs bind to 5-HT2 receptors could contribute significantly to the development of subtype-selective agents with enhanced therapeutic effects. We have begun to address this challenge by the combined approach of chemical synthesis and molecular modeling. 9-(Aminomethyl)-9,10-dihydroanthracene (AMDA) a novel, selective 5-HT2 antagonist that also has modest affinity for the histamine (H1) receptor has been reported by Westkaemper et al. A structure-affinity relationships (SAFIR) study of AMDA and its analogs was carried out by studying the effects of N-alkylation, variation of the amine-ring system linker chain length and constraint of the aromatic rings on the binding affinities of the compounds for the 5-HT2A and H1 receptors. The results of the docking studies carried out on the homology models of 5-HT2A and H1 receptors were consistent with the observed binding affinity data for both receptors. In order to explore the additional binding site interactions of 5-HT2A receptor, synthesis and testing of the ring-annulated analogs of AMDA were carried out. A 3-methoxytetraphen analog of AMDA (26) showed high affinity (Ki = 21 nM) and selectivity (126-fold) for 5-HT2A receptor as compared to H1 receptor (Ki = 2640 nM). Further, to test the utility of our homology models, and investigate the binding site specific interaction, a compound was synthesized and tested that lacks a basic amine and contains an acidic functionality designed specifically to interact with lysine K1915.39 found in H1 but not in 5-HT2A receptor. This compound would thus be both H1-selective and demonstrate that a basic amine-D3.32 interaction is not necessary for high affinity. The synthesized compound (34) lacking the nitrogen atom showed moderate affinity at the H1 receptor (Ki = 250 nM), and lacked affinity for 5-HT2A receptors. The modeled ligand orientations in combination with the observed affinity data provide another example of a successful structure-based design strategy.

Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two vitamin B6 salvage enzymes involved in metabolism of the primary inactive vitamin B6 (pyridoxal, pyridoxine and pyridoxamine) into the active cofactor form, pyridoxal 5’-phosphate (PLP). PLP, arguably the most important vitamin, is required by numerous vitamin B6 (PLP-dependent) enzymes as a co-factor. These enzymes serve vital roles in the metabolism of glucose, lipids, amino acids, heme, DNA/RNA and many neurotransmitters. High levels of vitamin B6 are linked to neurotoxicity, due to the non-specific interactions of PLP with non-B6 proteins. This problem is controlled, in part, by maintaining a low in vivo concentration of free PLP (~1 μM); raising the intriguing question of how the cell regulates, as well as, supplies sufficient PLP to meet the requirements of B6 enzymes. Similar to PLP excess, PLP deficiency, due to mutations in PL kinase and PNP oxidase or drug-induced inhibition of their activity, has been implicated in many pathological conditions. The objective of this study is to elucidate the mechanisms underlying PLP regulation by PL kinase, and its subsequent transfer to dozens of PLP-dependent enzymes. A second objective is to gain valuable information into whether a missense mutation (S261F) in PL kinase could affect the enzyme activity and/or structure. A third objective is to understand how vitamin B6 metabolism by PL kinase is disrupted by the neurotoxic compound, ginkgotoxin. The mutant (hPL kinase S261F) was obtained using site-directed mutagenesis. It was then expressed, purified and analyzed by circular dichroism, fluorescence spectroscopy, enzyme kinetics and native-PAGE. Our results showed no considerable differences between wild-type enzyme and the mutant, suggesting the mutation to be non-pathogenic. PLP was found to inhibit PL kinase by binding to the substrate PL site in the presence of substrate MgATP to form an abortive ternary complex (PL kinase-PLP-MgATP). The physiological significance of this ternary complex was also analyzed and it was found to be a source of PLP transfer to apo B6 enzymes. Enzyme kinetics, affinity chromatography and fluorescence polarization techniques were used to test our hypothesis that the reactive PLP is transferred from PL kinase to apo-B6 enzymes via channeling. Channeling should provide an efficient and protected way for PLP transfer from the kinase or oxidase to apo-B6 enzymes. Our results provide a strong support to the channeling mechanism. Ginkgotoxin was found to be a competitive inhibitor of PL kinase with a Ki of 18 μM. X-ray crystallographic analysis of its binding mode to PL kinase confirmed its binding to the substrate PL site of the enzyme. A unique hydrophobic interaction between its lipophilic side chain 4’-OCH3 and nearby Tyr127 and Val231, in addition to the conserved PL binding interactions, was found to be responsible for its higher affinity to the enzyme.