Objective: The purpose of this study was to determine the fluoride recharging capability of Opal Seal, a fluoride releasing orthodontic primer, as compared to Transbond XT, the control. Material and Methods: 1mm x 5mm disks of Opal Seal and Transbond were prepared according to the respective manufacturer’s instructions. Initially, the samples were stored in deionized water (DI) for 8 weeks. The samples were then randomly divided into one of two groups: Over-the-counter (OTC) fluoride mouthwash and prescription strength (PS) fluoride mouthwash. The OTC group samples were immersed in 5mL of 0.0219% sodium fluoride containing mouthwash for one minute every day for seven days. The PS group samples were immersed in 5mL of 0.2% sodium fluoride containing mouthwash for one minute. All of the samples were suspended in 5mL fresh DI water and fluoride release measurements were taken at baseline (the end of initial 8 weeks of storage), 24 hours, 3 days, 5 days, 7 days, and 14 days. Results: Opal Seal samples treated with the OTC fluoride mouthwash exhibited significant fluctuation in fluoride ion release across time (p=0.0058). However, there were no statistically significant differences in fluoride ion release between the individual timepoints and baseline. Similarly, Opal Seal samples treated with the PS fluoride mouthwash exhibited significant variation in the fluoride ion concentration across time (p< 0.001), and a statistically significant increase over baseline was seen at 24 hours only (p= 0.0006). The control group samples treated either with the OTC or PS mouthwash did not exhibit any significant difference in fluoride ion release between any individual timepoint and baseline. Conclusion: For Opal Seal and Transbond XT, there were no statistically significant differences of fluoride concentration at any timepoint compared to baseline measurements when using OTC mouthwash. When using PS mouthwash, there was a small, statistically significant increase of fluoride concentration of the Opal Seal samples after 24 hours but no differences were seen at any other timepoints. Opal Seal did not demonstrate a substantial amount of fluoride recharge when fluoride mouthwash is used as a fluoride delivery vehicle. Future well-designed randomized controlled trials are needed to evaluate the efficacy of Opal Seal primer when coupled with the use of fluoride mouthwashes.

HIV-1 enters the CNS soon after initial systemic infection. HIV-1 can induce a wide range of neurological deficits, collectively known as HIV-1-associated neurocognitive disorders (HAND). Mature neurons are not infected by HIV-1; instead, infected and/or activated glial cells release various viral and cellular factors that induce direct and/or indirect neuronal toxicity, leading to HAND. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162 (R5-tropic)-infected differentiated-U937 cells (HIV+sup) was collected and p24 level was measured by ELISA to assess the infection. We assessed HIV+sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through µ-opioid receptors. Effects of HIV+sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV+sup caused dose-dependent toxicity over a range of p24 levels (10-500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml). In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented. Importantly, the arrest of neurite growth that occurred with exposure to HIV+sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. During early stage of HIV-infection R5-tropic viruses are predominant, but during later stages of disease X4-tropic viruses are more predominant; co-receptor usage switch from CCR5 to CXCR4 is crucial in disease progression to AIDS. Some previous studies have shown that drugs of abuse interact with virus or viral proteins in strain/tropism-dependent manner. Therefore, we also assessed neurotoxic effects and interactions with opiates by supernatant from HIV-1LAI (X4-tropic)-infected H9 cells. Neurotoxic effects and the interactions with opiates of HIV-1LAI-supernatant are quantitatively similar to that of HIV-1SF162. Surprisingly, the cytokine/chemokine release profile of HIV-1LAI-infected H9 cells is similar to that of HIV-1SF162-infected U937 cells. Only in the presence of glia, HIV-1LAI virion induced neurotoxic effects, but no interactions with morphine were seen. Also our studies have shown that HIV-1LAI virions are slightly more neurotoxic than HIV-1SF162. Altogether, largely our results suggest that HIV+sup mediated neurotoxicity and the interactions with opiates are majorly attributed to cytotoxic factors released from infected and activated cells instead of viral strain specific factors. Although there is a correlation between opiate drug abuse and progression of HAND, the mechanisms that underlie interactions between HIV-1 and opiates remain obscure. Previous studies have shown that HIV-1 induces neurotoxic effects through abnormal activation of GSK3β. Interestingly, expression of GSK3β has shown to be elevated in the brains of young opiate abusers suggesting that GSK3β is also linked to neuropathology seen with opiate abusing patients. Thus, we hypothesized that GSK3β activation is a point of convergence for HIV- and opiate-mediated interactive neurotoxic effects. Cultures of striatal neurons were treated with HIV+sup (R5-tropic), in the presence or absence of morphine and GSK3β inhibitors. Our results show that multiple GSK3β inhibitors significantly reduce HIV-1-mediated neurotoxic outcomes, and also negate interactions with morphine that result in cell death. This suggests that GSK3β-activation is an important point of convergence and a potential therapeutic target for HIV- and opiate-mediated neurocognitive deficits.

Objective. The aim of this study was to determine the effects of interleukin-10 on mast cell development from bone marrow progenitors.Materials and Methods. Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-10, and the use of Signal Transducer and Activator of Transcription (STAT)3-deficient bone marrow cells were employed to measure the effects of IL-10 and STAT3 expression on cell viability, proliferation, and differentiation. Bax-deficient and Bcl-2 transgenic bone marrow cells were used to determine the importance of the mitochondria in IL-10-mediated effects.Overview. Mast cells arise from hematopoietic stem cells and continue development in either connective tissue or mucosa. Th2 cytokines have been implicated in the regulation of mast cell development and subsequent function. Mast cells have also been shown to be essential players in many Th2 immune responses. In the following study we investigate the effects of the Th2 cytokine IL-10 on mast cell development from isolated bone marrow progenitors. The addition of IL-10 to whole murine bone marrow greatly reduced cell numbers and altered the phenotype of the developing progenitor cells. The reduction in cell numbers was due to apoptosis, as judged by DNA fragmentation and caspase activation. The apoptosis observed included alteration in mitochondrial membrane potential. Furthermore, apoptosis could be reduced by the overexpression of Bcl-2 or by ablating p53 expression. Utilizing a flox/cre system we found that IL-10 mediated apoptosis required expression of Stat-3, since Stat-3 deficient bone marrow cells did not undergo apoptosis in response to IL-10. In this study we also observed significant alterations in the mast cell growth factor receptors IL-3R and c-kit. The loss of these growth factor receptors may explain the apoptosis induced by IL-10. These data demonstrate the potent regulatory capabilities of Th2 cytokines on mast cells, a central effector in the Th2 response.

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.

Herpes simplex virus entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in herpes simplex virus entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on herpes simplex virus capsid transport. Herpes simplex virus can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, herpes simplex virus successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. Herpes simplex virus immediate-early protein ICP0 is a multifunctional regulator of herpes simplex virus infection. Late in infection ICP0 interacts dynamically with cellular proteasomes. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for its IE functions. The fundamental and functional properties of ICP0 that is present in the virion tegument layer have not been well characterized. For these reasons, I sought to characterize tegument ICP0 and determine the role of tegument ICP0 during proteasome-dependent entry of herpes simplex virus. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. Virions with mutations in the RING finger domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins. ICP0 mutations that resulted in the absence of ICP0 in the tegument layer, allow herpes simplex virus to enter cells independently of the proteasome activity. I propose that proteasomal degradation of virion and/or host proteins is regulated by ICP0 to allow for efficient delivery of incoming herpes simplex virus capsids to the nucleus.

The opportunistic pathogen P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. During chronic infection of the cystic fibrosis lung, P. aeruginosa undergoes conversion to a mucoid phenotype, constitutively producing the exopolysaccharide alginate, composed of the uronic acids D-mannuronate and L-guluronate. This alginate production contributes significantly to virulence in the cystic fibrosis lung. Evidence suggests that the acetylation state of the mannuronate component of the alginate influences the ability of components of the immune system to phagocytose the organism. To garner new and relevant information regarding the mechanism of alginate acetylation in Pseudomonas aeruginosa, a variety of approaches were undertaken. Analysis of the alginate produced by algX, algG, and algK alginate biosynthesis mutants revealed that the small oligouronides they produced were unacetylated. This strongly supports the hypothesis that the mannuronates are acetylated in periplasm, and that a polymer of at least some specific size is required. While three alginate biosynthesis gene products (AlgI, AlgJ, and AlgF) have been shown to be involved in alginate acetylation, another gene in the cluster, algX, shares 30% identity with one of them and thus generates speculation as to its potential involvement in the process. To test this possibility, an algX mutant was complemented with a plasmid carrying a mutation at a conserved residue shown to be required for alginate acetylation in the homologous protein. Analysis of alginate from this construct suggested that AlgX is not involved in alginate acetylation. To determine if changes in levels of alginate acetylation are accomplished at the transcriptional level, transcript levels of several alginate biosynthesis genes in different media were determined by real-time PCR. As qRT-PCR had not been previously performed on any of the alginate biosynthesis genes, this yielded important information about the transcription of the operon. In addition, beta-galactosidase assays on upstream regions of several biosynthesis genes identified two previously unrecognized promoters, one upstream of algG and one upstream of algI. The remaining approach was to examine protein interactions of AlgF, the protein product of one of the three acetylation genes. 2-D redox SDS-PAGE gels indicated that disulfide bonding may be important for interactions with this protein. While mass spectrometry was unable to identify the binding partners of AlgF, efforts are ongoing to create a mutation in the P. aeruginosa genome that changes the cysteine residue in AlgF to a serine residue. This would be a definitive method for determining the importance of disulfide bonding in AlgF.

Background: The evaluation process for listing a patient on the liver transplant list is complicated and involves multiple consultations from various specialists, as well as extensive imaging and physiological studies. Although there are data on the outcomes of those listed, we know little about those that are denied listing. This research project will identify the reasons for liver transplant listing denial and predictors of death following denial for this challenging group of patients. Methods: Data from all patients (n=1,500) evaluated for a liver transplant from 1997 to 2007 by the Department of Gastroenterology, Hepatology, and Nutrition located at Virginia Commonwealth University Health System’s (VCUHS) Hume-Lee Transplant Program were reviewed to identify patients denied listing (n=350). Simple descriptive characteristics were generated and the reasons for denial were assessed. The Social Security Death Index was used to determine and/or confirm mortality and multiple logistic regression was conducted to determine the predictors of death following denial of transplant listing. Results: The majority of the denied patients were white males and the mean age was 50.9, SE= 0.542). The primary liver disease diagnosis for those denied listing was Hepatitis C Virus (HCV) (33.6%). Study participants whose primary diagnosis was ethyl alcohol abuse or hepatocellular carcinoma had greater odds of dying after not being listed when compared to those diagnosed with HCV; however, these findings were not statistically significant. The majority of participants were denied listing for Hepatic-related (38.8%), psychosocial-related (21.7%), and cardiac-related (15.7%) reasons. Men were two times more likely to die after denial than women (OR= 2.18, CI= 1.03, 4.62). Patients with a MELD score less than 30 were less likely to die after being denied listing compared to those with MELD scores 31 to 40. The risk of dying after denial was not statistically different for patients who were denied listing for hepatic-related and cardiac-related reasons compared to subjects who were denied for cancer. Conclusions: Our findings have clear implications for the future of transplant medicine and raise additional questions. The analysis shows men, those 51 years of age and older and patients with MELD scores between 31 and 40 are more likely to die after not being listed for transplant. We did not find significant evidence that those with particular primary liver disease diagnoses were more likely to die following denial for listing. Other studies taking into account the population of patients that are listed as well as those denied listing are necessary in order to understand the patho-physiological mechanisms so that patient-specific therapies may be developed if appropriate.

Microtubule poisons have proven to be effective in the treatment of a variety of malignancies. Although taxol-based derivatives promote microtubule stabilization, there is continuing interest in compounds that, like colchicines, act as microtubule destabilizing agents. Previous work from this laboratory showed that the novel microtubule poison, JG- 03-14, was active against breast tumor cells, promoting autophagic cell death. In the current work, we studied the influence of JG-03-14 on p53 wild type HCT116 colon carcinoma cells. A crystal violet sensitivity assay indicated that JG-03-14 induced growth inhibition, with 75% suppression of growth evident at a concentration of 500 nM. Time course studies of drug effects on cell viability indicated that JG-03-14 also produced cell killing. FACS analysis demonstrated that the HCT-116 cells arrested in the G2/M stage; furthermore, there was evidence of a hyperdiploid population that would be consistent with failure of the cells to divide despite completion of DNA synthesis. Finally, there was evidence of a small sub G0/G1cell population, indicating that the cells were not dying primarily by apoptosis, and suggesting that JG-03-14 induces an alternative mode of cell death. In contrast to cell shrinkage and nuclear fragmentation that was evident after treatment with taxol ( a positive control for apoptosis), DAPI staining of HCT-116 cells treated with JG-03-14 showed intact and enlarged nuclei, again consistent with the absence of apoptosis. Furthermore, there was no evidence of mitotic catastrophe (micronuclei in binucleated cells). Based on previous studies in MCF-7 and MDA-MB231 cell lines that demonstrated a substantial population of autophagic cells, HCT-116 cells were subjected to staining with acridine orange and monodansylcadaverine after treatment with JG-03-14. While control cells tended to show a single large autophagic vesicle closely associated with the cell nucleus, treatment with JG-03-14 resulted in extensive distribution of small acidic vesicles within the cytoplasm, indicative of autophagy. GFP-LC3 transfected cells incubated with JG-03-14 showed punctuated patterns that were also consistent with the promotion of autophagy. Finally, activation of the DNA damage response pathway was ruled out by the lack of induction of p53 and p21 in cells treated with JG-03-14. In summary, our studies indicate that the JG-03-14 induces both growth arrest and autophagic cell death in HCT116 colon carcinoma cells. The possibility of an alternative mode of cell death induced by JG-03-14 makes it a potentially usefull candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. Our result also suggested that JG-03-14 failed to induce bone marrow toxicity adding to its potential for clinical use.

Ribosomes form the core of the protein biosynthesis machinery and are essential to life. Ribosome biogenesis is a complex cellular process involving transcription of rRNA, pre-rRNA processing, rRNA modification and simultaneous assembly of ribosomal proteins. RNA nucleotide modification is observed in all domains of life. While there is enormous conservation of ribosome structure, very few post-transcriptional rRNA modifications have been conserved throughout evolution. A notable example of such rare conservation is the dimethylation of two adjacent adenosines in the 3’-terminal helix, a highly conserved region of the small subunit rRNA. Enzymes that carry out these dimethylations are equally conserved and are collectively known as the KsgA/Dim1 family of methyltransferases. The first member of the family, KsgA, was identified in E. coli as the determinant for resistance to the aminoglycoside antibiotic Kasugamycin. Orthologs have since been described in organisms of wide spread evolutionary origins as well as in eukaryotic cellular organelles, thus underscoring the unprecedented conservation of this family of enzymes and the resultant rRNA modification. The higher evolutionary orthologs of KsgA have adopted secondary roles in ribosome biogenesis in addition to their dimethyltransferase role. The eukaryotic ortholog, Dim1, is essential for proper processing of the primary rRNA transcript. Recently, KsgA has been speculated to function as a late stage ribosome biogenesis factor and a ΔksgA genotype in E. coli has been linked to cold sensitivity and altered ribosomal profiles. This report focuses on the biochemical characterization of KsgA and its interaction with the 30S subunit. We have established the salt conditions required for optimal KsgA methyltransferase activity while confirming that KsgA recognizes a translationally inactive conformation of 30S subunit in vitro. Our study of the functional conservation of KsgA/Dim1 enzymes in the bacterial system revealed that KsgA and the evolutionarily higher orthologs could recognize a common ribosomal substrate. This indicates that the recognition elements of both, the protein and the small subunit, have remained largely unchanged during the course of evolution. Finally, based on our site directed mutagenesis and biochemical studies, we report that KsgA binds to structural components of 16S rRNA other than the helix containing the target nucleosides.

Drug abuse and addiction are considered to be a result, at least in part, of the rewarding effects produced by increasing dopamine levels. 5-HT3 serotonin receptors have been shown to indirectly affect dopamine levels. Therefore, the effect of the 5-HT3 receptor partial agonist, MD-354, on the actions of psychomotor stimulants was analyzed in mouse locomotor activity assays to determine whether MD-354 is working through a 5-HT3 receptor agonist or antagonist mode of action. Studies with (+)amphetamine and (+)methamphetamine in combination with MD-354 indicated MD-354 is either devoid of action or is behaving similar to the 5-HT3 receptor antagonist, ondansetron. This effect could be occurring centrally; however peripheral effects can not be discounted. In combination with cocaine, MD-354 behaved similar to the 5-HT3 receptor agonist, SR 57227A, known to act both centrally and peripherally. This difference between central and peripheral effects could account for the different modes of action observed with MD-354. Studies also involved synthesis of potentially brain-penetrant carbamate analogs of MD-354, and QSAR to assist in validating a 5-HT3 receptor agonist pharmacophore.