Abstract The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells. The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a biphasic decrease in cell viability. The capacity of the cells to proliferate was not restored upon removal of the drug after 6 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, stilbene 5c-treated HCT-116 cells displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells carrying an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. However, our studies indicated that stilbene 5c works in a p53-independent manner. Exposure of P53-null HCT116 cells to stilbene resulted in a similar sensitivity as in p53-wild type HCT116 cells. We found that autophagic vacuoles were formed in response to stilbene 5c in p53-null HCT116 cells as well. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against colon cancer through the promotion of multiple modes of cell death.

There continues to be a need for more effective analgesics. The α2-adrenoceptor (AR) agonist clonidine is an analgesic whose use is severely limited by undesirable side effects. meta-Chlorophenylguanidine (MD-354), an agent developed in our laboratory, selectively potentiates the antinociceptive effects of clonidine in a biphasic manner. Mechanistic studies suggest that both 5-HT3 receptor and α2-AR mechanisms are involved. To further evaluate mechanisms underlying the analgesia-potentiating effect of clonidine by MD-354, pharmacological studies using more established 5-HT3 receptor agonists: meta-chlorophenylbiguanide (mCPBG) and centrally-acting SR57227A, and non-selective α2-adrenoceptor ligand TDIQ, administered alone and in combination with clonidine, were conducted in mouse antinociceptive assays. None of the examined analogs produced an antinociceptive effect when administered alone. Nevertheless, mCPBG potentiated the antinociceptive actions of clonidine in a monophasic manner and the effect was antagonized by the 5-HT3 receptor antagonist tropisetron but not by tropisetron methiodide, suggesting that potentiation is, at least in part, due to a central 5-HT3 receptor mechanism. SR57227A did not alter the antinociceptive actions of clonidine. TDIQ was found to potentiate the analgesic actions of clonidine in a synergistic manner (as determined by an isobolographic analysis) and the effect was blocked by α2-AR antagonists (BRL-44408, imiloxan, ARC-239; α2A-, α2B-, and α2C-AR antagonists, respectively). This supports the hypothesis that MD-354 could be potentiating the analgesic actions of clonidine via an α2-AR agonist mechanism. In order to explore the role of the ring nitrogen atoms and the chloro substituent of conformationally-constrained rotamers of MD-354, analogs of 2-amino-7-chloro-3,4-dihydroquinazoline, with a varying number of nitrogen atoms in the ring were synthesized. Preliminary binding affinity results indicated that the ring nitrogen atoms are essential for 5-HT3 receptor binding. In attempt to explain the varied binding and functional activity of MD-354 at α2-ARs, 3D homology models of α2A-, α2B- and α2C-AR were generated and docking studies of the low-energy rotamers of MD-354 were conducted. The present studies support a role for the involvement of 5-HT3 receptors and α2-ARs in antinociception. Analgesic adjuvants with a dual mechanism of action such as MD-354 might represent a promising avenue to pain treatment.

For many years it has been known that the injection of antigen bound to an antibody leads to more than a 1000-fold increase in antigen specific antibody response. This observation holds true for IgE, which is dependent upon CD23 expression, as this enhancement is not present in mice deficient in CD23. It also has been shown that when mice are injected with IgE-antigen complexes also display an increase in antigen specific T cell proliferation. While there are published studies that demonstrate a role for B cell derived exosomes in the activation and proliferation of T cells, none have focused upon the potential role of CD23 as a molecular basis for this phenomenon, at least in the context of allergy and asthma. This thesis provides direct evidence that B cell-derived exosomes possess co-stimulatory molecules, including CD80 and CD86, which act in concert with CD23 to induce T cell proliferation, at least in vitro. This is due to, or enhanced by, the exosomal transfer of the antigen or peptide to T cells. Importantly, the antigen transfer is dependent upon the availability of IgE and the expression of CD23.

The objective of this study was to evaluate parents’ preferences of the appearance and attire of orthodontists. Six subjects were asked to pose for photographs wearing various combinations of attire (casual, scrubs, white coat, formal), hairstyle and nametag. Survey participants were presented with choice sets and asked to select the most and least preferred provider photographs. A total of 77 parents of orthodontic patients participated in the computer-based survey. The results indicated that there were significant differences due to provider sex (P = 0.0013), provider age (P < .0001), dress (P < .0001), nametag (P = 0.0065) and hair (P < .0001). The most preferred providers were the younger female and the older male. Formal attire or scrubs was the most preferred style of dress. There was also a preference for the use of a nametag and for the provider to have his/her hair in a controlled style.

As esthetic improvement continues to be a primary goal of orthodontic patients and practitioners, it is important to understand the soft tissue movements that occur during the transition from rest to smile. Advances in technology allow capture of 3-dimesional photographs. The purpose of this study was to quantify the soft tissue changes that occur upon smiling and to compare changes between males and females using 3-dimensional photography. Fifty-four participants had resting and smiling photographs taken with the 3dMDface camera system. The two images were registered on stable facial surfaces, landmarks were placed, and measurements were recorded. Three-dimensional changes occurred in the following regions of the face in both males and females: eyes, ears, nose and lips. Intercommissure width of the lips was shown to increase more in females than males, but males showed increased mobility in landmarks at the base of the nose.

The high co-morbidity between alcohol (ethanol) and nicotine abuse suggests that nicotinic acetylcholine receptors (nAChRs), which are thought to underlie nicotine dependence, may also be involved in alcohol dependence. A genomic region that encodes the Alpha5* nAChR subtype has recently been shown to be associated with alcohol dependence phenotypes in humans. Therefore, the aim of this study was to determine the role of Alpha5* nAChRs in ethanol-responsive behaviors upon acute administration in mice as well as in their drinking behavior. We conducted tests in mice lacking the Alpha5 coding gene (Chrna5) in ethanol-induced hypothermia, hypnosis, anxiolysis, and conditioned place preference. We also assessed drinking behavior in these mice using models of voluntary ethanol consumption, two-bottle choice preference and intermittent access, as well as acute binge drinking behavior in the Drinking-in-the-Dark paradigm. Our results showed that deletion of the Alpha5 gene enhanced acute behaviors, including ethanol-induced hypothermia, hypnosis recovery time, and the anxiolytic-like response in mice. We also found that Alpha5 gene deletion resulted in decreased ethanol CPP, but had no effect on ethanol consumption in either model of drinking behavior tested under normal conditions. However, we discovered that under conditions of stress from multiple daily injections of saline or nicotine, Drinking-in-the-Dark intake was reduced in Alpha5 null mutant mice. We also examined the role of Beta2* nAChRs due to the tendency of the Beta2 subunit to be co-expressed with this subtype, which also plays an important role in nicotine dependence. Our results showed that pharmacological and genetic manipulation of Beta2* nAChRs modulated some acute alcohol-responsive behaviors, namely, hypnosis, recovery-time and the anxiolytic-like response produced by ethanol, but did not modulate ethanol drinking behavior in mice. These studies provide evidence that Alpha5* subtypes and Beta2* subtypes, which play a critical role in nicotine dependence, also play a role in acute ethanol-responsive behaviors in vivo, thus supporting studies in humans that nicotine and alcohol dependence share common genetic components.

Axonal pathology is a major contributor to impaired motor, sensory and cognitive dysfunction associated with multiple sclerosis particularly with the progressive forms of the disease. However, the early pathologic events responsible for axonal deterioration remain unclear. It is well recognized that maintaining proper axonal function is intimately related to proper establishment and maintenance of axonal domains such as the node of Ranvier and the axon initial segment (AIS). Numerous laboratories, including ours, have investigated the mechanisms that regulate node of Ranvier formation and maintenance. These studies have shown that node of Ranvier formation and maintenance require myelin contact. Interestingly, many of the same proteins that cluster at the node of Ranvier also cluster in the AIS; however, the mechanisms responsible for AIS clustering appear to be unique to the AIS as myelin contact is not required and the mechanisms appear to be intrinsic to the neuron. Determining how the AIS is developmentally generated is vital to a complete understanding of the AIS function. However, more in line with understanding the pathobiology of MS, our laboratory is interested in identifying the mechanisms responsible for the maintenance and restoration of AIS integrity and function. To achieve this goal, we have exploited the cuprizone toxicity model. This model results in a consistent course of demyelination followed by remyelination of layer V of the cerebral cortex. Using a combination of immunocytochemistry and confocal microscopy, we have analyzed AIS integrity as evidenced by the clustering of ankyrinG, a prominent initial segment protein. Our findings indicate that the number of AIS is not decreased following myelin loss. In addition, AIS length and surface area are not changed following demyelination. These findings are important as they suggest that myelin is not required for the maintenance of initial segment organization.

Objective: The aim of this study was to (1) assess the orthodontic patient and practitioner use and preferences of social media, and (2) to investigate the potential benefit of social media in marketing and communication strategies in orthodontic practices. Material and Methods: A survey was distributed to all participants, which included orthodontists from the American Association of Orthodontists (AAO) and patients/parents from the VCU Orthodontic Clinic and Private Practices throughout the United States. The participants were asked to answer questions related to their use of social media as well as their perceptions of usage of social media in the orthodontic practice. Results: 76% of orthodontists, 71% of the VCU Practice participants and 89% of the Private Practice participants used social media, with the highest preference for Facebook among all of the participants. Orthodontist’s posed information more often in the morning and afternoon (40% and 56%, respectively) and patients used social media mainly in the evening (76%). The most commonly used marketing strategies in the orthodontic practice were social media and a practice website (76% and 59%, respectively). Social media and practice websites were positively related to new patient starts (P=0.0376, P=0.0035, respectively). Newspapers were negatively related to new patient starts (P=0.0003). Conclusions: Social media use was more common in females and younger adults and facebook was the most commonly used social media site among all of the participants. Orthodontists posted information on social media websites mainly in the morning and afternoon, while patients spend more time on social media sites in the evening. Newspaper advertisements were negatively related to new patient starts. Facebook and Twitter were positively related to new patient starts when used as a marketing and communication tool in the orthodontic practice.

The Reporting of Supplement Use by Dental Patients on Their Medical History Questionnaire Objectives: The goals of the study were three fold. Estimate the prevalence of supplement use by dental patients. Determine if the design of a medical history form influences the prevalence of supplement use reported. Determine whether or not patients are aware of supplement side effects and interactions with medications. Methods: Patients were randomly allocated to either a standard medical history form or the same form with additional questions about supplement use. After completing the initial forms, a survey containing questions about supplements was filled out by both groups. For investigating differences between groups, logistic regression and analysis of variance were used depending on the type of outcome variable. Results: Two hundred and nine patients participated in the study. The mean number of supplements reported by patients was influenced by the type of health history questionnaire given to the patient. Specifically asking about supplements versus not asking at all resulted in nearly double the number of supplements reported by the patient (mean of 1.53 when asked, 0.76 when not asked, p< 0.0001). Patient age and income were related to number of supplements used per patient. The two oldest age categories (50–65 and >65) reported a mean number of supplements used of 2.82 and 2.72, respectively versus the youngest age group (<30 years old) which reported a mean of 1.05 (p<0.05). The highest income level (>$75,000 per year) reported the lowest number of supplements per patient of 0.56 versus the other income levels (p<0.02), which reported mean supplemental use ranging from 2.28 to 2.71. Additionally, the majority of the subjects (69 %) were not aware of the side effects and interactions of supplements with medications. Conclusion: Patients tend not to report supplement use on the medical history questionnaire unless they are directly asked and the majority of patients are not aware of interactions with medications. Patient income and age have an effect on the frequency of supplement use.

The current understanding of the distribution and frequency of periapical pathoses include biopsies submitted by all specialists and general dentists. As a result, they do not accurately reflect the distribution seen by endodontists. This retrospective chart review aims to determine the prevalence of periapical pathoses and associated demographics from biopsies submitted by endodontists over 30 years. All biopsy reports submitted to the Virginia Commonwealth University Oral Pathology Diagnostic Service from January 1, 1983 to December 31, 2012 were reviewed. Only reports submitted by verified endodontists and those with a periapical location were included. The following data was recorded from each report: submission date, referring endodontist, sex, age, race, biopsy location, tooth number, and histologic diagnosis. Results were calculated using chi-square and logistic regression analysis (significance p<0.05). Meeting the inclusion criteria were 9,777 biopsy reports for an overall distribution of 24.11% radicular cysts, 73.54% periapical granulomas, 1.66% scars, and 0.70% other pathoses. Findings include a significant association between sex, location, and diagnosis. An association with race, age, or location (left/right) was not seen. Significantly more radicular cysts were seen in males and in the anterior maxilla. Conversely, significantly more periapical granulomas were seen in females and in the posterior quadrants. Significantly more other diagnoses were found in the anterior mandible and more scars in the anterior maxilla. Overall, approximately ¾ of biopsies submitted for evaluation by endodontists are diagnosed as periapical granulomas and ¼ as radicular cysts. Other pathoses and scars make up less than 3% of diagnoses. Funding was provided through the AAE Resident Research Grant.