Assessing the pipeline: Perceived barriers to applying to dental school among pipeline program alumni.

PURPOSE/OBJECTIVES: The aim of this study was to increase the understanding of perceived barriers to applying to dental school experienced by underrepresented minority (URM), pipeline program alumni. METHODS: A qualitative study of alumni of New York University College of Dentistry pipeline programs, aimed at increasing the number of URM and low-income students in the dental profession, was conducted in 2020. Focus groups were convened to examine perceived barriers to applying to dental school and identified through a combination inductive and deductive thematic analysis. RESULTS: Twenty-three pipeline program alumni, ranging in age from 18 to 30 years old, participated in focus groups held between January and April 2020. All students identified as underrepresented minorities and 78% were first generation college students. Fifteen (65%) of the participants had not yet applied to dental school. Eight participants (35%) had applied to dental school, 3 (13%) were currently enrolled in dental school, and 5 (22%) were matriculating into dental school in Fall 2020. The following themes emerged as the most prominent challenges to applying to dental school: pre-health advisors (e.g., lack of knowledge about the pre-dental process and discouragement), and the cost of the application process (e.g., application fees, DAT and DAT preparation course costs, and interview costs). CONCLUSION: Through pre-dental pipeline programs, participants have access to informational resources and mentorship; however, despite participation in these programs, perceived barriers are still prevalent. Identification of the alumni's perceived barriers offer targeted areas where increased intervention may be helpful to reduce challenges and strengthen the pipeline.

Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells.

Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by beta-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect. Disclosure of potential conflicts of interest is found at the end of this article.

A mechanism for the inhibition of neural progenitor cell proliferation by cocaine.

BACKGROUND: Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation. METHODS AND FINDINGS: Microarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2alpha and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A. CONCLUSIONS: Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development.

Dopaminergic neurons derived from BG01V2, a variant of human embryonic stem cell line BG01.

BACKGROUND AND PURPOSE: Human embryonic stem cells (hESC) are considered a renewable source of dopamine producing neurons, and are of particular interest for their potential clinical use in Parkinson's disease. In this study, we characterized human dopaminergic neurons generated by stromal-derived inducing activity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01, characterized by a chromosome 17 trisomy. Similar chromosomal changes have been repeatedly observed in hESC cultures in different laboratories, indicating the importance of chromosome 17 for growth and adaptation of hESC to culture. METHODS: We investigated in vitro proliferation of differentiating cells using a BrDU incorporation assay, and monitored the cell population in long term cultures. Despite the cytogenetic abnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 days of differentiation, cell division ceased in 91% of the overall population of cells in the culture, indicating intact cell cycle regulation. RESULTS: Expression of midbrain specific marker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neural progenitor cells into midbrain specific dopamine neurons. These neurons expressed the dopamine transporter (DAT), and displayed functional DAT activity and electrical excitability. CONCLUSIONS: TH+ cells derived from the BG01V2 hESC line using SDIA are postmitotic and have functional characteristics of normal dopaminergic neurons.

Gene expression profiling reveals distinct cocaine-responsive genes in human fetal CNS cell types.

OBJECTIVES: Prenatal exposure to cocaine causes cytoarchitectural alterations in the developing neocortex. Previously, we reported that cocaine inhibits neural progenitor cell proliferation through oxidative endoplasmic reticulum stress and consequent down-regulation of cyclin A, whereas cyclin A expression was increased in astrocytes. In the present study, cell type-specific responses to cocaine were further explored. METHODS: Gene expression profiles were examined in five types of cells obtained from the human fetal cerebral cortex at 20 weeks gestation. Cells were treated with 100 µM cocaine in vitro for 24 hr, followed by gene expression analysis using a human neural/stem cell/drug abuse-focused cDNA array, with verification by quantitative real-time RT-PCR. RESULTS: Cocaine influenced transcription of distinct categories of genes in a cell type-specific manner. Cocaine down-regulated cytoskeleton-related genes including ezrin, γ2 actin, α3d tubulin and α8 tubulin in neural and/or A2B5+ progenitor cells. In contrast, cocaine modulated immune and cell death-related genes in microglia and astrocytes. In microglia, cocaine up-regulated the immunoregulatory and pro-apoptotic genes IL-1ß and BAX. In astrocytes, cocaine down-regulated the immune response gene glucocorticoid receptor and up-regulated the anti-apoptotic genes 14-3-3 ε and HVEM. Therefore, cell types comprising the developing neocortex show differential responses to cocaine. CONCLUSIONS: These data suggest that cocaine causes cytoskeletal abnormalities leading to disturbances in neural differentiation and migration in progenitor cells, while altering immune and apoptotic responses in glia. Understanding the mechanisms of cocaine's effects on human CNS cells may help in the development of therapeutic strategies to prevent or ameliorate cocaine-induced impairments in fetal brain development.

Microsurgical enophthalmos correction after silent sinus syndrome.

A 66-year-old man with silent sinus syndrome, resulting in progressive enophthalmos and subclinical chronic maxillary sinusitis, presented after several failed attempts at reconstruction with conventional methods. A free fibula osteoseptocutaneous flap was used to recreate the orbital floor, obliterate the maxillary sinus, and augment the periorbital contour deformity in a single stage. This is a novel approach for the treatment of Silent Sinus Syndrome in a single stage.

GABAergic lineage differentiation of AF5 neural progenitor cells in vitro.

We have previously described an immortal rat central-nervous-system progenitor cell line, AF5, which is able to exit the cell cycle and assume a differentiated state with neuronal properties. The phenotypic specification of differentiated AF5 cells, however, is not known. In the present study, when induced to differentiate by serum starvation in Neurobasal medium, AF5 cells down-regulate glial fibrillary acidic protein and up-regulate expression of beta-III-tubulin, medium-molecular-weight neurofilament protein, and neuronal growth-associated protein 43. Expression of the gamma-aminobutyric acid (GABA) lineage marker, glutamic acid decarboxylase 67 (GAD67), increases during differentiation, suggesting that AF5 cells adopt a GABAergic lineage. Time-course analysis of the GABAergic neuron specification transcription factor, Pitx2, by reverse transcription/polymerase chain reaction, has shown an increase in the Pitx2 transcript 48 h after initiation of differentiation. In differentiated AF5 cells, expression of the Pitx2 target gene products GAD65 and GABA transporter-1 increases. Cellular GABA levels in differentiated AF5 cells increase by about 26-fold, and GABA release into the medium is 150-fold higher compared with that of undifferentiated cells. Therefore, AF5 cells can be induced to differentiate to a neuronal phenotype with a GABAergic lineage.