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Item Antisense Oligonucleotides from the Stage-specific Myeloid Zinc Finger Gene MZF-1 Inhibit Granulopoiesis In Vitro(Rockefeller University Press, 1991-11-01) Bavisotto, Linda; Kaushansk, Kenneth; Lin, Nancy; Hromas, Robert; Medicine, School of MedicineZinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.Item Human Pluripotent Stem Cells Serve as an Effective In Vitro Model for Studies of Early Stages of Retinogenesis(Office of the Vice Chancellor for Research, 2016-04-08) Miller, Casey A.; Sridhar, Akshayalakshmi; Ho-A-Lim, Kimberly; Cooke, Jessica A.; Meyer, Jason S.Specification of the neural retina is one of the first events in human development and thus, efforts to study the initial stages of retinal specification have been largely limited. This is particularly true for the earliest event in retinogenesis, the establishment of a definitive retinal fate from a more primitive neural progenitor source. With the advent of human pluripotent stem cells (hPSCs), the complex interplay of transcription factors involved in early events of retinal development from an unspecified pluripotent population can be studied in an in vitro model. To examine this potential, hPSCs were directed to differentiate to a neuroretinal lineage in which a subpopulation of cells adopts a definitive retinal fate whereas others develop toward a forebrain lineage. Samples were collected over the first month of differentiation, starting from the undifferentiated state through when cells acquired either retinal or nonretinal forebrain identities and gene expression patterns were characterized using immunocytochemistry and quantitative RT-PCR. Results demonstrated that while neural transcription factors such as PAX6, OTX2, and LHX2 were expressed early in development, definite retinal transcription factors such as CHX10 were expressed later in differentiation. Furthermore, the expression of CHX10 was found to be uniquely associated with retinal populations and remained absent from the other neural populations, thereby illustrating the ability of this protocol to uniquely identify and isolate retinal populations and further study them in vitro. Overall, these studies will serve to further understand the specification of a retinal fate from a pluripotent population. Such information will assist in the establishment of more efficient methods to generate retinal cells from hiPSCs for translational purposes, as well as establish hiPSCs as a unique in vitro model system for studies of the earliest stages of human development. Mentor: Jason S. Meyer1-3Item Performance of several diagnostic systems on detection of occlusal primary caries in permanent teeth(2014) Jallad, Mahmoud; Zero, Domenick T.; Chu, Tien-Min Gabriel; Cochran, Michael A. (Michael Alan), 1944-; Cook, Norman Blaine, 1954-; Zandoná, Andréa F.Detection of caries at an early stage is unequivocally essential for early preventive intervention. Longitudinal assessment of caries lesions, especially under the opaque preventive sealant, would be of utmost importance to the dental community. OBJECTIVES: The aim of this two-part in-vitro study is to evaluate the performance of multiple detection methods: The International Caries Detection and Assessment System (ICDAS); two quantitative light-induced fluorescence systems QLF; Inspektor™ Pro and QLF-D Biluminator™2 (Inspektor Research Systems B.V.; Amsterdam, The Netherlands); and photothermal radiometry and modulated luminescence (PTR/LUM) of The Canary System® (Quantum Dental Technologies; Toronto, Canada). All these are to be evaluated on their detection of caries on posterior human permanent teeth for 1) of primary occlusal lesions, and 2) under the sealant of primary occlusal lesions. METHODS: One hundred and twenty (N = 120) human posterior permanent teeth, selected in compliance with IU-IRB “Institutional Review Board” standards, with non-cavitated occlusal lesions ICDAS (scores 0 to 4) were divided into two equal groups. The second group (N = 60) received an opaque resin dental sealant (Delton® Light-Curing Pit and Fissure Sealant Opaque, Dentsply, York, PA). All lesions were assessed with each detection method twice in a random order except for ICDAS, which was not used following the placement of the sealant. Histological validation was used to compare methods in regard to sensitivity, specificity, % correct, and the area under receiver- operating characteristic curve (AUC). Intra-examiner repeatability and inter-examiner agreement were measured using intraclass correlation coefficient (ICC). RESULTS: 1) Of primary occlusal lesions, sensitivity, specificity, and AUC values were respectively: 0.82, 0.86 and 0.87 (ICDAS); 0.89, 0.60 and 0.90 (Inspektor Pro); 0.96, 0.57 and 0.94 (QLF-D Biluminator 2); and 0.85, 0.43 and 0.79 (The Canary System). Intra-examiner repeatability and inter-examiner agreement were respectively: 0.81 to 0.87: 0.72 (ICDAS); 0.49 to 0.97: 0.73 (Inspektor Pro); 0.96 to 0.99: 0.96 (QLF-D Biluminator 2); and 0.33 to 0.63: 0.48 (The Canary System). 2) Of primary occlusal lesions under the opaque dental sealants, sensitivity, specificity, and AUC values were respectively: 0.99, 0.03 and 0.67 (Inspektor Pro); 1.00, 0.00 and 0.70 (QLF-D Biluminator 2); and 0.54, 0.50 and 0.58 (The Canary System). Intra-examiner repeatability and inter-examiner agreement were respectively: 0.24 to 0.37: 0.29 (Inspektor Pro); 0.80 to 0.84: 0.74 (QLF-D Biluminator 2); and 0.22 to 0.47: 0.01 (The Canary System). CONCLUSION: Limited to these in-vitro conditions, 1) ICDAS remains the method of choice for detection of early caries lesion due to its adequately high accuracy and repeatability. QLF systems demonstrate potential in longitudinal monitoring due to an almost perfect repeatability of QLF-D Biluminator 2. The Canary System performance and repeatability were not acceptable as a valid method of early caries detection. 2) None of the methods demonstrated acceptable ability in detecting of occlusal caries under the opaque sealant. However, QLF-D Biluminator 2, with limitation to these in-vitro conditions and Delton opaque sealant, demonstrated a fair accuracy AUC (0.70) in detecting of caries under sealants at an experimental threshold of 12.5% ΔF.