RESUMEN

BACKGROUND/PURPOSE: There is limited literature on the materials of choice and their properties when repairing 3-D printed resin-based restorations. The objective of this in-vitro study is to determine the shear bond strength of various repair materials to 3D printed SLA (stereolithography) resin. MATERIALS AND METHODS: For Group A (control), fifteen cylinders of 3-D printing SLA resin were printed as one unit of a Ø6.8 × 8 mm (diameter and height) cylindrical block with a Ø3 × 5 mm cylindrical block at the center. For the test groups, forty-five specimen cylinders of 3-D printing SLA resin (Ø6.8 × 8 mm) were fabricated and the surfaces were treated with 3 different test materials: Group B: Poly-Methyl Methacrylate (PMMA); Group C: Bis-acrylic composite resin, and Group D: Bis-GMA composite All specimens were tested using an Instron machine at a crosshead speed of 0.5 mm/min. A Shapiro-Wilk test was used to assess normality within the data, then the data was statistically analyzed by a Mann-Whitney test. RESULTS: There were no statistically significant differences between testing groups, except Group A. Group B displayed mixed (87%) and adhesive (13%) failure at the fractured surface. Group C showed both mixed (60%) and adhesive failure at the fractured surface (40%). All Group D showed mixed fracture patterns, partly cohesive fractured surface within the base cylinder area and partly adhesive fractured surface at the bonded interface. CONCLUSION: No statistically significant differences in the shear bond strength of the different repair materials to 3D printed cylinders were observed. The 3D printed cylinder repaired with Bis-GMA composite demonstrated the most predictability from the fractography analysis.

RESUMEN

STATEMENT OF PROBLEM: Clinical studies evaluating the tissue surface adaptation of complete denture bases fabricated by digital light processing (DLP) are lacking. PURPOSE: The purpose of this clinical study was to assess the tissue surface adaptation of complete denture bases generated by the DLP technique and to compare the adaptation with that of denture bases manufactured by 5-axis milling (MIL) and pack-and-press (PAP) method. MATERIAL AND METHODS: A total of 9 participants with 12 edentulous arches (7 maxillary and 5 mandibular) were included in this study. For each edentulous arch, the complete denture bases with occlusion rims were prepared by 3 different techniques (PAP, MIL, and DLP). A virtual denture base with occlusion rim was designed by means of a digital subtraction tool and served to fabricate the DLP and MIL denture bases. The complete denture bases were placed intraorally with an indicator applied to the intaglio surfaces. The thickness of the indicator was measured within the denture-bearing areas and anatomic landmarks of the edentulous arch to obtain the absolute tissue surface adaptation (ATA) value. The relative tissue surface adaptation (RTA) value was calculated from the differences between the ATA values of DLP or MIL techniques and those of the PAP technique. The Kruskal-Wallis test and the McNemar test were used for statistical analysis (α=.05). RESULTS: No statistically significant differences were found among the 3 denture base fabrication techniques with respect to the ATA values of either arch (P>.05). In terms of the RTA values for the maxillary arch, the DLP base was significantly different from the MIL base in the RC and P areas (both P<.05). The DLP base exhibited a higher frequency of negative RTA values than the MIL base. Regarding the RTA values for the mandibular arch, no significant differences were detected between the DLP and MIL denture bases (P>.05). CONCLUSIONS: The DLP and MIL denture bases demonstrated clinically acceptable tissue surface adaptation to both edentulous the maxilla and mandible. The DLP denture base was likely to exhibit intimate tissue adaptation in the stress-bearing areas of maxillary arches compared with the PAP denture base. The maxillary MIL denture base was likely to exhibit small gaps between the supporting tissue and denture base. Both DLP and MIL mandibular denture bases were likely to show intimate adaptation on the lingual slope compared with the PAP base.

Asunto(s)

Diseño de Dentadura , Maxilar , Diseño Asistido por Computadora , Bases para Dentadura , Dentadura Completa , Humanos , Luz , Mandíbula

RESUMEN

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor with a pivotal role in physiological and pathological responses to hypoxia. While HIF-1α is known to be involved in hypoxia-induced upregulation of microRNA (miRNA) expression, HIF-1α is also targeted by miRNAs. In this study, miRNAs targeting HIF-1α were identified and their effects on its expression and downstream target genes under hypoxic conditions were investigated. Cell migration under the same conditions was also assessed. METHODS: microRNAs that target HIF-1α were screened using 3'-untranslated region luciferase (3'-UTR-luciferase) reporter assays. The expression levels of HIF-1α and its downstream target genes after transfection with miRNA were assessed using quantitative RT-PCR and western blot analyses. The effect of the miRNAs on the transcriptional activity of HIF-1α was determined using hypoxia-responsive element luciferase (HRE-luciferase) assays. Cell migration under hypoxia was examined using the wound-healing assay. RESULTS: Several of the 19 screened miRNAs considerably decreased the luciferase activity. Transfection with miR-200c had substantial impact on the expression level and transcription activity of HIF-1α. The mRNA level of HIF-1α downstream genes decreased in response to miR-200c overexpression. MiR-200c inhibited cell migration in normoxia and, to a greater extent, in hypoxia. These effects were partly reversed by HIF-1α expression under hypoxic conditions. CONCLUSION: miR-200c negatively affects hypoxia-induced responses by downregulating HIF-1α, a key regulator of hypoxia. Therefore, overexpression of miR-200c might have therapeutic potential as an anticancer agent that inhibits tumor hypoxia.

Asunto(s)

Movimiento Celular/genética , Regulación hacia Abajo/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Hipoxia de la Célula/genética , Línea Celular Tumoral , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Luciferasas/metabolismo , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Regulación hacia Arriba/genética , Cicatrización de Heridas

RESUMEN

Development of stable rCHO cell lines is still time consuming and labor intensive, although it is a critical step in the commercial development of recombinant antibodies. The current work demonstrates, for the first time, that electroporation of CHO cells with DMSO can enhance stable expression of recombinant antibodies in rCHO cells. Electroporation with DMSO resulted in an average 3.7-fold and 2.8-fold increases in expression levels of aflibercept and pembrolizumab, respectively, in pools of stable rCHO cells. It also resulted in an average of 2.2-fold and 2.6-fold increases in the expression of aflibercept and pembrolizumab, respectively, in single-cell derived rCHO clones. Simple batch cultures of rCHO cell clones with the highest expression produced 1.0 g/l for aflibercept and 1.4 g/l for pembrolizumab without a time-consuming gene amplification process. Electroporation with DMSO also shortened the development of rCHO cell lines to 2-3 months, allowing rapid establishment of stable rCHO cell lines with a desirable expression level antibodies.

Asunto(s)

Anticuerpos/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Dimetilsulfóxido , Electroporación , Animales , Anticuerpos/genética , Células CHO , Técnicas de Cultivo de Célula , Cricetulus , Expresión Génica , Técnicas de Transferencia de Gen , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Bone morphogenetic protein-7 is a multifunctional growth factor involved in various cellular processes such as osteogenesis, kidney and eye development, brown adipogenesis, and bone metastasis, and thus has been considered to have therapeutic potential for treating various diseases. In this study, we established a Chinese hamster ovary (CHO) cell line stably overexpressing recombinant human BMP-7 (rhBMP-7). Over the course of a 14-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 188 mg/l of rhBMP-7 protein. The rhBMP-7 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a recovery rate of approximately 55%, with protein purity greater than 95%. The purified rhBMP-7 was further demonstrated to be functionally active by measuring the proliferation of MC3T3-E1 cells, revealing a half-maximal effective concentration of 28.31 ng/ml.

Asunto(s)

Proteína Morfogenética Ósea 7 , Animales , Reactores Biológicos , Células CHO , Cromatografía , Clonación Molecular , Cricetulus/genética , Humanos , Proteínas Recombinantes

RESUMEN

Micro(mi)RNAs play important and varied roles in tumorigenesis; however, the full repertoire of miRNAs that affect cancer cell growth is not known. In this study, an miRNA library was screened to identify those that affect the growth of A549 tumor cells. Among 300 miRNAs, miR-28-5p, -323-5p, -510-5p, -552-3p, and -608 were the most effective in inhibiting cell growth. More specifically, overexpressing miR-28-5p, -323-5p, and -510-5p induced G1 arrest, as determined by flow cytometry, whereas that of miR-608 induced cell death in a caspase-dependent manner. Moreover, several genes involved in apoptosis and cell cycle progression were downregulated upon overexpression of each of the five miRNAs, with the functional targets of miR-552-3p and miR-608 confirmed by microarray, quantitative real-time PCR, and luciferase reporter assay. In miR-608-transfected cells, B cell lymphoma 2-like 1 (BCL2L1), D-type cyclin 1 (CCND1), CCND3, cytochrome b5 reductase 3 (CYB5R3), phosphoinositide 3-kinase regulatory subunit 2 (PIK3R2), specificity protein 1 (SP1), and phosphorylated Akt were all downregulated, while Bcl-2-interacting killer (BIK) was upregulated. Moreover, miR-608 was determined to have a suppressive function on tumor growth in an NCI-H460 xenograft model. These findings provide insights into the roles of five miRNAs in growth inhibition and their potential function as cancer therapeutics.

Asunto(s)

Apoptosis/genética , Ciclo Celular/genética , Biblioteca de Genes , MicroARNs/análisis , MicroARNs/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma.

Asunto(s)

Proteínas Inhibidoras de la Apoptosis/fisiología , MicroARNs/fisiología , Neuroblastoma/patología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , Masculino , Ratones , Proteína Proto-Oncogénica N-Myc , Nanopartículas , Neuroblastoma/prevención & control , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Survivin

RESUMEN

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3'-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.

Asunto(s)

Quinasa 2 Dependiente de la Ciclina/metabolismo , MicroARNs/metabolismo , Neoplasias/terapia , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Ciclo Celular/genética , Movimiento Celular/genética , Proliferación Celular/genética , Quinasa 2 Dependiente de la Ciclina/genética , Regulación hacia Abajo/genética , Células HeLa , Humanos , MicroARNs/genética , Análisis por Micromatrices , Neoplasias/genética , Células Madre Neoplásicas , Fosfatidilinositol 3-Quinasas/genética , ARN Interferente Pequeño/genética , Transgenes/genética , Proteína de Unión al GTP rac1/genética

RESUMEN

Vascular endothelial growth factor (VEGF) signaling plays an important role in angiogenesis. In the VEGF signaling pathway, the key components are VEGF and its receptors, Flt-1 and KDR. In this study, we show that transfection of synthetic miR-200b reduced protein levels of VEGF, Flt-1, and KDR. In A549 cells, miR-200b targeted the predicted binding sites in the 3'-untranslated region (3'-UTR) of VEGF, Flt-1, and KDR as revealed by a luciferase reporter assay. When transfected with miR-200b, the ability of HUVECs to form a capillary tube on Matrigel and VEGF-induced phosphorylation of ERK1/2 were significantly reduced. Taken together, these results suggest that miR-200b negatively regulates VEGF signaling by targeting VEGF and its receptors and that miR-200b may have therapeutic potential as an angiogenesis inhibitor.

Asunto(s)

Inhibidores de la Angiogénesis/genética , MicroARNs/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Patológica/metabolismo , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Regiones no Traducidas 3'/genética , Western Blotting , Línea Celular Tumoral , Colágeno , Combinación de Medicamentos , Endotelio Vascular , Ensayo de Inmunoadsorción Enzimática , Genes Reporteros , Humanos , Laminina , Luciferasas/análisis , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Neoplasias/genética , Neoplasias/metabolismo , Neovascularización Patológica/genética , Fosforilación/genética , Plásmidos , Proteoglicanos , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Survivin is a protein which functions as a mitotic regulator as well as apoptosis inhibitor. In this study, we show that introduction of synthetic miR-542-3p mimetic reduced both mRNA and protein levels of survivin. In A549 cells, luciferase reporter assay revealed that miR-542-3p targeted predicted binding sites in the 3'-untranslated region (3'-UTR) of survivin. We also demonstrate that ectopic expression of miR-542-3p inhibited cell proliferation by inducing Gap 1 (G1) and Gap 2/Mitosis (G2/M) cell cycle arrest. Collectively, these results suggest that survivin is a direct target of miR-542-3p and growth inhibition by miR-542-3p may have a potential utility as an anti-cancer therapy.

Asunto(s)

Ciclo Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Línea Celular Tumoral , Humanos , Proteínas Inhibidoras de la Apoptosis , MicroARNs/genética , Mitosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Survivin