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BACKGROUND: Augmented reality (AR) technology has been shown to be effective in displaying information and presenting three-dimensional objects. Although AR applications are commonly used by learners via mobile devices, plastic models or two-dimensional images are still commonly used in tooth carving practice. Learners practicing tooth carving face a challenge due to the three-dimensional features of teeth as there is a lack of tools available that provide sequential guidance. In this study, we developed an AR-based tooth carving practice tool (AR-TCPT) and compared it to a plastic model to evaluate its potential as a practice tool as well as its user experience. METHODS: To model tooth carving, we created a three-dimensional object from sequential steps that included the maxillary canines and maxillary first premolars (16 steps), mandibular first premolars (13 steps), and mandibular first molars (14 steps). Image markers, created using Photoshop software, were assigned to each tooth. An AR-based mobile application was developed using the Unity engine. For tooth carving, 52 participants were randomly assigned to a control group (n = 26; using a plastic tooth model) or an experimental group (n = 26; using the AR-TCPT). User experience was evaluated using a 22-item questionnaire. Data were comparatively analyzed using the nonparametric Mann-Whitney U test via the SPSS program. RESULTS: The AR-TCPT detects image markers with the mobile device camera and displays three-dimensional objects for tooth fragmentation. Users can manipulate the device to view each step or examine the shape of a tooth. The results of the user experience survey revealed that the AR-TCPT experimental group scored significantly higher in tooth carving experience compared with the control group that used the plastic model. CONCLUSION: Compared with the conventional plastic model, the AR-TCPT provided a better user experience for tooth carving. The tool is highly accessible as it is designed to be used on mobile devices by users. Further studies are required to determine the educational impact of the AR-TCTP on quantitative scoring of carved teeth as well as individual user's carving abilities.
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Realidade Aumentada , Educação em Odontologia , Aplicativos Móveis , Dente , Computadores de Mão , Estudos Prospectivos , Dente/anatomia & histologia , Modelos Anatômicos , Educação em Odontologia/métodos , Estudantes de Odontologia , HumanosRESUMO
BACKGROUND: Acquiring adequate theoretical knowledge in the field of dental radiography (DR) is essential for establishing a good foundation at the prepractical stage. Currently, nonface-to-face DR education predominantly relies on two-dimensional (2D) videos, highlighting the need for developing educational resources that address the inherent limitations of this method. We developed a virtual reality (VR) learning medium using 360° video with a prefabricated head-mounted display (pHMD) for nonface-to-face DR learning and compared it with a 2D video medium. METHODS: Forty-four participants were randomly assigned to a control group (n = 23; 2D video) and an experimental group (n = 21; 360° VR). DR was re-enacted by the operator and recorded using 360° video. A survey was performed to assess learning satisfaction and self-efficacy. The nonparametric statistical tests comparing the groups were conducted using SPSS statistical analysis software. RESULTS: Learners in the experimental group could experience VR for DR by attaching their smartphones to the pHMD. The 360° VR video with pHMD provided a step-by-step guide for DR learning from the point of view of an operator as VR. Learning satisfaction and self-efficacy were statistically significantly higher in the experimental group than the control group (p < 0.001). CONCLUSIONS: The 360° VR videos were associated with greater learning satisfaction and self-efficacy than conventional 2D videos. However, these findings do not necessarily substantiate the educational effects of this medium, but instead suggest that it may be considered a suitable alternative for DR education in a nonface-to-face environment. However, further examination of the extent of DR knowledge gained in a nonface-to-face setting is warranted. Future research should aim to develop simulation tools based on 3D objects and also explore additional uses of 360° VR videos as prepractical learning mediums.
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Radiologia , Realidade Virtual , Humanos , Autoeficácia , Software , Satisfação PessoalRESUMO
For an enhanced understanding of the biological mechanisms of human disease, it is essential to investigate protein functions. In a previous study, we developed a prediction method of gene ontology (GO) terms by the I-TASSER/COFACTOR result, and we applied this to uPE1 in chromosome 11. Here, to validate the bioinformatics prediction of C11orf52, we utilized affinity purification and mass spectrometry to identify interacting partners of C11orf52. Using immunoprecipitation methods with three different peptide tags (Myc, Flag, and 2B8) in HEK 293T cell lines, we identified 79 candidate proteins that are expected to interact with C11orf52. The results of a pathway analysis of the GO and STRING database with candidate proteins showed that C11orf52 could be related to signaling receptor binding, cell-cell adhesion, and ribosome biogenesis. Then, we selected three partner candidates of DSG1, JUP, and PTPN11 for verification of the interaction with C11orf52 and confirmed them by colocalization at the cell-cell junctions by coimmunofluorescence experiments. On the basis of this study, we expect that C11orf52 is related to the Wnt signaling pathway via DSG1 from the protein-protein interactions, given the results of a comprehensive analysis of the bioinformatic predictions. The data set is available at the ProteomeXchange consortium via PRIDE repository (PXD026986).
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Biologia Computacional , Proteínas , Cromatografia de Afinidade , Ontologia Genética , Humanos , Espectrometria de Massas , Proteínas/genéticaRESUMO
In chromosome 11, 71 out of its 1254 proteins remain functionally uncharacterized on the basis of their existence evidence (uPE1s) following the latest version of neXtProt (release 2020-01-17). Because in vivo and in vitro experimental strategies are often time-consuming and labor-intensive, there is a need for a bioinformatics tool to predict the function annotation. Here, we used I-TASSER/COFACTOR provided on the neXtProt web site, which predicts gene ontology (GO) terms based on the 3D structure of the protein. I-TASSER/COFACTOR predicted 2413 GO terms with a benchmark dataset of the 22 proteins belonging to PE1 of chromosome 11. In this study, we developed a filtering algorithm in order to select specific GO terms using the GO map generated by I-TASSER/COFACTOR. As a result, 187 specific GO terms showed a higher average precision-recall score at the least cellular component term compared to 2413 predicted GO terms. Next, we applied 65 proteins belonging to uPE1s of chromosome 11, and then 409 out of 6684 GO terms survived, where 103 and 142 GO terms of molecular function and biological process, respectively, were included. Representatively, the cellular component GO terms of CCDC90B, C11orf52, and the SMAP were predicted and validated using the overexpression system into 293T cells and immunofluorescence staining. We will further study their biological and molecular functions toward the goal of the neXt-CP50 project as a part of C-HPP. We shared all results and programs in Github (https://github.com/heeyounh/I-TASSER-COFACTOR-filtering.git).
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Cromossomos Humanos Par 11 , Biologia Computacional , Bases de Dados de Proteínas , Ontologia Genética , Humanos , Proteínas/genéticaRESUMO
PURPOSE: Stem cell factor (SCF) has been recently acknowledged as a novel endothelial permeability factor. However, the mechanisms by which SCF-induced activation of the SCF cognate receptor, cKit, enhances endothelial permeability have not been fully elucidated. This study aimed to investigate the role of Src in SCF-induced breakdown of the blood-retinal barrier (BRB). METHODS: In vitro endothelial permeability and in vivo retinal vascular permeability assays were performed to investigate the role of Src in SCF-induced breakdown of the BRB. Immunofluorescence staining experiments were performed to analyze the cellular distribution of phosphorylated Src and vascular endothelial (VE)-cadherin. RESULTS: SCF markedly reduced electric resistance across the human retinal vascular endothelial monolayer in vitro and enhanced extravasation of dyes in murine retinal vasculature in vivo. Inhibition of cKit activation using cKit mutant mice and chemical inhibitor substantially diminished the ability of SCF to increase endothelial permeability and retinal vascular leakage. In human retinal vascular endothelial cells, SCF induced strong phosphorylation of Src and distinct localization of phosphorylated Src in the plasma membrane. Inhibition of Src activation using chemical inhibitors abolished the SCF-induced hyperpermeability of human retinal vascular endothelial cells and retinal vascular leakage in mice. In addition, treatment with Src inhibitors restored junctional expression of VE-cadherin that disappeared in SCF-treated retinal endothelial cells and retinal vasculature. CONCLUSIONS: These results showed the important role of Src in mediating SCF-induced breakdown of the BRB and retinal vascular leakage. Given that increased retinal vascular permeability is a common manifestation of various ocular diseases, the SCF/cKit/Src signaling pathway may be involved in the development of the hyperpermeable retinal vasculature in many ocular disorders.
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Barreira Hematorretiniana/efeitos dos fármacos , Vasos Retinianos/metabolismo , Fator de Células-Tronco/farmacologia , Quinases da Família src/fisiologia , Animais , Barreira Hematorretiniana/metabolismo , Western Blotting , Caderinas/metabolismo , Permeabilidade Capilar/fisiologia , Células Endoteliais , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. APPROACH AND RESULTS: We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase-mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF-neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. CONCLUSIONS: Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.
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Permeabilidade Capilar , Células Endoteliais/metabolismo , Fator de Células-Tronco/metabolismo , Junções Aderentes/metabolismo , Animais , Anticorpos Neutralizantes/administração & dosagem , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/prevenção & controle , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , Vasos Retinianos/metabolismo , Transdução de Sinais , Fator de Células-Tronco/genética , Fator de Células-Tronco/imunologia , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Aortic valve replacement (AVR) has recently been performed at many centers using a minimally invasive approach to reduce postoperative mortality, morbidity, and pain. Most previous reports on minimally invasive AVR (MiAVR) have mainly focused on aortic stenosis, and those exclusively dealing with aortic regurgitation (AR) are few. The purpose of this study was to investigate early surgical results and review our experience with patients with chronic severe AR who underwent AVR via right anterior mini-thoracotomy (RAT). METHODS: Data were retrospectively collected in this single-center study. Eight patients who underwent RAT AVR between January 2020 and January 2024 were enrolled. Short-term outcomes, including the length of hospital stay, in-hospital mortality, postoperative complications, and echocardiographic data, were analyzed. RESULTS: No in-hospital mortalities were observed. Postoperative atrial fibrillation occurred temporarily in three patients (37.5%). However, none required permanent pacemaker implantation or renal replacement therapy. The median values of ventilator time, length of intensive care unit stay, and hospital stay were 17 hours, 34.5 hours, and 9 days, respectively. Preoperative and postoperative measurements of left ventricular ejection fraction were similar. However, the left ventricular end systolic and diastolic diameters significantly decreased postoperatively from 42 mm to 35.5 mm (p=0.018) and 63 mm to 51 mm (p=0.012), respectively. CONCLUSION: MiAVR via RAT is a safe and reproducible procedure with acceptable morbidity and complication rates in patients with chronic severe AR. Despite some limitations such as a narrow surgical field and demanding learning curve, MiAVR is a competent method for AR.
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BACKGROUND: The aim of this study was to compare the early outcomes of octogenarians undergoing minimally invasive right anterior mini-thoracotomy aortic valve replacement (RAT-AVR) with those undergoing transcatheter aortic valve implantation (TAVI) for aortic valve disease. METHODS: In this single-center retrospective study, data were collected from octogenarians before and after RAT-AVR and TAVI between January 2021 and July 2022. Short-term outcomes, including the length of hospital stay, in-hospital mortality, all-cause mortality, and other major postoperative complications, were compared and analyzed. RESULTS: There were no significant differences in in-hospital mortality, stroke, acute kidney dysfunction requiring renal replacement therapy, length of intensive care unit stay, or length of hospital stay. However, the TAVI group had a higher incidence of permanent pacemaker insertion (10% vs. 0%, p=0.54) and paravalvular leaks (75% vs. 0%, p<0.001). CONCLUSION: In the present study on octogenarians, both TAVI and RAT-AVR showed comparable short-term results. Although both procedures were considered safe and effective in the selected group, RAT-AVR had a lower incidence of complete atrioventricular block and paravalvular regurgitation.
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Owing to a lack of reliable markers and therapeutic targets, pancreatic ductal adenocarcinoma (PDAC) remains the most lethal malignant tumor despite numerous therapeutic advances. In this study, we utilized cell-SELEX to isolate a DNA aptamer recognizing the natural conformation of the target on the cell surface. PAp7T8, an aptamer optimized by size and chemical modification, exhibited specific targeting to pancreatic cancer cells and orthotopic xenograft pancreatic tumors. To confer therapeutic functions to the aptamer, we adopted a drug-conjugated oligobody (DOligobody) strategy. Monomethyl auristatin E was used as a cytotoxic drug, digoxigenin acted as a hapten, and the humanized anti-digoxigenin antibody served as a universal carrier of the aptamer. The resulting PAp7T8-DOligobody showed extended in vivo half-life and markedly inhibited tumor growth in an orthotopic pancreatic cancer xenograft model without causing significant toxicity. Therefore, PAp7T8-DOligobody represents a promising novel therapeutic delivery platform for PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Preparações Farmacêuticas , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Anticorpos , Oligonucleotídeos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
BACKGROUND/AIM: Invasive papillary cholangio-carcinoma (IPC) is a minor subtype of extrahepatic cholangiocarcinoma. However, its etiology and characteristics remain unknown because of the unavailability of in vitro and in vivo models. We aimed to establish a novel preclinical model for translational research of IPC. MATERIALS AND METHODS: A patient-derived xenograft (PDX) was engrafted in NOG mice and the cell line National Cancer Center human IPC (NCChIPC) was subsequently established from the PDX tumors. Immunohistochemistry and RNA-sequencing were used to determine the retention of original characteristics of patient tissues. RESULTS: PDX tumors showed successful amplification, and the NCChIPC-derived xenograft largely retained the histopathological features of the original tumor with CK19, MUC1 and MUC5AC expression. Transcriptome analysis showed a high correlation between patient and preclinical models. Additionally, anticancer drugs response was analyzed in the NCChIPC PDX. CONCLUSION: These novel preclinical models here will help elucidate IPC etiology and facilitate translational research.
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Carcinoma Papilar/genética , Colangiocarcinoma/genética , Queratina-19/genética , Mucina-5AC/genética , Mucina-1/genética , Idoso , Animais , Antineoplásicos/farmacologia , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Transcriptoma/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report the fabrication of a novel titania membrane of the dual-pore system that is strategically designed and prepared by a two-step replication process and sol-gel reaction. The primary nanoporous channel structure is fabricated by the cage-like PMMA template (CPT) obtained from the nanoporous alumina membrane and the secondary mesoporous structure is formed by the sol-gel reaction of the lyotropic precursor solution within the CPT. Furthermore the mesoporous titania membrane (MTM) frame consists of the titania nanoparticles of 10-12 nm in diameter. Morphology and structural properties of the MTM are investigated by field emission scanning electron microscopy, high resolution transmission electron microscopy, x-ray diffraction and Brunauer-Emmett-Teller surface area. The photocatalytic activity and the solar energy properties of the MTM are characterized by UV-vis spectrophotometer, spectrofluorometer and photoinduced I-V measurement. The photocatalytic test indicates that the MTM has higher efficiency than the commercial P25 with a good recyclability due to its large-scale membrane style and the preliminary result on the solar cell application shows a solar energy conversion efficiency of 3.35% for the dye-sensitized solar cell utilizing the MTM.
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PURPOSE: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein. METHODS: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry. RESULTS: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER. CONCLUSIONS: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.
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Proteínas do Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Espaço Extracelular/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Linhagem Celular , Proliferação de Células , Proteínas do Citoesqueleto/química , Chaperona BiP do Retículo Endoplasmático , Proteínas do Olho/química , Feminino , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/química , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico/biossíntese , Humanos , Chaperonas Moleculares/biossíntese , Oligopeptídeos , Peptídeos , Dobramento de Proteína , Proteínas Recombinantes de Fusão , Via Secretória , Fatores de Tempo , Malha Trabecular/metabolismo , Regulação para CimaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignant tumor and more than 50% patients are diagnosed at metastatic stage. The preclinical model systems that reflect the genetic heterogeneity of metastatic tumors are urgently needed to guide optimal treatment. This study describes the development of patient-derived preclinical platform using very small sized-percutaneous liver gun biopsy (PLB) of metastatic pancreatic cancer, based on patient-derived xenograft (PDX)-mediated tissue amplification and subsequent organoid generation. To increase the success rate and shorten the tumor growth period, patient-derived orthotopic xenograft (PDOX) model was developed to directly implant threadlike PLB samples into the pancreas. The engraftment success rate of PDOX samples from 35 patients with metastatic PDAC was 47%, with these samples showing the potential to metastasize to distant organs, as in patients. The PDOX models retained the genetic alterations and histopathological features of the primary tumors. Tumor organoids were subsequently generated from first passage cancer cells isolated from F1 tumor tissue of PDOX that preserve the epithelial cancer characteristics and KRAS mutations of primary tumors. The response to gemcitabine of PDOX-derived organoids correlated with clinical outcomes in corresponding patients as well as PDOX models in vivo, suggesting that this PDOX-organoid system reflects clinical conditions. Collectively, these findings indicate that the proposed PDOX-organoid platform using PLB samples assessed both in vitro and in vivo could predict drug response under conditions closer to those found in actual patients, as well as enhancing understanding of the complexity of metastatic PDAC.
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PURPOSE: Stem cell factor (SCF) has recently demonstrated activity as a novel endothelial permeability factor that contributes to the development of diabetes-induced hyperpermeable retinal vasculature. This study investigated the therapeutic potential of masitinib, a pharmacologic inhibitor of the SCF receptor cKit, for prevention of diabetes-induced breakdown of blood retinal barrier (BRB). METHODS: Permeability assays were performed with human retinal microvascular endothelial cells (HRMECs) and murine retinal vasculature. Localization of vascular endothelial (VE)-cadherin and activation of SCF signaling pathway was determined by immunofluorescence and Western blotting assays. Mice and rats with streptozotocin (STZ)-induced diabetes were used to investigate the role of cKit and masitinib in diabetes-induced retinal vascular hyperpermeability. RESULTS: Masitinib substantially blocked SCF-induced phosphorylation of cKit in HRMECs. In vitro and in vivo vascular permeability assays showed that masitinib significantly inhibited SCF-induced endothelial hyperpermeability and junctional loss of VE-cadherin. Streptozotocin-induced diabetes was induced in cKit-mutant mice with low cKit expression in their endothelial cells. Although diabetic wild-type mice exhibited enhanced retinal vascular leakage, diabetic cKit-mutant mice showed no increase in retinal vascular leakage or alteration in the distribution of VE-cadherin; this indicates the crucial role of cKit in diabetes-induced breakdown of BRB. Moreover, in vivo prevention experiments showed that an intravitreal injection of masitinib substantially inhibited the development of hyperpermeable retinal vasculature. CONCLUSIONS: These results provide the first demonstration that cKit inhibitors, such as masitinib, might be promising therapeutics for prevention of diabetes-induced breakdown of the BRB.
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Permeabilidade Capilar/efeitos dos fármacos , Diabetes Mellitus Experimental , Retinopatia Diabética/complicações , Endotélio Vascular/efeitos dos fármacos , Doenças Retinianas/prevenção & controle , Tiazóis/administração & dosagem , Resistência Vascular/efeitos dos fármacos , Animais , Benzamidas , Barreira Hematorretiniana/efeitos dos fármacos , Western Blotting , Células Cultivadas , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Piridinas , RNA/genética , Ratos , Ratos Sprague-Dawley , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/biossíntese , Fator de Células-Tronco/genéticaRESUMO
This study demonstrates the application feasibility of electrochemical impedance spectroscopy (EIS) in measuring estrogen (17ß-estradiol) in gas phase. The present biosensor gives a linear response (R2 = 0.999) for 17ß-estradiol vapor concentration from 3.7 ng/L to 3.7 × 10-4 ng/L with a limit of detection (3.7 × 10-4 ng/L). The results show that the fabricated biosensor demonstrates better detection limit of 17ß-estradiol in gas phase than the previous report with GC-MS method. This estrogen biosensor has many potential applications for on-site detection of a variety of endocrine disrupting compounds (EDCs) in the gas phase.
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PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.
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Morte Celular/genética , DNA/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP72/genética , Degeneração Retiniana/genética , Células Ganglionares da Retina/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Etoposídeo/toxicidade , Proteínas de Choque Térmico HSP72/biossíntese , Imuno-Histoquímica , Ratos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologiaRESUMO
We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and α-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both α-SMA and SM22-α proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.
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Células Endoteliais/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Células-Tronco Pluripotentes/citologia , Espermatogônias/citologia , Testículo/citologia , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Células-Tronco Pluripotentes/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Espermatogônias/fisiologia , Testículo/fisiologiaRESUMO
To understand the regulatory mechanisms governing glucocorticoid-mediated MYOC induction in human trabecular meshwork (HTM) cells, the expression and degradation of MYOC mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative MYOC promoters was assessed by luciferase reporter assay. Here, we confirmed that MYOC is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that MYOC mRNA is degraded very slowly, with approximately half persisting for at least 4 days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of MYOC revealed that luciferase induction by DEX was increased by 280 ± 34% in HTM cells. Moreover, DEX induction required the region between base pairs -2548 and -1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and human retinal pigment epithelium (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5'-flanking sequences of MYOC. It will be a major step towards understanding the expression pattern of MYOC in HTM cells and TM tissue.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Dexametasona/farmacologia , Proteínas do Olho/metabolismo , Glucocorticoides/farmacologia , Glicoproteínas/metabolismo , Malha Trabecular/metabolismo , Northern Blotting , Células Cultivadas , Cicloeximida/farmacologia , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glicoproteínas/genética , Humanos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Malha Trabecular/efeitos dos fármacosRESUMO
Transplantation of human cord blood-derived endothelial progenitor cells (EPCs) is reported to contribute to neovascularization in various ischemic diseases. However, the possible beneficial role and underlying mechanisms in diabetes-impaired wound healing have been less well characterized. In this study, EPC transplantation stimulated keratinocyte and fibroblast proliferation substantially as early as 3 days after injury, leading to significantly accelerated wound closure in streptozotocin-induced diabetic nude mice, compared to PBS control. RT-PCR analysis showed that EPCs secreted various wound healing-related growth factors. Among them, keratinocyte growth factor and platelet-derived growth factor were highly expressed in the EPCs and were present at substantial levels in the EPC-injected dermal tissue. Using EPC-conditioned medium (CM), we found that paracrine factors from EPCs directly exerted mitogenic and chemotactic effects on keratinocytes and fibroblasts. Moreover, injection of EPC-CM alone into the same diabetic wound mice promoted wound healing and increased neovascularization to a similar extent as achieved with EPC transplantation. These results indicate that the beneficial effect of EPC transplantation on diabetic wounds was mainly achieved by their direct paracrine action on keratinocytes, fibroblasts, and endothelial cells, rather than through their physical engraftment into host tissues (vasculogenesis). In addition, EPC-CM was shown to be therapeutically equivalent to EPCs, at least for the treatment of diabetic dermal wounds, suggesting that conditioned medium may serve as a novel therapeutic option that is free from allograft-associated immune rejection concern.