Autophagy Impairment Induces Premature Senescence in Primary Human Fibroblasts

BACKGROUND:Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells. METHODOLOGY/PRINCIPAL FINDINGS:Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways. CONCLUSION:Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts

Prediction of cognitive impairment via deep learning trained with multi-center neuropsychological test data

Background Neuropsychological tests (NPTs) are important tools for informing diagnoses of cognitive impairment (CI). However, interpreting NPTs requires specialists and is thus time-consuming. To streamline the application of NPTs in clinical settings, we developed and evaluated the accuracy of a machine learning algorithm using multi-center NPT data. Methods Multi-center data were obtained from 14,926 formal neuropsychological assessments (Seoul Neuropsychological Screening Battery), which were classified into normal cognition (NC), mild cognitive impairment (MCI) and Alzheimers disease dementia (ADD). We trained a machine learning model with artificial neural network algorithm using TensorFlow (https://www.tensorflow.org) to distinguish cognitive state with the 46-variable data and measured prediction accuracies from 10 randomly selected datasets. The features of the NPT were listed in order of their contribution to the outcome using Recursive Feature Elimination. Results The ten times mean accuracies of identifying CI (MCI and ADD) achieved by 96.66 ± 0.52% of the balanced dataset and 97.23 ± 0.32% of the clinic-based dataset, and the accuracies for predicting cognitive states (NC, MCI or ADD) were 95.49 ± 0.53 and 96.34 ± 1.03%. The sensitivity to the detection CI and MCI in the balanced dataset were 96.0 and 96.0%, and the specificity were 96.8 and 97.4%, respectively. The time orientation and 3-word recall score of MMSE were highly ranked features in predicting CI and cognitive state. The twelve features reduced from 46 variable of NPTs with age and education had contributed to more than 90% accuracy in predicting cognitive impairment. Conclusions The machine learning algorithm for NPTs has suggested potential use as a reference in differentiating cognitive impairment in the clinical setting.The publication costs, design of the study, data management and writing the manuscript for this article were supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2017S1A6A3A01078538), Korea Ministry of Health & Welfare, and from the Original Technology Research Program for Brain Science through the National Research Foundation of Korea funded by the Korean Government (MSIP; No. 2014M3C7A1064752)

The Efficacy of Porous Hydroxyapatite Granule as a Carrier of E.coli-derived Recombinant Human Bone Morphogenetic Protein-2

Porous hydroxyapatite (HA) has great osteoconductivity, which allows it to be used as a bone graft extender. Porous HA enables bone formation by binding with recombinant human bone morphogenetic protein-2 (rhBMP-2). This research is to assess the capability of porous HA granules as a carrier of E.coli-derived rhBMP-2 (E.BMP-2). We compared the release of E.BMP-2 from porous HA of both block and granular forms for 120 min by using an ELISA. Using alkaline phosphatase (ALP) activity assay of human mesenchymal stem cells (hMSC), and the rat abdominal ectopic bone formation model, we also examined the osteogenic capacity of granular HA containing E.BMP-2.The result has shown that the rate of E.BMP-2 released from porous HA for 120 min indicated 42% of HA granules and 27.9% of HA block; the amount of HA granules was larger than HA block. At each set time interval of 10 min, 20 min, 40 min, 80 min, and 120 min, the amount of E.BMP-2 released from HA granules were significantly higher than those from HA block. The ALP activity on the third day of porous HA granules treated with 100 mu g E.BMP-2 was significantly higher than that of pure HA granules. In addition, in the rat in vivo model, bony tissue was confirmed in the all of the cases in the E.BMP-HA group but not in the pure HA group, according to the micro-CT and the histology 8 weeks after the surgery. Moreover, in contrast to the pure HA group, the E.BMP-HA group showed a higher level of bone volume, percent bone volume, structure model index, trabecular thickness, and trabecular number in the micro-CT scan. In conclusion, porous HA granules can be bound with E.BMP-2 and can properly release E.BMP-2, so that it can increase the osteoblastic differentiation of hMSC and induce the ectopic bone formation, consequently qualifying as a carrier of E.BMP-2.This study was supported by a Grant-in-Aid(No.03-2011-300) from the SNUH Research Fund.OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000007273/7SEQ:7PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:0000007273ADJUST_YN:YEMP_ID:A077517DEPT_CD:801CITE_RATE:.345FILENAME:첨부된 내역이 없습니다.DEPT_NM:의학과EMAIL:[email protected]_YN:NCONFIRM:

Fabrication and Evaluation of Osteoblastic Differentiation of Human Mesenchymal Stem Cells on Novel CaO-SiO2-P2O5-B2O3 Glass-Ceramics

Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2-B2O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2-P2O5-B2O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new bone graft extender.This work was supported by a Grant-in-Aid from the Seoul Development Institute (Seoul R&BD Program, No. ST100012).OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000007273/3SEQ:3PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:0000007273ADJUST_YN:YEMP_ID:A077517DEPT_CD:801CITE_RATE:1.964FILENAME:첨부된 내역이 없습니다.DEPT_NM:의학과EMAIL:[email protected]_YN:YCONFIRM:

In vitro and in vivo evaluation of the bioactivity of hydroxyapatite-coated polyetheretherketone biocomposites created by cold spray technology

Polyetheretherketone (PEEK) is a material that is widely used in medicine because its mechanical properties show excellent similarity to those of human bone. However, because it is bioinert, PEEK shows limited ability to bind to natural bone tissue. Here, we applied a cold spray method to make a hydroxyapatite (HA)-coated PEEK hybrid material and evaluated its osteointegration in vitro and in vivo. With the cold spray method, the HA coating formed a homogeneous layer and adhered strongly to the PEEK disk implant. When the material was tested in vitro, early cell adhesion and viability improved. Alkaline phosphatase (ALP) activity and calcium concentration were also higher in cells cultured on HA-coated PEEK disks. In addition, the expression of osteoblast differentiation markers, such as ALP, bone sialoprotein and runt-related transcription factor 2, increased in these cells. For the in vivo test, we designed and implanted HA-coated PEEK cylinders into a rabbit ilium model by the press-fit method. The bone-implant contact ratio, trabecular number and trabecular thickness were determined using either three-dimensional microcomputed tomography or general two-dimensional histomorphometric analysis. This report demonstrates that the HA coating on the PEEK implant added with the cold spray method increased biocompatibility in vitro and promoted osteointegration in vivo, which suggests that the HA coating may improve the biofunctionality of various medical devices used in clinical applications.OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000007273/2SEQ:2PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:0000007273ADJUST_YN:YEMP_ID:A077517DEPT_CD:801CITE_RATE:5.093FILENAME:첨부된 내역이 없습니다.DEPT_NM:의학과EMAIL:[email protected]_YN:YCONFIRM:

Microstructure evolution in nanoporous gold thin films made from sputter-deposited precursors

We fabricate almost crack-free 1.5 ??m thick nanoporous gold thin films using free-corrosion dealloying and transfer processes from sputter-deposited precursors. By controlling the temperature and the concentration of the nitric acid solution during free-corrosion dealloying, we obtain ligament sizes in nanoporous gold between 22 and 155 nm. We investigate the effects of dissolution rate of Ag atoms, surface diffusivity of Au atoms and formation of Ag oxide on nanoporosity evolution.close1

In Vivo Evaluation of CaO SiO2 P2O5 B2O3 Glass-ceramics Coating on Steinman Pin

Surface coating using ceramics improves the bone bonding strength of an implant. We questioned whether a new type of glass-ceramics (BGS-7) coating (CaO-SiO2-P2O5-B2O3) would improve the osseointegration of Steinman pins (S-pins) both biomechanically and histomorphometrically. An in vivo study was performed using rabbits by inserting three S-pins into each iliac bone. The pins were 2.2-mm S-pins with a coating of 30-μm-thick BGS-7 and 550-nm-thick hydroxyapatite (HA), as opposed to an S-pin without coating. A tensile strength test and histomorphometrical evaluation was performed. In the 2-week group, the BGS-7 implant showed a significantly higher tensile strength than the S-pin. In the 4- and 8-week groups, the BGS-7 implants had significantly higher tensile strengths than the S-pins and HA implants. The histomorphometrical study revealed that the BGS-7 implant had a significantly higher contact ratio than the S-pin and HA implants in the 4-week group. The biomechanical and histomorphometrical tests showed that the BGS-7 coating had superior bone bonding properties than the groups without the coating from the initial stage of insertion. The BGS-7 coating of an S-pin will enhance the bone bonding strength, and there might also be an advantage in human bone bonding.This work was supported by a Grant-in-Aid from the SNUH Research Fund (No. 03-2011-300).OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000007273/4SEQ:4PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:0000007273ADJUST_YN:NEMP_ID:A077517DEPT_CD:801CITE_RATE:1.964FILENAME:첨부된 내역이 없습니다.DEPT_NM:의학과EMAIL:[email protected]_YN:YCONFIRM:

Effects of porous beta-tricalcium phosphate-based ceramics used as an E-coli-derived rhBMP-2 carrier for bone regeneration

Recombinant human bone morphogenetic protein-2 (rhBMP-2) requires carriers for clinical effectiveness. In this study, whether porous beta-tricalcium phosphate (beta-TCP)-based ceramics are ideal carriers for rhBMP-2 was investigated. Hydroxyapatite (HA), beta-TCP, TCP/HA (80 %/20 %), HA with rhBMP-2, TCP with rhBMP-2, and TCP/HA (80 %/20 %) with rhBMP-2 were manufactured by a sponge method with a pore size of 300 mu m or more and macro-porosity of 83 %. The alkaline phosphatase (ALP) activity and ALP expression of the cells with 100 % beta-TCP granules were more increased than the those of cells with 100 % HA and TCP/HA (80 %/20 %) at the baseline or when treated with 15 ng/ml of rhBMP-2. In an SD rat calvarial defect model, new bone formation was evidently shown in the TCP 100 %-rhBMP-2 and TCP/HA (80 %/20 %)-rhBMP-2 groups, showing that the most affected area was filled with newly-formed bone, that the percent bone volume and trabecular number were larger when compared to the groups without rhBMP-2 treatment at both 4 and 8 weeks after surgery using micro-CT and histology. Porous TCP-based ceramic granules enhanced the osteoblastic differentiation in the hMSC system when treated with 15 ng/ml of rhBMP-2 and accelerated bone-healing by trabecular number in a rat calvarial defect model. Thus, in this study it was proposed that TCP-based ceramics might be useful carriers of rhBMP-2.This study was supported by a Grant-in-Aid from the Korean Ministry of Knowledge Economy/KEIT (No.10033623).OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000007273/8SEQ:8PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:0000007273ADJUST_YN:YEMP_ID:A077517DEPT_CD:801CITE_RATE:2.141FILENAME:첨부된 내역이 없습니다.DEPT_NM:의학과EMAIL:[email protected]_YN:YCONFIRM: