scholarly journals Tripterine Treatment Improves Endothelial Progenitor Cell Function via Integrin-Linked Kinase

2015 ◽  
Vol 37 (3) ◽  
pp. 1089-1103 ◽  
Author(s):  
Chenhui Lu ◽  
Xixiang Yu ◽  
Keqiang Zuo ◽  
Xiaoping Zhang ◽  
Chuanwu Cao ◽  
...  
Keyword(s):  
Vascular Function ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Tube Formation ◽  
Dominant Negative ◽  
Endothelial Progenitor ◽  
Integrin Linked Kinase ◽  
Gsk 3Β ◽  
Green Fluorescent

Background/Aims: Atherosclerosis is associated with dysfunction of endothelial progenitor cells (EPCs). Tripterine, a chemical compound derived from the Chinese medicinal plant Tripterygium wilfordii Hook, displays anti-inflammatory properties in several animal models. We hypothesized that tripterine can improve EPC function and thus the efficiency of EPC transplantation. Methods and Results: Tripterine preconditioning (2.5 μM, 4 h) improved EPC proliferation, tube formation, migration, and adhesion, and reduced apoptosis in cells cultured in ox-LDL (200 µg/ml). Tripterine restored integrin-linked kinase (ILK) levels downregulated by ox-LDL in EPCs, suggesting the involvement of the ILK/Akt pathway. Small interfering RNA-mediated depletion of ILK and dominant-negative ILK transduction inhibited the phosphorylation of the ILK downstream signaling targets protein kinase B/Akt and glycogen synthase kinase 3-beta (GSK-3β), and reduced β-catenin and cyclin D1 expression. In atherosclerotic mice injected with green fluorescent protein-labeled EPCs to evaluate EPC function, tripterine decreased aortic lesions and plaque deposition, and injection of tripterine-treated EPCs restored ILK levels. Conclusion: The present results suggest that tripterine improves vascular function in atherosclerosis by enhancing EPC function through a mechanism involving the ILK signaling pathway.

scholarly journals MicroRNA-126 enhances the biological function of endothelial progenitor cells under oxidative stress via PI3K/Akt/GSK-3β and ERK1/2 signaling pathways

2020 ◽  
Author(s):  
Qinqin Wu ◽  
Benling Qi ◽  
Xiaoyu Duan ◽  
Xiaoyan Ming ◽  
Fengqin Yan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Biological Function ◽  
Glycogen Synthase ◽  
Early Stage ◽  
Tube Formation ◽  
Dependent Manner ◽  
Endothelial Progenitor ◽  
H2o2 Treatment ◽  
Reverse Transcription Pcr ◽  
Gsk 3Β

Endothelial progenitor cell (EPC) transplantation is a safe and effective method to treat acute myocardial infarction (AMI). However, oxidative stress leads to the death of a large number of EPCs in the early stage of transplantation, severely weakening the therapeutic effect. Previous studies demonstrated that microRNAs (miRNAs) regulate the biological function of EPCs. The aim of the current study was to investigate the effect of miRNA on the biological function of EPCs under oxidative stress. Quantitative reverse transcription PCR was performed to detect the expression of miR-126, miR-508-5p, miR-150, and miR-16 in EPCs from rats, among which miR-126 showed a relatively higher expression. Treatment with H2O2 decreased miR-126 expression in EPCs in a dose-dependent manner. EPCs were further transfected with miR-126 mimics or inhibitors, followed by H2O2 treatment. Overexpression of miR-126 enhanced the proliferation, migration, and tube formation of H2O2-treated EPCs. MiR-126 overexpression also inhibited reactive oxygen species and malondialdehyde levels and enhanced superoxide dismutase levels, as well as increased angiopoietin (Ang)1 expression and decreased Ang2 expression in H2O2-treated EPCs. Moreover, miR-126 participated in the regulation of phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt)/ glycogen synthase kinase-3β (GSK-3β) and extracellular signal-regulated kinase (ERK)1/2 signaling in EPCs, where both pathways were activated after miR-126 overexpression in H2O2-treated EPCs. Overall, we showed that miR-126 promoted the biological function of EPCs under H2O2-induced oxidative stress by activating the PI3K/Akt/GSK-3β and ERK1/2 signaling pathway, which may serve as a new therapeutic approach to treat AMI.

scholarly journals Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Takuya Tomita ◽  
Shoshiro Hirayama ◽  
Yasuyuki Sakurai ◽  
Yuki Ohte ◽  
Hidehito Yoshihara ◽  
...  
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Embryonic Fibroblasts ◽  
Α Subunit ◽  
Biochemical Alterations ◽  
Cellular Processes ◽  
Responsible Gene ◽  
Intracellular Protein Degradation ◽  
Green Fluorescent

ABSTRACT The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α′ subunit of casein kinase II (CK2α′) as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin

2013 ◽  
Vol 304 (5) ◽  
pp. F553-F564 ◽  
Author(s):  
Richard Bouley ◽  
Paula Nunes ◽  
Billy Andriopoulos ◽  
Margaret McLaughlin ◽  
Matthew J. Webber ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Target Cells ◽  
Intracellular Camp ◽  
Coated Pits ◽  
Parathyroid Hormone Receptor ◽  
Body Fluid Homeostasis ◽  
Green Fluorescent ◽  
G Protein Coupled

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling

2011 ◽  
Vol 301 (4) ◽  
pp. H1531-H1539 ◽  
Author(s):  
Hidekazu Ishida ◽  
Shigetoyo Kogaki ◽  
Jun Narita ◽  
Hiroaki Ichimori ◽  
Nobutoshi Nawa ◽  
...  
Keyword(s):  
Late Stage ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Morphological Changes ◽  
Cardiac Cells ◽  
The Other ◽  
Cardiomyocyte Differentiation ◽  
Cardiac Progenitors ◽  
P19cl6 Cells ◽  
Gsk 3Β

LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.

Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2977-2986 ◽  
Author(s):  
Janet F. Staab ◽  
Yong-Sun Bahn ◽  
Paula Sundstrom
Keyword(s):  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Germ Tube ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Tube Formation ◽  
Developmental Expression ◽  
Specific Gene ◽  
Expression Of Genes ◽  
Green Fluorescent

The authors have engineered plasmid constructs for developmental and constitutive expression of yeast-enhanced green fluorescent protein (yEGFP3) in Candida albicans. The promoter for the hyphae-specific gene Hyphal Wall Protein 1 (HWP1) conferred developmental expression of yEGFP3 in germ tubes and hyphae but not in yeasts or pseudohyphae when targeted to the ENO1 (enolase) locus in single copy. The pHWP1GFP3 construct allows for the easy visualization of HWP1 promoter activity in individual cells expressing true hyphae without having to prepare RNA for analysis. Constitutive expression of yEGFP was seen in all cell morphologies when the HWP1 promoter was replaced with the ENO1 promoter region. The use of the plasmids for expression of genes other than yEGFP3 was examined by substituting the putative C. albicans BCY1 (SRA1) gene, a component of the cAMP signalling pathway involved in yeast to hyphae transitions, for yEGFP3. Strains overexpressing BCY1 from the ENO1 promoter were inhibited in germ tube formation and filamentation in both liquid and solid media, a phenotype consistent with keeping protein kinase A in its inactive form by association with Bcy1p. The plasmids are suitable for studies of germ tube induction or assessing germ tube formation by measuring yEGFP3 expression, for inducible expression of genes concomitant with germ tube formation by the HWP1 promoter, for constitutive expression of genes by the ENO1 promoter, and for expressing yEGFP3 using a promoter of choice.

Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors

2007 ◽  
Vol 292 (2) ◽  
pp. H1170-H1180 ◽  
Author(s):  
Jeremiah S. Hinson ◽  
Matthew D. Medlin ◽  
Kashelle Lockman ◽  
Joan M. Taylor ◽  
Christopher P. Mack
Keyword(s):  
Smooth Muscle ◽  
Transcription Factors ◽  
Nuclear Localization ◽  
Transforming Growth Factor ◽  
Serum Response Factor ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
Specific Gene ◽  
Green Fluorescent

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22α expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (ΔB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM α-actin, SM22α, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-β on SMC-specific transcription. Our data confirmed the importance of the NH2-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM α-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

Ser9 phosphorylation of mitochondrial GSK-3β is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

2008 ◽  
Vol 295 (5) ◽  
pp. H2079-H2086 ◽  
Author(s):  
Katsuhiko Ohori ◽  
Tetsuji Miura ◽  
Masaya Tanno ◽  
Takayuki Miki ◽  
Takahiro Sato ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Oxidant Stress ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
H9c2 Cardiomyocytes ◽  
Gsk 3Β ◽  
Induced Apoptosis ◽  
Green Fluorescent ◽  
Interfering Rna

The aim of this study was to determine the role of GSK-3β in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3β and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 ± 2.7% to 26.0 ± 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3β knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3β (K85R). The level of colocalization of intracellular GSK-3β with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3β or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3β colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3β knockdown. These results suggest that the suppression of GSK-3β activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.

scholarly journals Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

2002 ◽  
Vol 13 (12) ◽  
pp. 4401-4413 ◽  
Author(s):  
Ewa Markiewicz ◽  
Thomas Dechat ◽  
Roland Foisner ◽  
Roy. A Quinlan ◽  
Christopher J. Hutchison
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Lamin A ◽  
Dependent Manner ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Green Fluorescent

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathioneS-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.

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