scholarly journals Downregulation of POFUT1 Impairs Secondary Myogenic Fusion Through a Reduced NFATc2/IL-4 Signaling Pathway

2019 ◽  
Vol 20 (18) ◽  
pp. 4396
Author(s):  
Audrey Der Vartanian ◽  
Julien Chabanais ◽  
Claire Carrion ◽  
Abderrahman Maftah ◽  
Agnès Germot
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Time Course ◽  
Myogenic Differentiation ◽  
Cell Types ◽  
C2c12 Cells ◽  
Wild Type ◽  
Blocking Antibody ◽  
The Impact ◽  
Nuclei Number

Past work has shown that the protein O-fucosyltransferase 1 (POFUT1) is involved in mammal myogenic differentiation program. Pofut1 knockdown (Po –) in murine C2C12 cells leads to numerous elongated and thin myotubes, suggesting significant defects in secondary fusion. Among the few pathways involved in this process, NFATc2/IL-4 is described as the major one. To unravel the impact of POFUT1 on secondary fusion, we used wild-type (WT) C2C12 and Po – cell lines to follow Myf6, Nfatc2, Il-4 and Il-4rα expressions during a 120 h myogenic differentiation time course. Secreted IL-4 was quantified by ELISA. IL-4Rα expression and its labeling on myogenic cell types were investigated by Western blot and immunofluorescence, respectively. Phenotypic observations of cells treated with IL-4Rα blocking antibody were performed. In Po –, we found a decrease in nuclei number per myotube and a downexpression of Myf6. The observed downregulation of Nfatc2 is correlated to a diminution of secreted IL-4 and to the low level of IL-4Rα for reserve cells. Neutralization of IL-4Rα on WT C2C12 promotes myonuclear accretion defects, similarly to those identified in Po –. Thus, POFUT1 could be a new controller of myotube growth during myogenesis, especially through NFATc2/IL-4 signaling pathway.

Abstract 160: Pim-1 Preserves Cardiac Stem Cell Proliferation with Age

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Michael McGregor ◽  
Shabana Din ◽  
Natalie Gude ◽  
Mark A Sussman
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Time Course ◽  
Protein A ◽  
Cell Types ◽  
Tissue Homeostasis ◽  
Dominant Negative ◽  
Repair Capacity ◽  
Tissue Samples ◽  
The Impact

Rationale Cardiac stem cells (CSC) regulate cardiomyogenesis and support regenerative processes in the heart, but aging adversely affects stem cell repair capacity. Aging is a primary cause of impaired cardiac function characterized by accumulation of senescent cells. CSC senescence is associated with permanent growth arrest that decreases survival signaling and cellular replacement, inevitably diminishing the capacity of the heart to maintain tissue homeostasis. Therefore, promoting CSC growth may improve cardiac performance with age. Pim-1 kinase exhibits protective and proliferative effects in the myocardium but the role of Pim-1 in cardiac aging has not been thoroughly studied. Objective Demonstrate that Pim-1 promotes stem cell growth in the aged myocardium correlating with increased expression of centromere protein A (CENP-A), a kinetochore-associated protein known to support cell proliferation in numerous species and cell types. Methods & Results CENP-A expression levels were evaluated from murine myocardial tissue samples ranging in age from 11 days post coitum to 4 months of age with analysis by immunoblot as well as quantitative PCR. CENP-A expression was colocalized with c-kit as a marker of CSC by immunohistochemical labeling, revealing a decline in CENP-A expression over the time course of postnatal myocardial maturation. The impact of Pim-1 upon CENP-A level was assessed by comparative analysis of non-transgenic mice versus genetically modified transgenic mouse lines expressing either Pim-1 (wild type) or a dominant negative functionally dead Pim-1 mutant. Pim-1 overexpression increases persistence of CENP-A in CSCs with age, as well as the prevalence of cycling CSCs as marked by phosph-H3 expression, while the functionally dead mutant accelerates CENP-A diminution and decreases CSC proliferation. Conclusion CENP-A decline in c-kit positive cells with age provides intriguing evidence of a potential mechanism for the diminished capacity of CSCs to maintain tissue homeostasis. Pim-1 mitigates CENP-A diminution, demonstrating the promising potential of Pim-1 to promote cardiac growth and repair with age.

Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway

2013 ◽  
Vol 304 (2) ◽  
pp. C128-C136 ◽  
Author(s):  
Miriam Hoene ◽  
Heike Runge ◽  
Hans Ulrich Häring ◽  
Erwin D. Schleicher ◽  
Cora Weigert
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Myogenic Differentiation ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Wild Type ◽  
C2c12 Myoblasts ◽  
Sequence Of Events ◽  
Primary Mouse

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6−/− skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6−/− myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6−/− cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6−/− cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

Time-dependent platelet-vessel wall interactions induced by intestinal ischemia-reperfusion

2003 ◽  
Vol 284 (6) ◽  
pp. G1027-G1033 ◽  
Author(s):  
Dianne Cooper ◽  
Keith D. Chitman ◽  
Matthew C. Williams ◽  
D. Neil Granger
Keyword(s):  
Time Course ◽  
Platelet Adhesion ◽  
Ischemia Reperfusion ◽  
Time Dependent ◽  
Wild Type ◽  
Blocking Antibody ◽  
Low Levels ◽  
Intestinal Ischemia Reperfusion ◽  
Deficient Mice ◽  
Platelet Depletion

Platelets roll and adhere in venules exposed to ischemia-reperfusion (I/R). This platelet-endothelial adhesion may influence leukocyte trafficking because platelet depletion decreases I/R-induced leukocyte emigration. The objectives of this study were 1) to assess the time course of platelet adhesion in the small bowel after I/R and 2) to determine the roles of endothelial and/or platelet P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) in this adhesion. The adhesion of fluorescently labeled platelets was monitored by intravital microscopy in postcapillary venules exposed to 45 min of ischemia and up to 8 h of reperfusion. Peak platelet adhesion was observed at 4 h of reperfusion. To assess the contributions of platelet and endothelial cell P-selectin, platelets from P-selectin-deficient and wild-type mice were infused into wild-type and P-selectin-deficient mice, respectively. Platelets deficient in P-selectin exhibited low levels of adhesion comparable to that in sham-treated animals. In the absence of endothelial P-selectin, platelet adhesion was reduced by 65%. Treatment with a blocking antibody against PSGL-1 reduced adhesion by 57%. These results indicate that I/R induces a time-dependent platelet-endothelial adhesion response in postcapillary venules via a mechanism that involves PSGL-1 and both platelet and endothelial P-selectin, with platelet P-selectin playing a greater role.

Combined Carfilzomib and Selective PI3Kδ Inhibition (TGR1202) Results In Enhanced Myeloma Cell Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3224-3224
Author(s):  
Claire Torre ◽  
Yanyan Gu ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Feedback Loop ◽  
Myeloma Cell ◽  
Pi3k Signaling ◽  
Mtor Signaling Pathway ◽  
The Impact

Abstract Introduction The PI3K signaling pathway plays a vital role in regulating cell growth, proliferation and survival in multiple myeloma (MM) as well as in many other cancers. TGR-1202, an isoform-specific PI3Kδ inhibitor, with efficacy in preclinical models of hematologic malignancies, is currently in Phase I clinical development. In multiple myeloma, PI3K signaling appears to be very important for many extracellular signals, yet inhibition with -pan PI3K inhibitors have not exhibited significant activity. However, literature reports indicate that there are several MM cell lines that express PI3K -δ, and do appear to have differential sensitivity to specific isoform inhibition as opposed to pan PI3K inhibition. In this report, we sought to evaluate the effects of TGR-1202 alone and in combination with the proteasome inhibitor carfilzomib, with the intent of further understanding the mechanism of action and evaluating the impact of the combination. Methods Human myeloma cell lines (MM.1S, MY5, RPMI8226, U266, KMS11, ARH-77, OPM1, OPM2, LP1, JJN3 and L363) were treated with TGR-1202 alone, carfilzomib alone, or with the combination of TGR-1202 and carfilzomib. Annexin V/PI staining and Western blot were used to identify the cellular and molecular sequelae of the combination. Result 10 µM TGR-1202 alone did not cause significant cell death in the MM cell lines tested at 48 hours. When cells were treated with the combination of TGR-1202 and 3 nM carfilzomib, we observed enhanced apoptosis in all of the tested cell lines. In the U266 cell line, 3 nM carfilzomib and 10 µM TGR-1202 induced 16% and 14% cell apoptosis respectively. In the combination treatment, apoptosis increased to 75%. To explore the molecular mechanisms underlying the combination, we used a Western blot assay to evaluate the impact of the combination on the mTOR signaling pathway, a known reciprocal feedback loop when PI3K is blocked. TGR-1202 alone did not have an obvious effect on the mTOR signaling pathway, yet combining TGR-1202 with carfilzomib, significantly inhibited phospho-mTOR, suggesting total pathway blockade. Conclusion The combination of TGR-1202 with carfilzomib induces synergistic apoptosis in MM cell lines. Preliminary data suggests that this occurs through blockade of the entire reciprocal feedback loop of mTOR activation. Additional data from primary patient samples and underlying mechanisms will be presented at the meeting. These findings support the rationale for future clinical studies of TGR-1202, a selective PI3K-δ inhibitor in combination with the proteasome inhibitor carfilzomib. Disclosures: Boise: Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells

2001 ◽  
Vol 360 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Kirill KISELYOV ◽  
Dong Min SHIN ◽  
Nikolay SHCHEYNIKOV ◽  
Tomohiro KUROSAKI ◽  
Shmuel MUALLEM
Keyword(s):  
Time Course ◽  
Ryanodine Receptors ◽  
Cell Types ◽  
Ruthenium Red ◽  
General Mechanism ◽  
Inward Rectification ◽  
Wild Type ◽  
Ip3 Receptor ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dt40 Cells

Persistence of capacitative Ca2+ influx in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-deficient DT40 cells (DT40IP3R-/−) raises the question of whether gating of Ca2+-release activated Ca2+ current (Icrac) by conformational coupling to Ca2+-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of Icrac Ca2+ current in wild-type and DT40IP3R-/− cells. In both cell types passive depletion of internal Ca2+ stores by infusion of EGTA activated a Ca2+ current with similar characteristics and time course. The current was highly Ca2+-selective and showed strong inward rectification, all typical of Icrac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of Icrac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited Icrac activation in DT40IP3R-/− cells, even after complete depletion of intracellular Ca2+ stores by ionomycin. Wild-type and DT40IP3R-/− cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40IP3R-/− cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP3Rs and RyRs can efficiently gate Icrac in DT40 cells and explain the persistence of Icrac gating by internal stores in the absence of IP3Rs.

scholarly journals NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

2020 ◽  
Author(s):  
Qiao Zhang ◽  
Zhe Yang ◽  
Yueli Ni ◽  
Honggang Bai ◽  
Qiaoqiao Han ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Xenograft Model ◽  
Metabolic Reprogramming ◽  
Regulatory Function ◽  
Rt Pcr ◽  
Repressive Effect ◽  
The Impact

Abstract Background: Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods: The impact of ROS accumulation on NF-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NF-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NF-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NF-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NF-κB, pSTAT3, G6PD expression level and proliferation functions. Results: ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion: ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

scholarly journals Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis

2021 ◽  
Vol 11 ◽  
Author(s):  
Xin Yan ◽  
Sen Hu ◽  
Yan Yang ◽  
Da Xu ◽  
Wenxing Liu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Time Course ◽  
Nitrosative Stress ◽  
Brucella Melitensis ◽  
No Production ◽  
Wild Type ◽  
Twin Arginine Translocation ◽  
Raw264.7 Macrophages ◽  
Mutant Strains ◽  
Related Proteins

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

287 PORCINE ADIPOSE-DERIVED STEM CELLS IN CO-CULTURE FUSE ACTIVELY WITH MOUSE MYOTUBES AND EXPRESS MYOGENIC MARKERS

2013 ◽  
Vol 25 (1) ◽  
pp. 291
Author(s):  
S. Jain ◽  
D. J. Milner ◽  
M. Bionaz ◽  
J. A. Cameron ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Myogenic Differentiation ◽  
Primary Source ◽  
Cell Types ◽  
C2c12 Cells ◽  
Immunofluorescent Staining ◽  
C2c12 Myotubes ◽  
Adipose Derived Stem Cells ◽  
Myogenic Markers

Mesenchymal stem cells (MSC) have been shown to be useful in regenerative medicine with their capability to give rise to various different cell types, including osteoblasts, adipocytes, chondrocytes, muscle cells, and neurons. Among MSC, bone marrow-derived stem cells (BMSC) are considered the primary source. Recently, many studies have shown the regenerative capabilities of adipose-derived stem cells (ASC). The ASC, with their greater abundance and ease of harvest, provide clear advantages over BMSC. We have previously demonstrated the myogenic differentiation of porcine ASC when co-cultured with differentiating C2C12 myoblasts in a myogenic differentiation-promoting medium. In this study, we sought to examine the myogenic potential of porcine ASC when co-cultured with fully differentiated murine myotubes. For the present study, we used porcine ASC isolated from the back fat of a transgenic gree fluorescent protein (GFP)-expressing pig at passage 3. The ASC were added to mouse C2C12 myotube cultures that had been induced towards myogenesis for 72 h. As controls, we co-cultured, in the same conditions, GFP-expressing endothelial cells (ENDO) from the aorta of the same pig. Additionally, we cultured ASC, ENDO, and C2C12 cells alone in myogenic-differentiation medium. Cultures were harvested at 12, 24, and 48 h after addition of porcine cells to myotube cultures for measurement of mouse- or porcine-specific myogenic markers by quantitative RT-PCR and immunohistochemistry. We were able to observe fusion of ASC GFP-expressing cells with pre-formed mouse myotubes by detection of myotubes expressing GFP. Additionally, immunofluorescent staining of co-cultures with an antibody specific for porcine nuclear Lamin A demonstrated the presence of ASC nuclei incorporated into myotubes. We observed large increases in gene expression of porcine-specific myogenin (MYOG; >900-fold) and desmin (DES; 8-fold). Unexpectedly, ENDO in co-culture with myotubes also had increased expression of DES (4-fold) and MYOG (400-fold), possibly indicating their de-differentiation and adaptation to a myogenic phenotype. In addition, expression of mouse-specific DES and MYOG were boosted in C2C12 myotubes when co-cultured for 48 h compared with C2C12 alone, suggesting enhanced myogenesis or prolonged survival of myotubes in co-culture. Cultures of ASC and ENDO alone did not display increased expression of myogenic markers. These results provide support for the use of ASC for muscle regeneration strategies, as in the case of damaged muscles and muscular dystrophy. In addition, compared with our previous observations, where ASC were co-cultured with undifferentiated C2C12, the ASC co-cultured with myotubes appeared to have an enhanced fusion and expression of myogenic markers. Finally, the capacity of ENDO to fuse and actively expressed muscle-specific genes deserves further investigation.

scholarly journals Low-molecular-weight fucoidan regulates myogenic differentiation through the mitogen-activated protein kinase pathway in C2C12 cells

2011 ◽  
Vol 106 (12) ◽  
pp. 1836-1844 ◽  
Author(s):  
Kui-Jin Kim ◽  
Ok-Hwan Lee ◽  
Boo-Yong Lee
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Morphological Changes ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Mitogen Activated Protein Kinase ◽  
Low Molecular Weight ◽  
Mitogen Activated Protein ◽  
The Impact

Low-molecular-weight fucoidan (LMWF) has been broadly studied in recent years due to its numerous biological properties. Nevertheless, there have been no reports about the effects of LMWF on myogenic differentiation (MyoD). The objective of the present study was to demonstrate the impact of LMWF on myogenesis in C2C12 cells. The ultimate aim was to establish whether LMWF regulates myogenesis similar to heparin as a partial regulator of myogenesis. LMWF was prepared at a minimal size by ultra-filtration compared with crude fucoidan. We treated C2C12 cells with LMWF and/or heparin during MyoD. The data from the present study are the first to suggest that LMWF suppresses the expression of the myogenic regulatory factors and the myocyte enhancer factors as well as the morphological changes that occur during differentiation. Additionally, the expression of the mitogen-activated protein kinase (MAPK) family was significantly inhibited by LMWF when compared with controls. The LMWF-treated group showed significantly decreased expression of reactive oxygen species (ROS) enzymes compared with control cells. Heparin was used as a positive control because it has been reported to activate MyoD. Taken together, these results suggest that LMWF might regulate MyoD through the MAPK pathway and by regulating ROS activity in C2C12 cells.

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