Measurement of Cellular Adhesion Ratios Under Shear Flow on Various Substrates

2011 ◽  
Author(s):  
Ryo Shirakashi ◽  
Kiyoshi Takano ◽  
Christophe Provin ◽  
Yasuyuki Sakai ◽  
Teruo Fujii
Keyword(s):  
Tissue Culture ◽  
Shear Stress ◽  
Type I Collagen ◽  
Hepatoma Cell ◽  
Glass Plate ◽  
Perfusion Culture ◽  
Cellular Adhesion ◽  
Hepatoma Cell Line ◽  
Type I ◽  
Polystyrene Plate

Perfusion culture is an effective method to enhance the oxygen and nutrient mass transfer for the culture of highly metabolic cells and/or the culture at a high cell density. However, the flow rate of culture medium induces a shear stress that may lead to the death of cells if it is too high. In this study, we measured the cellular adhesion ratio on various materials coated with type-I collagen under Poiseuille flow with flow rates in the range 1–21 mL/min. Hepatoma cell line, HepG2 cells, attached better on a polystyrene plate for tissue culture coated with type-I collagen (with τ0.5, the shear stress required to detach 50% of cells, equal to 42.2 Pa) followed by a collagen coated glass plate (τ0.5 of 40.5Pa), then a polystyrene plate for tissue culture without collagen coating (τ0.5 of 33.8Pa), and finally on a PDMS (τ0.5 of 24.8Pa) plate coated with collagen. The fluorescence staining of the collagen suggests that clumps of cells and collagen were detached from the surface, which implies that the cell-collagen bonds are stronger than collagen-substrate bonds. Accounting these results, it can be concluded that by reinforcing the bonds between collagen and substrate, it might be possible for the cellular monolayer to stay attached on the substrate until τ0.5 reaches ∼40Pa. This conclusion suggests the importance of carefully choosing the cell substrate, which has a strong binding with the coated extracellular matrix, for the cell culture under a high shear stress.

Changes in the transferrin requirement of cultured chick embryo mesoderm cells during early differentiation

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 81-93
Author(s):  
E. J. Sanders
Keyword(s):  
Chick Embryo ◽  
Type I Collagen ◽  
Specific Effect ◽  
Primitive Streak ◽  
Cellular Adhesion ◽  
Type I ◽  
Surface Binding ◽  
Surface Receptors ◽  
Early Differentiation ◽  
Species Specific

Mesodermal tissue from the chick embryo at various stages of early differentiation was cultured in hydrated gels of type I collagen in the presence and absence of transferrin. Primary mesoderm explants from primitive-streak-stage embryos responded to the presence of avian transferrin by significantly improved outgrowth which appeared to be related to the ability of the cells to attach to, and migrate in, the collagen. No evidence was obtained which suggested that this observation was dependent on increased cell proliferation. This outgrowth enhancement was not duplicated by transferrin of human origin. The avian transferrin did not produce this effect on cells cultured on plastic substrata, suggesting that the species-specific effect involves modulation by the extracellular matrix. Mesoderm explants from somite stages of development showed no increase in outgrowth in the presence of either avian or human transferrin as judged by counting the number of outwandering cells. Ultrastructural immunocytochemistry indicated surface binding of transferrin by cells in the gels, and the presence of endogenous transferrin on the surfaces of mesoderm cells in situ and in their extracellular environment. It is suggested that by binding to cell surface receptors, transferrin may be able to influence the strength of cellular adhesion to collagen and hence the capacity for cell locomotion.

scholarly journals Biomimetic Properties of Force-Spun PHBV Membranes Functionalised with Collagen as Substrates for Biomedical Application

Coatings ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 350 ◽  
Author(s):  
Kegan McColgan-Bannon ◽  
Sarah Upson ◽  
Piergiorgio Gentile ◽  
Muhammad Tausif ◽  
Stephen Russell ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Biomedical Application ◽  
Cell Metabolism ◽  
Polymer Concentration ◽  
Cell Activity ◽  
Cellular Adhesion ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Appreciable Change ◽  
Adhesion Properties

The force-spinning process parameters (i.e., spin speed, spinneret-collector distance, and polymer concentration), optimised and characterised in previous work by this group, allowed the rapid fabrication of large quantities of high surface area poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) polymeric fibre membranes. This paper examined the potential application for force-spun PHBV fibres functionalised with type I collagen for tissue regeneration applications. PHBV fibre scaffolds provide a biologically suitable substrate to guide the regeneration of dermal tissues, however, have poor cellular adhesion properties. The grafting of collagen type-I to PHBV fibres demonstrated improved cell adhesion and growth in Neo-NHDF (neonatal human dermal fibroblasts) fibroblasts. The examination of fibre morphology, thermal properties, collagen content, and degradability was used to contrast the physicochemical properties of the PHBV and PHBV-Collagen fibres. Biodegradation models using phosphate buffered saline determined there was no appreciable change in mass over the course of 6 weeks; a Sirius Red assay was performed on degraded samples, showing no change in the quantity of collagen. Cell metabolism studies showed an increase in cell metabolism on conjugated samples after three and 7 days. In addition, in vitro cytocompatibility studies demonstrated superior cell activity and adhesion on conjugated samples over 7 days.

Shear stress enhances human endothelial cell wound closure in vitro

2000 ◽  
Vol 279 (1) ◽  
pp. H293-H302 ◽  
Author(s):  
Maria Luiza C. Albuquerque ◽  
Christopher M. Waters ◽  
Ushma Savla ◽  
H. William Schnaper ◽  
Annette S. Flozak
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Wound Closure ◽  
Type I Collagen ◽  
Umbilical Vein ◽  
Type I ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Closure Rate

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm2 to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm2 under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm2 closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.

scholarly journals Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain

2005 ◽  
Vol 73 (9) ◽  
pp. 5402-5409 ◽  
Author(s):  
Babita Mahajan ◽  
Dewal Jani ◽  
Rana Chattopadhyay ◽  
Rana Nagarkatti ◽  
Hong Zheng ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Plasmodium Knowlesi ◽  
Evaluation Studies ◽  
Hepatoma Cell ◽  
Plasmodium Yoelii ◽  
Host Cells ◽  
Hepatoma Cell Line ◽  
Type I ◽  
Efficacy Evaluation ◽  
Repeat Domain

ABSTRACT Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias.

scholarly journals Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges

PLoS ONE ◽  
2016 ◽  
Vol 11 (9) ◽  
pp. e0161479 ◽  
Author(s):  
Nathalie Mayer ◽  
Silvia Lopa ◽  
Giuseppe Talò ◽  
Arianna B. Lovati ◽  
Marielle Pasdeloup ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Perfusion Culture ◽  
Type I ◽  
Clinical Grade ◽  
Soluble Factors ◽  
Collagen Production ◽  
Osteoarthritic Chondrocytes

scholarly journals Development of a Hybrid Polymer-Based Microfluidic Platform for Culturing Hepatocytes towards Liver-on-a-Chip Applications

Polymers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 3215
Author(s):  
Gulsim Kulsharova ◽  
Akbota Kurmangaliyeva ◽  
Elvira Darbayeva ◽  
Luis Rojas-Solórzano ◽  
Galiya Toxeitova
Keyword(s):  
Cell Line ◽  
Volume Fraction ◽  
Hepatoma Cell ◽  
Perfusion Culture ◽  
Bottom Layer ◽  
Hepatoma Cell Line ◽  
Dynamics Modeling ◽  
Microfluidic Platform ◽  
Huh7 Cells

The drug development process can greatly benefit from liver-on-a-chip platforms aiming to recapitulate the physiology, mechanisms, and functionalities of liver cells in an in vitro environment. The liver is the most important organ in drug metabolism investigation. Here, we report the development of a hybrid cyclic olefin copolymer (COC) and polydimethylsiloxane (PDMS) microfluidic (HCP) platform to culture a Huh7 hepatoma cell line in dynamic conditions towards the development of a liver-on-a-chip system. The microfluidic platform is comprised of a COC bottom layer with a microchannel and PDMS-based flat top layer sandwiched together. The HCP device was applied for culturing Huh7 cells grown on a collagen-coated microchannel. A computational fluid dynamics modeling study was conducted for the HCP device design revealing the presence of air volume fraction in the chamber and methods for optimizing experimental handling of the device. The functionality and metabolic activity of perfusion culture were assessed by the secretion rates of albumin, urea, and cell viability visualization. The HCP device hepatic culture remained functional and intact for 24 h, as assessed by resulting levels of biomarkers similar to published studies on other in vitro and 2D cell models. The present results provide a proof-of-concept demonstration of the hybrid COC–PDMS microfluidic chip for successfully culturing a Huh7 hepatoma cell line, thus paving the path towards developing a liver-on-a-chip platform.

Filamin Binding to Glycoprotein Ibα Signals Talin-Directed α IIbβ 3 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 383-383
Author(s):  
Shuju Feng ◽  
Xin Lu ◽  
Michael H. Kroll
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Wall Shear Stress ◽  
Type I Collagen ◽  
Thrombus Formation ◽  
Cytoplasmic Domain ◽  
Platelet Glycoprotein ◽  
Type I ◽  
Wall Shear

Abstract von Willebrand factor (VWF) binding to platelet glycoprotein (Gp) Ib-IX-V triggers platelet activation. Under conditions of pathologically elevated arterial wall shear stress, VWF-dependent platelet adhesion is coupled to aggregation and thrombus formation principally through GpIbα-induced signaling to α IIbβ 3. To elucidate the mechanism of GpIbα signaling to α IIbβ 3, we have examined molecular interactions involving structural proteins that bind to the cytoplasmic domains of GpIbα and β 3. In CHO cells co-expressing human GpIb-IX and α IIbβ 3, the activation of α IIbβ 3 as reported by monoclonal antibody PAC-1 binding is stimulated by ristocetin (1 mg/ml) + purified human VWF(5 μg/ml). When filamin binding to the cytoplasmic domain of GpIbα is eliminated by deleting GpIbα residues 560-570, PAC-1 binding is eliminated. When human platelets in reconstituted whole blood are treated with a peptide that interferes with filamin binding to GpIbα (as reported in Blood2003;102:2122–2129), shear-dependent (1500 sec−1 shear rate or 60 dynes/cm2 shear stress) platelet deposition onto bovine type I collagen is inhibited. In washed resting platelets and platelets activated by 120 dynes/cm2 shear stress, filamin co-immunoprecipitates with both GpIbα and α IIbβ 3; only its association with β 3 is eliminated by DNaseI (1 mg/ml), demonstrating that filamin binds indirectly to α IIbβ 3 through other cytoskeletal elements. One such element is observed to be talin, which co-immunoprecipitates with filamin, α IIbβ 3 and small amounts of GpIbα in resting platelets. When platelets are sheared for two minutes at 120 dynes/cm2, talin’s DNaseI-resistant association with filamin is decreased and its DNaseI-sensitive association with α IIbβ 3 is increased. These changes are prevented when shear-dependent VWF binding to GpIb-IX-V is blocked by monoclonal antibody AK2. Shear-dependent VWF binding to GpIb-IX-V also results in the proteolysis of talin, which is considered to be one mechanism by which the N-terminal head domain of talin regulates α IIbβ 3 activation. Shear-dependent talin proteolysis is not affected by blocking VWF binding to α IIbβ 3 with a RGD peptide. These results demonstrate that the cytoplasmic domain of GpIbα transduces signals to activate α IIbβ 3 through its interactions with filamin. These signals depend only on VWF binding to GpIb-IX-V. Under pathologically elevated wall shear stress in vitro, the mechanism of signaling may be the release of talin by filamin, thus permitting the proteolysis of talin and enhancing talin’s interaction with α IIbβ 3.

scholarly journals Dissecting the Mechanisms of Hepcidin and BMP-SMAD Pathway Regulation By FKBP12

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2008-2008
Author(s):  
Alessia Pagani ◽  
Mariateresa Pettinato ◽  
Alessandro Dulja ◽  
Silvia Colucci ◽  
Mariam Aghajan ◽  
...  
Keyword(s):  
Hepatoma Cell ◽  
Hereditary Hemochromatosis ◽  
Hepatoma Cell Line ◽  
Main Role ◽  
Type I ◽  
Primary Hepatocytes ◽  
Smad Pathway ◽  
Smad Signaling

Abstract The BMP-SMAD pathway is activated when a dimeric ligand (BMP) interacts with a dimeric serine threonine kinase receptor (BMPRII) and triggers the activation of a dimeric BMP type I receptor (BMPRI). Catalytically active BMPRIs phosphorylate SMAD1/5/8 that, upon SMAD4 binding, translocate to the nucleus to regulate the expression of BMP target genes, including hepcidin. Hepcidin is the main regulator of iron homeostasis that controls body iron levels by binding and blocking the sole iron exporter ferroportin. In agreement, hepcidin expression is homeostatically activated by serum and liver iron, and its deficiency is a common hallmark of Hereditary Hemochromatosis (HH) and the major cause of iron overload in beta thalassemia. The components of the BMP-SMAD pathway relevant for hepcidin regulation are ALK2 and ALK3 (BMPRI); BMPR2 and ACVR2A (BMPRII), BMP2 and BMP6 (BMP ligands). Recently, we have identified the immunophilin FKBP12 as an inhibitor of hepcidin and demonstrated that FKBP12 binds ALK2 to avoid ligand-independent activation of the BMP-SMAD pathway. To investigate the mechanism of BMP-SMAD pathway and hepcidin regulation by FKBP12, we performed in vitro, ex vivo and in vivo studies. We found that FKBP12 sequestration by the immunosuppressive drug Tacrolimus (TAC) stabilizes ALK2-ALK2 homodimers and ALK2-ALK3 heterodimers in a transfected human hepatoma cell line. In addition, it increases the interaction of ALK2 with ACVR2A and BMPR2. To investigate the role of FKBP12 on BMP-SMAD signaling, BMPRI and II were silenced in murine primary hepatocytes. Despite FKBP12 co-immunoprecipitates only with ALK2, silencing of Alk2 and Alk3 completely blunts TAC-mediated BMP-SMAD pathway activation, suggesting that FKBP12 functionally interacts also with ALK3. Acvr2a silencing impairs TAC-dependent hepcidin upregulation, whereas Bmpr2 silencing does not. As expected, Fkbp12 silencing abrogates hepcidin upregulation by TAC, confirming the main role of this immunophilin in hepcidin regulation. In vivo, TAC treatment upregulates hepcidin in wild type and HH mouse models, but surprisingly, Fkbp12 mRNA downregulation by ASOs does not. Interestingly, Fkbp 2, 4 and 8 are highly expressed in murine hepatocytes and, according to literature data, are able to bind to TAC. Of note, Fkbp12 is the least expressed immunophilin in murine primary hepatocytes. To further investigate the FKBPs involved in TAC-dependent hepcidin regulation, Fkbp2, 4 and 8 were knockdown in murine primary HCs that were then treated with TAC. The TAC effect is preserved in siFkbp2- and siFkbp4-derived HCs, but abolished when Fkbp8 was downregulated. Overall these data suggest that: 1) FKBP12 regulates BMP-SMAD signaling by favoring ALK2-ALK3 homo and heterodimerization, and interaction with BMPRII in the absence of ligands; 2) TAC-mediated hepcidin upregulation is dependent upon ALK2, ALK3, ACVR2A, FKBP12 and FKBP8. 3) In vivo, TAC treatment upregulates hepcidin whereas Fkbp12 silencing does not, suggesting the existence of redundancy between the different FKBPs. Further studies are needed to dissect the role of FKBP8 in BMP-SMAD pathway and hepcidin regulation. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Muckenthaler: Silence Therapeutics: Research Funding. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

scholarly journals Attachment of Human Endothelial Cells to Polyester Vascular Grafts: Pre-Coating With Adhesive Protein Assemblies and Resistance to Short-Term Shear Stress

2014 ◽  
pp. 167-177 ◽  
Author(s):  
J. CHLUPÁČ ◽  
E. FILOVÁ ◽  
T. RIEDEL ◽  
M. HOUSKA ◽  
E. BRYNDA ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Type I Collagen ◽  
Type I ◽  
Laminar Shear Stress ◽  
Protein Assemblies ◽  
Vascular Prostheses ◽  
Thrombotic Risk ◽  
Adhesive Protein ◽  
Fibrin Network

Cardiovascular prosthetic bypass grafts do not endothelialize spontaneously in humans, and so they pose a thrombotic risk. Seeding with cells improves their performance, particularly in small-caliber applications. Knitted tubular polyethylene-terephthalate (PET) vascular prostheses (6 mm) with commercial type I collagen (PET/Co) were modified in the lumen by the adsorption of laminin (LM), by coating with a fibrin network (Fb) or a combination of Fb and fibronectin (Fb/FN). Primary human saphenous vein endothelial cells were seeded (1.50 × 105/cm2), cultured for 72 h and exposed to laminar shear stress 15 dyn/cm2 for 40 and 120 min. The control static grafts were excluded from shearing. The cell adherence after 4 h on PET/Co, PET/Co +LM, PET/Co +Fb and PET/Co +Fb/FN was 22 %, 30 %, 19 % and 27 % of seeding, respectively. Compared to the static grafts, the cell density on PET/Co and PET/Co +LM dropped to 61 % and 50 %, respectively, after 120 min of flow. The cells on PET/Co +Fb and PET/Co +Fb/FN did not show any detachment during 2 h of shear stress. Pre-coating the clinically-used PET/Co vascular prosthesis with LM or Fb/FN adhesive protein assemblies promotes the adherence of endothelium. Cell retention under flow is improved particularly on fibrin-containing (Fb and Fb/FN) surfaces.

Hepcidin Expression Is Regulated by a Complex of Hemochromatosis-Associated Proteins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 267-267 ◽  
Author(s):  
Paul J. Schmidt ◽  
Franklin W. Huang ◽  
Diedra M. Wrighting ◽  
Paul T. Toran ◽  
Nancy C. Andrews
Keyword(s):  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Hepatoma Cell ◽  
Bmp Signaling ◽  
Hepatoma Cell Line ◽  
Type I ◽  
Mutant Form ◽  
Iron Release ◽  
Hepcidin Expression ◽  
Bmp Receptors

Abstract Hemochromatosis is a common genetic disease resulting from increased dietary iron absorption and tissue iron deposition. Mutations in five unrelated genes are known to cause hemochromatosis in humans and mice. These encode the classic hemochromatosis protein (HFE), transferrin receptor 2 (TFR2), the iron exporter ferroportin (FPN), hemojuvelin (HJV), and the circulating anti-microbial peptide hepcidin (HAMP). Hepcidin binds to FPN, causing its internalization and degradation, thus decreasing cellular iron release. A basic understanding of the pathophysiology of FPN and hepcidin mutations has recently been elucidated; however, it was still unclear how mutations in HFE, TFR2, and HJV cause hemochromatosis. All are associated with decreased hepcidin production and inappropriately high levels of ferroportin activity. HFE, TFR2 and HJV are normally expressed in the hepatic cells that produce hepcidin. With collaborators, we showed that HJV acts as a bone morphogenetic protein (BMP) co-receptor. HJV binds to the BMP ligands and forms a complex with Type I BMP receptors, resulting in signaling through a SMAD pathway and induction of hepcidin expression. Disease causing mutations in HJV abrogate BMP co-receptor activity, and hepatocytes from Hjv−/ − mice have a blunted response to BMP2. HFE was known to form a complex with the classical transferrin receptor, TFR1. Several models have been proposed implicating this complex in the regulation of normal iron homeostasis, but they have not taken the role of hepcidin into account. To examine the HFE/TFR1 interaction in vivo, we developed mice expressing a mutant form of TFR1 that should constitutively interact with HFE. We found that these transgenic animals have a phenotype similar to Hfe−/ − mice, suggesting that TFR1 serves to sequester HFE to silence its activity. We next asked whether HFE might also participate in BMP signaling. We found that forced expression of HFE in a hepatoma cell line induces transcription of a reporter gene linked to the hepcidin promoter. It also induces transcription from a heterologous promoter containing BMP-responsive elements, suggesting that HFE works through the BMP pathway. In contrast, forced expression of TFR2 did not amplify expression of either reporter, but it prevented cellular release of a soluble cleavage product of HJV. Furthermore, we showed that both HFE and TFR2 are associated with HJV in a stable protein complex that can be isolated by co-immunoprecipitation or Ni-affinity chromatography. TFR2 appears to aid in the recruitment of HFE to this complex. We conclude that HFE and TFR2 thus serve to amplify BMP signaling through an HJV/BMP receptor pathway. Our findings provide a compelling explanation for the similar clinical hemochromatosis phenotypes resulting from mutations in these genes.

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