Null Mutation of Pleckstrin2, a PI3K Effector, Impairs Platelet Secretion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3522-3522
Author(s):  
Yanfeng Wang ◽  
Lurong Lian ◽  
Charles S. Abrams
Keyword(s):  
Cell Membrane ◽  
Inositol Phosphates ◽  
Cell Spreading ◽  
Lipid Binding ◽  
Jurkat Cells ◽  
Null Mutation ◽  
Filamentous Actin ◽  
Dense Granules ◽  
Ph Domains ◽  
Platelet Secretion

Abstract Pleckstrin-2, a widely expressed paralog of pleckstrin, is composed of two Pleckstrin Homology (PH) domains and Disheveled-Egl 10-Pleckstrin (DEP) domain. Although the activity of pleckstrin is regulated by its’ phosphorylation state, pleckstrin-2 is not a phospho-protein suggesting that it possesses a different mechanism of regulation. Previous reports have shown that many PH domains mediate protein binding to inositol phosphates and phospholipids, thus regulating protein function. Therefore, we speculated that localized production of specific polyphosphatidylinositols might bind, and activate, pleckstrin-2. Using a lipid-binding assay, we found that pleckstrin-2 bound with greatest affinity to the products of phosphatidylinositol 3-kinase and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bound the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. GFP-tagged pleckstrin-2 had a cytoplasmic distribution in non-adherent Jurkat cells, but through a pathway dependent on the phospholipid-binding pocket of its PH domains, rapidly moved to the cell membrane following adhesion to immobilized fibronectin. Once bound to the cell membrane, pleckstrin-2 enhanced Jurkat cell spreading 2-fold and increased membrane ruffling. The membrane association of pleckstrin-2, and its resultant cell spreading, were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. To investigate the role of this protein within platelets, we generated mice containing a null mutation within the pleckstrin-2 gene. Pleckstrin-2 null mice were born at the expected frequency, and had no overt spontaneous hemorrhagic events. Mice lacking pleckstrin-2 had normal platelet counts and morphologic appearance of their megakaryocytes. Following stimulation of the PAR-4 (thrombin) receptor, pleckstrin-2 knockout platelets displayed normal assembly of filamentous actin. However, pleckstrin-2 null platelets had impaired aggregation following stimulation by collagen or submaximal doses of the PAR-4 activating peptide. Since pleckstrin-2 deficient platelets aggregated normally in response to ADP, these results suggested that these platelets might have an impaired ability to secrete dense granules. Accordingly, we found that pleckstrin-2 null platelets had a defect in their ability to secrete ATP in response to stimulation by 5μM collagen or 200μM of the PAR-4 activating peptide. However, pleckstrin-2 knockout platelets did incorporate 14C-serotonin as efficiently as wild type platelets. This latter observation suggested that the secretion defect in pleckstrin-2 null platelets was not attributable to a deficiency of dense granules, but instead is due to a defect in exocytosis of granules. Together, these data suggest that the PH domains of pleckstrin-2 cooperatively bind PI3K generated phospholipids on the cell membrane, and help mediate platelet secretion.

PI3K Regulates Pleckstrin-2 in T-Cell Cytoskeletal Re-Organization.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3305-3305
Author(s):  
Tami L. Bach ◽  
Wesley T. Kerr ◽  
Charles S. Abrams
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Membrane Trafficking ◽  
Inositol Phosphates ◽  
Point Mutations ◽  
Cell Spreading ◽  
Lipid Binding ◽  
Membrane Association ◽  
Ph Domains

Abstract Pleckstrin-2, a paralog of pleckstrin-1, is composed of two Pleckstrin Homology (PH) domains and a Disheveled-Egl 10-Pleckstrin (DEP) domain. Several studies have shown that PH domains mediate binding of their host proteins to inositol phosphates and phospholipids, and thus regulate protein function. PH domains are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. Proteins containing the DEP domain also regulate a broad range of cellular functions and evidence is emerging that several signaling proteins may rely on their DEP domains for membrane association. We speculated that the function of pleckstrin-2 is dependent upon its ability to bind to specific polyphosphatidylinositols. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to the products of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bind to the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. To examine the effect of pleckstrin-2 in human T-cells, Jurkat T-cells were transfected with GFP-tagged plasmids that direct the expression of pleckstrin-2 variants. Using confocal video microscopy we demonstrated that upon activation of the T-cell antigen receptor or the integrin α4β1, pleckstrin-2 rapidly moves from the cytoplasm to the cellular membrane and enhances membrane ruffling. Quantitation of cell footprint size revealed a two-fold increase in cell spreading. Furthermore, the membrane association of pleckstrin-2 and its resultant cell spreading were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. Consistent with this observation, a pleckstrin-2 variant containing point mutations in both of its PH domains failed to associate with the cell membrane and had no effect on spreading under the same conditions, suggesting that pleckstrin-2 membrane association occurs through a pathway dependent on the phospholipid-binding pocket of its PH domains. Although still membrane-bound, a pleckstin-2 variant containing point mutations in the second β-turn and second α-helical coil of the DEP domain demonstrated a decreased ability of pleckstrin-2 to induce membrane ruffles and lamellipodia, without decreasing filopodia formation. The cell footprint size of the DEP domain mutants was also decreased compared to that of wild type pleckstrin-2. These results suggest that the pleckstrin-2 DEP domain may function to promote actin-rich membrane extensions and ruffling. The localization of receptors, signaling intermediates, and cytoskeletal components at the T-cell/APC interface is thought to be a major determinant of efficient T-cell activation. Our data indicate that in T-lymphocytes, pleckstrin-2 uses modular motifs to bind to membrane-associated phosphatidylinositols, such as those generated by PI3K, to organize the actin cytoskeleton and to promote lymphocyte spreading.

Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts

1995 ◽  
Vol 269 (5) ◽  
pp. C1093-C1104 ◽  
Author(s):  
M. Glogauer ◽  
J. Ferrier ◽  
C. A. McCulloch
Keyword(s):  
Cell Membrane ◽  
Magnetic Force ◽  
Calcium Influx ◽  
Single Cells ◽  
Cell Spreading ◽  
Calcium Flux ◽  
Filamentous Actin ◽  
Physical Force ◽  
Physical Stimuli ◽  
Applied Forces

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

scholarly journals Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 869-877 ◽  
Author(s):  
Qiansheng Ren ◽  
Christian Wimmer ◽  
Michael C. Chicka ◽  
Shaojing Ye ◽  
Yi Ren ◽  
...  
Keyword(s):  
Wild Type Mouse ◽  
Limiting Factor ◽  
Attachment Protein ◽  
Altered Expression ◽  
Dense Granules ◽  
Protein Receptors ◽  
Snare Regulators ◽  
Protein Attachment ◽  
Granule Release ◽  
Platelet Secretion

Abstract Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13dJinx mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from α-granules and lysosomes was severely compromised. Unc13dJinx platelets showed attenuated aggregation and, consequently, Unc13dJinx mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13dJinx platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13dJinx platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis.

Abstract 583: Characterization of Cell Membrane Microdomains Facilitating HDL Biogenesis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Hong Choi ◽  
Isabelle Ruel ◽  
Rui Hao Leo Wang ◽  
Jacques Genest
Keyword(s):  
Cell Membrane ◽  
Cell Surface ◽  
Protease Inhibitors ◽  
Binding Proteins ◽  
Lipid Binding ◽  
Cholesterol Homeostasis ◽  
Scaffolding Protein ◽  
Membrane Microdomains ◽  
Discontinuous Sucrose Gradient ◽  
Lipid Binding Proteins

High-density lipoprotein (HDL) particles, generated in the process of removing excess cellular cholesterol, play crucial roles in maintaining cholesterol homeostasis in arterial cells and in protecting the cardiovascular system from the development of atherosclerosis. Cholesterol-loaded cells increase their binding capacity to the HDL scaffolding protein, apolipoprotein A-I (ApoA-I), however, cell surface factors necessary for ApoA-I binding remains to be elucidated. To characterize cell membrane microdomains interacting with ApoA-I, primary human skin fibroblasts were incubated with ApoA-I for 1h at 4°C. After linking protein-protein interactions with a membrane-impermeable crosslinker, DTSSP, cells were subjected to homogenization. The cell homogenate was separated by a discontinuous sucrose gradient centrifugation and ten fractions were collected. ApoA-I-associated cell membrane fraction was located by immunoblotting for ApoA-I and organelle markers. Membrane-containing fractions were fragmented using sonication prior to immunoprecipitation of ApoA-I-associated microdomains using an anti-ApoA-I antibody. Major lipid classes present in the microdomains are phosphatidylcholine, phosphatidylserine, sphingomyelin and cholesterol. Two cell membrane proteins, caveolin and ABCA1, were excluded from the microdomains. These data suggest that ApoA-I bind to cholesterol-rich cell surface microdomains that are different from ABCA1 and caveolae domains. LC-MS/MS analysis identified the presence of 26 proteins in the microdomains. Among these, several desmosomal proteins, lipid binding proteins and protease inhibitors were identified. Overall, our results suggest that the initial binding of ApoA-I to cell surface occurs on the lateral sides of cell membranes where desmosomal proteins provide a binding site for ApoA-I, and that lipid binding proteins facilitate lipidation of ApoA-I while protease inhibitors protect ApoA-I and related proteins from degradation. In conclusion, we established a new method to isolate cell membrane microdomains interacting with ApoA-I. Using this method, we found that ApoA-I associates with desmosomal proteins for the formation of HDL.

scholarly journals HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy

2020 ◽  
Vol 295 (45) ◽  
pp. 15183-15195 ◽  
Author(s):  
Amanda E. Ward ◽  
Volker Kiessling ◽  
Owen Pornillos ◽  
Judith M. White ◽  
Barbie K. Ganser-Pornillos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Membrane Fusion ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Lipid Binding ◽  
Plasma Membrane Vesicles ◽  
Tirf Microscopy ◽  
Host Restriction ◽  
Whole Cells

To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.

scholarly journals Lipid utilization by human lymphocytes is correlated with high-density-lipoprotein binding site activity

1992 ◽  
Vol 285 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Q Xu ◽  
G Jürgens ◽  
L A Huber ◽  
G Böck ◽  
H Wolf ◽  
...  
Keyword(s):  
Cell Membrane ◽  
High Density Lipoprotein ◽  
Binding Site ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Density Lipoprotein ◽  
Lipid Binding ◽  
High Density ◽  
Cell Protein ◽  
Significant Difference

The nature and physiological importance of high-density lipoprotein (HDL) binding sites on unstimulated (resting) and mitogen-activated (blast) human peripheral blood lymphocytes were investigated. Specific HDL binding on resting and blast T-lymphocytes was saturable at 50 micrograms of 125I-HDL/ml and of high affinity, with Kd values of 8.1 x 10(-8) M and 6.5 x 10(-8) M, respectively, and Bmax. values of 79 ng and 180 ng/mg of cell protein respectively at 4 degrees C. Binding of HDL double-labelled with fluorescent dioctadecylindocarbocyanine (Dil) and isotope (125I) as well as of single fluorescence- or isotope-labelled HDL was inhibited competitively by HDL apoproteins. Studies of the cholesterol flux between the cells and HDL showed that HDL, low-density lipoprotein (LDL) or BSA at a concentration of 100 micrograms/ml in the tissue culture medium did not result in a significant difference in exogenous [3H]cholesterol efflux from the cell membrane at 37 degrees C. Proliferating T-blasts incorporated more cholesterol from HDL or LDL than did resting lymphocytes. When the cells were pulsed with 125I-HDL and chased in fresh lipid-free medium, up to 80% of the radioactivity released was not precipitable with trichloroacetic acid. This percentage decreased in a competitive manner when unlabelled HDL was present in the chase incubation medium. Finally, cultivation of lymphocytes with conditioned medium from macrophages increased Dil-HDL binding/uptake, while it was decreased by mevinolin-induced inhibition of hydroxymethylglutaryl-coA reductase. In conclusion, human lymphocytes possess a HDL binding site (receptor) responsible for lipid binding/uptake and concomitant internalization and degradation of apoproteins from HDL, but not for reverse cell membrane cholesterol transport. The activity of the binding site is up-regulated during cell proliferation and down-regulated during cell growth suppression.

DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2200-2200
Author(s):  
Guoying Zhang ◽  
Binggang Xiang ◽  
Ye Shaojing ◽  
Magdalena Chrzanowska-Wodnicka ◽  
Andrew J. Morris ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Wild Type ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Dense Granules ◽  
Activation Mechanisms ◽  
Platelet Spreading ◽  
Inside Out ◽  
Platelet Secretion

Abstract Abstract 2200 Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

Trogocytosis in Multiple Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1681-1681
Author(s):  
Ross D. Brown ◽  
Karieshma Kabani ◽  
Shihong Yang ◽  
Aklilu Esther ◽  
Phoebe Joy Ho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Membrane ◽  
Plasma Cells ◽  
Costimulatory Molecules ◽  
Jurkat Cells ◽  
Cell Contact ◽  
Immune Recognition ◽  
Tumor Escape

Abstract Trogocytosis is the transfer of cell membrane material from one cell to another during short term cell-cell contact. Recent studies have suggested that the transfer of cell membrane patches across the immunological synapse is at least partly responsible for immune tolerance, tumor escape and the production of adaptive T regulatory cells (Tregs). We sought to identify the extent of tumor-related trogocytosis in patients with multiple myeloma and have developed an in vitro model for further study. Trogocytosis in patients with multiple myeloma was demonstrated by flow cytometry, immunohistochemistry, confocal microscopy, lack of mRNA expression and by the failure to upregulate costimulatory molecules in vitro after stimulation by IL2 and huCD40LT. Of the costimulatory molecules CD80, CD86, B7-H1, B7-H3 and PD-L2, only CD80 and CD86 showed significant transfer to T cells. Increased CD80 expression was found on the T cells of 9% of patients and CD86 on 13% of patients (n= 95). Both CD4 and CD8 memory (CD45RO+) cells were involved but not naïve T cells (CD45RA+). HLA-G expression was found on less than 1% of T cells in 69/70 different myeloma blood samples. Following biotinylation of plasma cells (CD38++) using an in vitro culture model, trogocytosis was demonstrated in up to 36% of T cells. The presence of trogocytosis on CD3- TCRαβ-mutant Jurkat cells (J.RT3-T3.3) and on normal T cells with HLA incompatibility infers that trogocytosis is independent of immune recognition and TCR engagement. In vitro stimulation with IL-2 and huCD40LT and mRNA studies showed that T cells acquire CD80 and CD86 cell surface antigen but unlike B cells do not produce CD80 and CD86 mRNA (n=5) and cannot be stimulated to express CD80 and CD86 (n=6). Trogocytosis was not evident in age-matched control lymphocytes but could be induced in normal lymphocytes in vitro after exposure to malignant plasma cells. Trogocytosis is common in patients with multiple myeloma and involves the transfer of costimulatory molecules and other cell membrane proteins from malignant plasma cells to T cells. Tumor-induced trogocytosis may be a common cause of the failure of cytotoxic T cells and provide a mechanism of tumor escape.

scholarly journals Structural Refinement of BAR-PH Domains Remodeling Cell Membrane Using MDFF

2016 ◽  
Vol 110 (3) ◽  
pp. 537a-538a
Author(s):  
Chun Chan ◽  
Xiaoyun Pang ◽  
Yan Zhang ◽  
Victor W. Hsu ◽  
Fei Sun ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Structural Refinement ◽  
Ph Domains

scholarly journals Computational Analysis of the Binding Specificities of PH Domains

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Zhi Jiang ◽  
Zhongjie Liang ◽  
Bairong Shen ◽  
Guang Hu
Keyword(s):  
Inositol Phosphates ◽  
Structural Aspect ◽  
Genomic Research ◽  
Ph Domain ◽  
Domain Structures ◽  
Ph Domains ◽  
Cellular Signal ◽  
Structure Relationship ◽  
Insight Into ◽  
Structure Properties

Pleckstrin homology (PH) domains share low sequence identities but extremely conserved structures. They have been found in many proteins for cellular signal-dependent membrane targeting by binding inositol phosphates to perform different physiological functions. In order to understand the sequence-structure relationship and binding specificities of PH domains, quantum mechanical (QM) calculations and sequence-based combined with structure-based binding analysis were employed in our research. In the structural aspect, the binding specificities were shown to correlate with the hydropathy characteristics of PH domains and electrostatic properties of the bound inositol phosphates. By comparing these structure properties with sequence-based profiles of physicochemical properties, PH domains can be classified into four functional subgroups according to their binding specificities and affinities to inositol phosphates. The method not only provides a simple and practical paradigm to predict binding specificities for functional genomic research but also gives new insight into the understanding of the basis of diseases with respect to PH domain structures.

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