scholarly journals Stimulation of Phospholipid Scrambling of the Erythrocyte Membrane by 9-Cis-Retinoic Acid

2017 ◽  
Vol 41 (2) ◽  
pp. 543-554 ◽  
Author(s):  
Majed Abed ◽  
Kousi Alzoubi ◽  
Florian Lang ◽  
Abdulla  Al Mamun Bhuayn
Keyword(s):  
Retinoic Acid ◽  
Cell Membrane ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling

Background/Aims: The endogenous retinoid 9-cis-retinoic acid has previously been shown to trigger apoptosis in a wide variety of cells including several tumor cells and has thus been suggested for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the accomplishment of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and formation of ceramide. The present study explored, whether 9-cis-retinoic acid induces eryptosis and whether the effect involves Ca2+ and/or ceramide. Methods: Flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Exposure to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased Fluo3-fluorescence, and the effect of 9-cis-retinoic acid on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Exposure to 9-cis-retinoic acid (1 µg/ml) further significantly increased the ceramide abundance at the erythrocyte surface and significantly increased hemolysis. Conclusions: 9-cis-retinoic acid triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and ceramide.

scholarly journals Triggering of Suicidal Erythrocyte Death by Fascaplysin

2016 ◽  
Vol 39 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Indole Alkaloid ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation

Background/Aims: The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from the hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Stimulation of Suicidal Erythrocyte Death by the CDC25 Inhibitor NSC-95397

2016 ◽  
Vol 40 (3-4) ◽  
pp. 597-607 ◽  
Author(s):  
Mohamed Jemaà ◽  
Morena Mischitelli ◽  
Myriam Fezai ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation ◽  
Protein Kinase C Inhibitor ◽  
Ros Formation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The CDC25B inhibitor NSC-95397 triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The substance is effective in part by modification of gene expression. Similar to apoptosis of nucleated cells erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of protein kinases. The present study explored, whether NSC-95397 induces eryptosis and, if so, to shed some light on the mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to NSC-95397 significantly increased the percentage of annexin-V-binding cells (≥ 1 µM), significantly decreased forward scatter (≥ 2.5 µM), and significantly increased Fluo3-fluorescence (≥ 1 µM), DCFDA fluorescence (5 µM) and ceramide abundance (≥ 5 µM). The effect of NSC-95397 (5 µM) on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+ and by addition of the protein kinase C inhibitor staurosporine (1 µM). Conclusions: NSC-95397 triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring entry of Ca2+ and activation of staurosporine sensitive kinase(s).

scholarly journals Edelfosine Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2221-2230 ◽  
Author(s):  
Marilena Briglia ◽  
Antonella Fazio ◽  
Elena Signoretto ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The anti-inflammatory, anti-autoimmune, antiparasitic, and anti-viral ether phospholipid edelfosine (1-O-octadecyl-2-O-methylglycero-3-phosphocholine) stimulates apoptosis of tumor cells and is thus considered for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i) and oxidative stress. The present study explored, whether and how edelfosine induces eryptosis. Methods: Flow cytometry and photometry, respectively, were employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 6 hours exposure of human erythrocytes to edelfosine (5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence. The effect of edelfosine on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Edelfosine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

scholarly journals Enhanced Eryptosis Following Exposure to Carnosic Acid

2015 ◽  
Vol 37 (5) ◽  
pp. 1779-1791 ◽  
Author(s):  
Katja Stockinger ◽  
Rosi Bissinger ◽  
Ghada Bouguerra ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Carnosic Acid ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca2+ entry and increased ceramide abundance.

scholarly journals Taurolidine Sensitivity of Eryptosis, the Suicidal Erythrocyte Death

2018 ◽  
Vol 51 (2) ◽  
pp. 501-512 ◽  
Author(s):  
Madeline Fink ◽  
Abdulla  Al Mamun Bhuyan ◽  
Nefeli Zacharopoulou ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling ◽  
Induced Apoptosis

Background/Aims: The taurine derivative Taurolidine is effective against diverse bacteria and tumor growth. In the treatment of cancer, the substance is effective in part by triggering suicidal death or apoptosis of tumor cells. The Taurolidine-induced apoptosis involves mitochondria. Erythrocytes lack mitochondria but are nevertheless able to enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explores, whether Taurolidine induces eryptosis and, if so, which cellular mechanisms are involved. Methods: Phosphatidylserine exposure at the cell surface was estimated using annexin-V-binding, cell volume using forward scatter, [Ca2+]i using Fluo3-fuorescence, reactive oxygen species (ROS) formation using 2’,7’-dichlorodihydrofuorescein (DCF)-dependent fluorescence, and ceramide abundance using specific antibodies. Results: A 48 hours exposure of human erythrocytes to Taurolidine (60 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence and ceramide abundance, but not DCF-fluorescence. The effect of Taurolidine on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Taurolidine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by increase of ceramide abundance.

scholarly journals Stimulation of Suicidal Erythrocyte Death by Phosphatase Inhibitor Calyculin A

2016 ◽  
Vol 40 (1-2) ◽  
pp. 163-171 ◽  
Author(s):  
Mustafa Almasry ◽  
Mohamed Jemaà ◽  
Morena Mischitelli ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Calyculin A ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The serine/threonine protein phosphatase 1 and 2a inhibitor Calyculin A may trigger suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+] i). Eryptosis is fostered by activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, and D4476 sensitive casein kinase. Eryptosis may further involve zVAD sensitive caspases. The present study explored, whether Calyculin A induces eryptosis and, if so, whether its effect requires Ca2+ entry, kinases and/or caspases Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, and [Ca2+] i from Fluo-3 fluorescence, as determined by flow cytometry. Results: A 48 hours exposure of human erythrocytes to Calyculin A (≥ 2.5 nM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo-3 fluorescence. The effect of Calyculin A on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by staurosorine (1 µM), SB203580 (2 µM), D4476 (10 µM), and zVAD (10 µM). Conclusions: Calyculin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part requiring Ca2+ entry, kinase activity and caspase activation.

scholarly journals Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes

2017 ◽  
Vol 41 (2) ◽  
pp. 731-741 ◽  
Author(s):  
Mustafa Almasry ◽  
Mohamed Jemaà ◽  
Morena Mischitelli ◽  
Florian Lang ◽  
Caterina Faggio
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitors ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM). Conclusions: Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca2+ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).

scholarly journals Stimulation of Eryptosis by Narasin

2015 ◽  
Vol 37 (5) ◽  
pp. 1807-1816 ◽  
Author(s):  
Ghada Bouguerra ◽  
Rosi Bissinger ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Subsequent Increase ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell ◽  
Stimulation Of

Background/Aims: Narasin, an ionophore used for the treatment of coccidiosis, has been shown to foster apoptosis of tumor cells. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by Ca2+ entry with subsequent increase of cytosolic Ca2+ activity ([Ca2+]i), and by ceramide. The present study explored, whether and how narasin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to narasin (10 and 25 ng/ml) significantly increased the percentage of annexin-V-binding cells. Forward scatter was decreased by 1 ng/ml narasin but not by higher narasin concentrations (10 and 25 ng/ml). Narasin significantly increased Fluo3-fluorescence (10 and 25 ng/ml) and slightly, but significantly increased ceramide abundance (25 ng/ml). The effect of narasin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Narasin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled and partially dependent on Ca2+ entry. Narasin further leads to cell shrinkage and slight increase of ceramide abundance.

scholarly journals Enhanced Eryptosis Following Auranofin Exposure

2015 ◽  
Vol 37 (3) ◽  
pp. 1018-1028 ◽  
Author(s):  
Kousi Alzoubi ◽  
Jasmin Egler ◽  
Majed Abed ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Methods: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. Results: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). Conclusions: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

scholarly journals Stimulating Effect of Manumycin A on Suicidal Erythrocyte Death

2016 ◽  
Vol 38 (3) ◽  
pp. 1147-1156 ◽  
Author(s):  
Jasmin Egler ◽  
Jens Zierle ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Human Erythrocyte ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Binding Cell

Background/Aims: The streptomycete derived farnesyltransferase inhibitor Manumycin A triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The present study explored whether Manumycin A could similarly stimulate eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry as well as activation of staurosporine sensitive protein kinase C and SB203580 sensitive p38 kinase. The present study explored, whether Manumycin A induces eryptosis and, if so, to shed some light on the mechanisms involved. Methods: Phosphatidylserine abundance at the human erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, and hemolysis from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Manumycin A (≥ 5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly incrased hemolysis. The effect of Manumycin A on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by addition of staurosporine (1 µM) and by addition of SB203580 (2 µM). Conclusions: Manumycin A triggers hemolysis, cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane. The effect on cell membrane scrambling was in part but not fully dependent on entry of extracellular Ca2+, as well as activity of staurosporine and SB203580 sensitive kinases.

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