scholarly journals Modulation of ferritin H-chain expression in Friend erythroleukemia cells: transcriptional and translational regulation by hemin.

1992 ◽  
Vol 12 (7) ◽  
pp. 3015-3022 ◽  
Author(s):  
E M Coccia ◽  
V Profita ◽  
G Fiorucci ◽  
G Romeo ◽  
E Affabris ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Protoporphyrin Ix ◽  
Mrna Translation ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Iron Storage ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Friend Erythroleukemia Cells ◽  
Ferritin H

The mechanisms that regulate the expression of the H chain of the iron storage protein ferritin in Friend erythroleukemia cells (FLCs) after exposure to hemin (ferric protoporphyrin IX), protoporphyrin IX, and ferric ammonium citrate (FAC) have been investigated. Administration of hemin increases the steady-state level of ferritin mRNA about 10-fold and that of ferritin protein expression 20-fold. Experiments with the transcriptional inhibitor actinomycin D and transfection studies demonstrate that the increment in cytoplasmic mRNA content results from enhanced transcription of the ferritin H-chain gene and cannot be attributed to stabilization of preexisting mRNAs. In addition to transcriptional effects, translational regulation induces the recruitment of stored mRNAs into functional polyribosomes after hemin and FAC administration, resulting in a further increase in ferritin synthesis. Administration of protoporphyrin IX to FLCs produces divergent transcriptional and translational effects. It increases transcription but appears to suppress ferritin mRNA translation. FAC treatment increases the mRNA content slightly (about twofold), and the ferritin levels rise about fivefold over the control values. We conclude that in FLCs, hemin induces ferritin H-chain biosynthesis by multiple mechanisms: a transcriptional mechanism exerted also by protoporphyrin IX and a translational one, not displayed by protoporphyrin IX but shared with FAC.

Modulation of ferritin H-chain expression in Friend erythroleukemia cells: transcriptional and translational regulation by hemin

1992 ◽  
Vol 12 (7) ◽  
pp. 3015-3022
Author(s):  
E M Coccia ◽  
V Profita ◽  
G Fiorucci ◽  
G Romeo ◽  
E Affabris ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Protoporphyrin Ix ◽  
Mrna Translation ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Iron Storage ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Friend Erythroleukemia Cells ◽  
Ferritin H

The mechanisms that regulate the expression of the H chain of the iron storage protein ferritin in Friend erythroleukemia cells (FLCs) after exposure to hemin (ferric protoporphyrin IX), protoporphyrin IX, and ferric ammonium citrate (FAC) have been investigated. Administration of hemin increases the steady-state level of ferritin mRNA about 10-fold and that of ferritin protein expression 20-fold. Experiments with the transcriptional inhibitor actinomycin D and transfection studies demonstrate that the increment in cytoplasmic mRNA content results from enhanced transcription of the ferritin H-chain gene and cannot be attributed to stabilization of preexisting mRNAs. In addition to transcriptional effects, translational regulation induces the recruitment of stored mRNAs into functional polyribosomes after hemin and FAC administration, resulting in a further increase in ferritin synthesis. Administration of protoporphyrin IX to FLCs produces divergent transcriptional and translational effects. It increases transcription but appears to suppress ferritin mRNA translation. FAC treatment increases the mRNA content slightly (about twofold), and the ferritin levels rise about fivefold over the control values. We conclude that in FLCs, hemin induces ferritin H-chain biosynthesis by multiple mechanisms: a transcriptional mechanism exerted also by protoporphyrin IX and a translational one, not displayed by protoporphyrin IX but shared with FAC.

Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1548-1548
Author(s):  
Yasumasa Okazaki ◽  
Hong Yin ◽  
Yuxiang Ma ◽  
Mary Yeh ◽  
Kwo-yih Yeh ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Iron Content ◽  
Iron Status ◽  
Protoporphyrin Ix ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Down Regulation ◽  
Cellular Iron

Abstract The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood, Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.

Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3813-3822 ◽  
Author(s):  
Erika M. Becker ◽  
Judith M. Greer ◽  
Prem Ponka ◽  
Des R. Richardson
Keyword(s):  
Friedreich Ataxia ◽  
Protoporphyrin Ix ◽  
Erythroid Differentiation ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Mrna Levels ◽  
Synthesis Inhibitor ◽  
Protein Levels ◽  
Friend Cells

Friedreich ataxia (FA) is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (β-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and β-globinmRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis.

scholarly journals Translational regulation of ferritin synthesis in rat liver. Effects of chronic dietary iron overload

1989 ◽  
Vol 264 (3) ◽  
pp. 925-928 ◽  
Author(s):  
G Cairo ◽  
L Tacchini ◽  
L Schiaffonati ◽  
E Rappocciolo ◽  
E Ventura ◽  
...  
Keyword(s):  
Iron Overload ◽  
Translational Regulation ◽  
Dietary Iron ◽  
Iron Storage ◽  
Subunit Mrna ◽  
Ferritin Synthesis ◽  
Parenteral Iron ◽  
Liver Ferritin ◽  
Ferritin H

In rats with chronic dietary iron overload, a higher amount of liver ferritin L-subunit mRNA was found mainly engaged on polysomes, whereas in control rats ferritin L-subunit mRNA molecules were largely stored in ribonucleoprotein particles. On the other hand, ferritin H-subunit mRNA was unchanged by chronic iron load and remained in the inactive cytoplasmic pool. In agreement with previous reports, in rats acutely treated with parenteral iron, only the ferritin L-subunit mRNA increased in amount, whereas both ferritin subunit mRNAs shifted to polysomes. This may indicate that, whereas in acute iron overload the hepatocyte operates a translation shift of both ferritin mRNAs to confront rapidly the abrupt entry of iron into the cell, during chronic iron overload it responds to the slow iron influx by translating a greater amount of L-subunit mRNA to synthesize isoferritins more suitable for long-term iron storage.

scholarly journals Ferritin translation by interleukin-6: the role of sequences upstream of the start codons of the heavy and light subunit genes

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2525-2537 ◽  
Author(s):  
JT Rogers
Keyword(s):  
Translational Regulation ◽  
Umbilical Vein ◽  
Start Codon ◽  
Moderate Increase ◽  
Human Hepatoma Cells ◽  
Iron Storage ◽  
Translational Enhancer ◽  
Umbilical Vein Endothelial Cells ◽  
Ferritin H

Interleukin-1beta (IL-1beta) elevates H- and L-ferritin subunit synthesis in both human hepatoma cells (HepG2) and primary human umbilical vein endothelial cells. Ferritin induction is greater than the increase in total HepG2 protein synthesis in response to IL-1. IL-6 causes a moderate increase in L-subunit synthesis. The levels of the mRNAs for the ferritin H-subunits (H-mRNA) and light subunits (L-mRNA) remain unchanged, indicating that expression of the iron storage protein, ferritin, is regulated by translational mechanisms during inflammation. We have found a translational enhancer region in the L- ferritin mRNA 5′UTR that confers two-fold baseline and twofold IL-1- dependent translational regulation to a CAT reporter message. The L- mRNA motif is related to a 61 nucleotide (nt) G+C-rich translational enhancer within 70 nt of the H-ferritin start codon. Sequences upstream of the start codons (SUS elements) in both H-mRNA and L-mRNAs confer IL- 1beta but not IL-6-dependent translation to hybrid ferritin/CAT reporter mRNAs. The H- and L-ferritin mRNA SUS elements contain a motif similar to a consensus reported for the 5′ leaders of other acute phase response mRNAs. Transfected hybrid H-mRNA SUS/CAT mRNAs with a three nucleotide deleted version of the H-mRNA SUS displays an eightfold reduced level of translation and no longer confer IL-1beta-dependent translation.

scholarly journals Study on Sucrosomial® Iron Endocytosis-Mediated Uptake and Enterocytes Release

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4892-4892
Author(s):  
Elisa Brilli ◽  
Asperti Michela ◽  
Magdalena Gryzik ◽  
Alessandro Lucchesi ◽  
Giovanni Martinelli ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Iron Absorption ◽  
Phospholipid Bilayer ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Iron Release ◽  
Iron Storage

Abstract Introduction: iron homeostasis is maintained by regulating the iron levels in plasma which is maintained by four coordinated processes: duodenal iron absorption, macrophage iron recycling, hepatic iron storage and erythropoiesis. Iron in the Fe2+ form is transported across the apical duodenal membrane by DMT1 and subsequently transferred to the blood via the iron exporter, Ferroportin the only know cell membrane iron exporter. Due to the presence of two check points at cellular levels, iron absorption and release are mainly regulated, because of this iron containing oral formulations are poorly absorbed and bioavailable. To overcome cellular barriers and increasing the bioavailability of supplemented iron forms, there is a need for new carriers that work protecting the iron as well as enhancing its intestinal absorption and release into the blood stream. Moreover thus reducing dosage and side effects. Sucrosomial® Iron (SI) represents an innovative oral iron-containing carrier in which ferric pyrophosphate is protected by a phospholipid bilayer membrane plus a sucrester matrix. To date, in vitro studies have shown that SI is mostly absorbed as vesicle-like structure, bypassing the conventional iron absorption pathway. Due to its behaviour at the gastrointestinal tract, SI is well tolerated and highly bioavailable compared to conventional iron salts. To deeply understand involvement of endocytosis in SI absorption and release, in vitro experiments using endocytosis and ferroportin inhibitors were carried out Aim: to study Sucrosomial® Iron uptake and release in different in vitro systems. Materials and Methods: CACO-2 and THP1 cells were used to investigate the role of FPN in Sucorsomial Iron release from cells. For release study, CACO-2 cells were exposed for 18h to quercetin (150mmol/L) in order to downregulate FPN expression. CACO-2 quercetin pre-treated cells were co-cultured with TPH1 cells, and SI or FAC were added. However, prior to measure cell Ferritin content, the incubation medium was discarded and cells were washed to remove quercetin. Iron uptake-release analysis was performed using co-culture transwell system between CACO-2 cells and TPH1. To investigate the cellular fate of cellular iron in quercetin treated CACO-2 and TPH1 cells we measured cell ferritin content. To inhibit endocytosis absorption pathway, CACO-2, THP1 and HepG2 cells were pre-treated with PitStop2 and Dyngo 4a inhibitors and then treated with SI, or Ferrous Sulfate (FS) or ferric ammonium citrate (FAC). Cellular Ferritin content was measured. Results: in order to understand the effect of quercetin on iron storage, we used CACO2 and TPH1 cells pre-treated with quercetin and then treated with SI, FAC or nothing (control). Quercetin-SI treated CACO-2 cells showed no differences in Ferritin expression compared to control cells (3,94 ngFTL/mg proteins Vs 4,56 ngFTL/mg proteins) while in quercetin-FAC treated cells ferritin expression was decreased compare to control cells (16,3 ngFTL/mg proteins Vs 27,55 ngFTL/mg proteins). In a similar manner, quercetin-SI treated TPH1 cells didn't show increase in Ferritin expression compared to control cells (20 ngFTL/mg proteins Vs 15,15 ngFTL/mg proteins), only in quercetin-FAC treated cells we observed a Ferritin expression increase compared to control untreated cells (16 ngFTL/mg proteins Vs 24 ngFTL/mg proteins). Results from experiments using endocytosis inhibitors showed that SI absorption in CACO-2 cells is inhibited using Dyngo4a (from 4ngFTL/mg proteins to 0,36 ngFTL/ mg proteisn) while PitStop3 seems to reduce SI absorption in THP1 (from 396 ngFL/mg protein to 199,91 ngFTL/mg proteins) and HepG2 cells (from 26,86 ngFL/mg proteins to 3,93 ngFTL/mg proteins), since ferritin expression significantly decrease only in SI treated cells. Conclusions: endocytosis pathway seems to be involved in SI cellular uptake but this process is regulated in different manner probably due to different cell types. Release experiments showed that cells treated with quercetin could reduce for a negative feedback DMT1 expression as well, affecting iron uptake from cells treated with FAC but not with SI and consequently, if SI is able to bypass commonly iron uptake mechanism, FPN inhibition did not show iron release perturbation from cells treated with SI. Disclosures Brilli: Pharmanutra s.p.a.: Consultancy. Martinelli:Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy; Abbvie: Consultancy; Novartis: Speakers Bureau; Amgen: Consultancy; Ariad/Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Tarantino:Pharmanutra s.p.a.: Employment.

scholarly journals Regulation of ferritin H-chain expression in differentiating Friend leukemia cells

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1570-1579 ◽  
Author(s):  
EM Coccia ◽  
E Stellacci ◽  
R Orsatti ◽  
U Testa ◽  
A Battistini
Keyword(s):  
Translational Control ◽  
Translational Regulation ◽  
State Level ◽  
Cell Types ◽  
Coding Region ◽  
Iron Storage ◽  
Human Ferritin ◽  
Responsive Element ◽  
Differentiation Inducers ◽  
Ferritin H

The mechanisms that regulate the expression of ferritin, the iron storage protein, have been investigated in Friend erythroleukemia cells (FLCs) induced to differentiate by several chemical compounds. In differentiating FLCs, administration of hemin increases the steady- state level of ferritin mRNA about 15-fold and the ferritin content about 20- to 25-fold. Conversely, iron salts have only mild stimulatory effects on these parameters and iron chelators only slightly inhibited the stimulatory effect of hemin. Transient transfection experiments with a construct in which the human ferritin H-chain promoter drives the expression of the indicator chloramphenicol acetyltransferase (CAT) gene show that the increase in mRNA content is mainly due to enhanced transcription. In addition to transcriptional effects, translational regulation resulting in the further increase in ferritin synthesis is shown by CAT assays from cells transiently transfected with a construct containing the coding region for the indicator CAT mRNA under the translational control of the mRNA ferritin iron-responsive element. We conclude that, in FLCs induced to differentiate, hemin acts synergistically with the differentiation inducers, increasing ferritin expression. Both transcriptional and translational mechanisms are responsible for this synergistic effect, which appears to be characteristic of differentiated erythroid cells because it is not observed in other cell types (ie, fibroblastic cell lines).

CALM Insufficiency Impairs Leukemia Cell Proliferation by Limiting Iron Uptake

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1478-1478
Author(s):  
Catherine P. Lavau ◽  
Jessica L Heath ◽  
Paula B. Scotland ◽  
Daniel S. Wechsler
Keyword(s):  
Cell Proliferation ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Iron Chelation ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Iron Storage ◽  
Iron Deficient

Abstract Abstract 1478 Background: The CALM-AF10 fusion protein that arises from the t(10;11) chromosomal translocation is found in both acute myeloid leukemia and 8–10% of T-cell acute lymphoblastic leukemia and is associated with a poor clinical outcome. The interaction of AF10 with the DOT1L histone methyltransferase has been shown to be necessary for CALM-AF10 mediated transformation. However, the role of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein in leukemogenesis remains uncertain. CALM plays a role in clathrin-mediated endocytosis. This process mediates the entry of various growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. Objective: We hypothesize that loss of CALM function, as a result of CALM haploinsufficiency and/or a dominant negative CALM-AF10 effect, may contribute to transformation. Design/Methods: To study the effects of CALM deficiency, we used genetically modified fit1 mice that harbor a Calm gene mutation that results in a severely truncated and nonfunctional Calm protein. Fetal liver (FL) cells from Calm−/−, Calm+/− or CalmWT/WT E14 embryos were retrovirally transduced with MLL-ENL or CALM-AF10 and studied in vitro or transplanted into syngeneic mice. Interleukin-3 dependent cell lines were established from Calm−/− or CalmWT control FL cells immortalized by CALM-AF10 or MLL-ENL. Results: CALM-AF10 leukemias induced in Calm−/− or Calm+/− precursors were considerably delayed compared with those induced in CalmWT controls. Clathrin-mediated endocytosis of fluorescently labeled transferrin receptor (TfR) was impaired in Calm−/− cells. This was accompanied by a 2–4 fold increase in surface expression of TfR by flow cytometry and an increase in total TfR protein as measured by Western blot in Calm−/− cells. Calm -deficient cells also displayed lower levels of the iron storage protein ferritin, suggestive of an iron deficient state. Each of these features was reversed by re-expression of CALM via retroviral transduction of Calm−/− cells with a CALM expression vector. Paralleling the prolonged latency of Calm−/− leukemias, we found that Calm -deficient cells proliferated at a considerably slower rate than CALM -rescued cells. This cell proliferation defect could be rescued by supplementing the cells with iron (ferric ammonium citrate, 50 μM), indicating that reduced iron availability limits the expansion of Calm -deficient cells. Intriguingly, Calm−/− cells were significantly more sensitive to the growth inhibitory effect of iron chelation (deferoxamine, 5 μM) than CALM -rescued cells. Conclusions: These observations suggest that loss of CALM function impairs iron import and consequently limits the rate of cell proliferation. This raises the possibility that CALM haploinsufficiency present in CALM-AF10 leukemias might render cells particularly responsive to iron chelation therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Unconventional endocytosis and trafficking of Transferrin Receptor induced by iron

2020 ◽  
pp. mbc.E20-02-0129
Author(s):  
Elena Gammella ◽  
Irene Schiano Lomoriello ◽  
Alexia Conte ◽  
Stefano Freddi ◽  
Alessandra Alberghini ◽  
...  
Keyword(s):  
Translational Control ◽  
Transferrin Receptor ◽  
Translational Regulation ◽  
Iron Homeostasis ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Protein Levels ◽  
Ferric Ammonium ◽  
Clathrin Adaptor ◽  
Proteasomal Inhibitor

The post-translational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knock-down experiments showed that TfR1 lysosomal degradation is independent of: 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-Ferritin and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional post-translational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis often not considering its function in iron homeostasis.

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