The Ubiquitin Ligase Triad1 Is Involved in Myelopoiesis and Interacts with the Transcriptional Repressors Gfi1 and Gfi1b.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 64-64 ◽  
Author(s):  
Bert A. Van der Reijden ◽  
Jurgen A.F. Marteijn ◽  
Liesbeth Van Emst ◽  
Theo De Witte ◽  
Joop H. Jansen
Keyword(s):  
Bone Marrow ◽  
Zinc Finger ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
26S Proteasome ◽  
Specific Substrate ◽  
Granulocytic Differentiation ◽  
Substrate Proteins ◽  
Zinc Finger Region ◽  
Marrow Cells

Abstract We identified Triad1 as a gene that is upregulated by retinoic acid during the granulocytic differentiation of acute promyelocytic leukemia cells. In normal hematopoiesis, we show that Triad1 is weakly expressed in immature CD34+ bone marrow cells, and highly expressed in mature monocytes and granulocytes. Together, this suggests that Triad1 plays a role in the differentiation of hematopoietic cells. Triad1 contains a tripartite domain including two RING fingers, indicating that this protein might function as a ubiquitin E3 ligase, catalyzing the the conjugation of ubiquitin to substrate proteins thereby marking them for targeted degradation by the 26S proteasome. Using GST pull down experiments, we show that Triad1 binds to the ubiquitin conjugating (E2) enzymes UbcH6 and 7. In addition, immunoprecipitation of Triad1 in cells that were transfected with FLAG-tagged ubiquitin shows that Triad1 binds to ubiquitinated proteins, and that Triad1 is capable of self-ubiquitination, further corroborating the assumption that Triad1 acts as a E3 ubiquitin ligating enzyme. To study the role of Triad1 in hematopoiesis we overexpressed the gene in primary murine bone marrow cells using a retroviral vector that contains Triad1 in front of an IRES-GFP cassette. GFP positive cells were FACS sorted and used in colony assays (CFU-GM). Compared to empty vector controls (GFP alone), Triad1 expression resulted in more than 80% inhibition of clonogenic growth. Importantly, addition of the proteasome inhibitor MG132 (10E-8 M) reversed the Triad1-induced suppression of colony formation. Furthermore, three Triad1 expression constructs in which one of the conserved cys/his residues of the TRIAD domain (essential for function) were mutated did not show the suppressive effect on colony formation. Together, these data show that Triad1 is involved in myelopoiesis and acts through the ubiquitination of specific substrate proteins. To identify these substrates, a yeast-two-hybrid screen of a human bone marrow cDNA library was performed using the Triad1 protein as a bait. Interestingly, the transcriptional repressor Gfi1b was found to bind to Triad1. The interaction was confirmed by immunoprecipitation using GFP-Triad1 and FLAG-tagged Gfi1b transfections in mammalian cells. We show that Triad1 binds to the zinc finger region of Gfi1b. This region is very (>98%) homologous to the paralogue Gfi1. Further immunoprecipitation analyses showed that Triad1 also binds to the zinc finger region of Gfi1. Gfi1 plays an essential role in neutrophil development and Gfi1 pointmutations result in neutropenia in man. Currently, we are studying the direct ubiquitination of Gfi and Gfi1b by Triad1 in in vitro ubiquitination assays. In addition, we are studying the effect of Triad1 on the transcriptional repression of the ELA2 and other promoters by Gfi1.

scholarly journals Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1).

1996 ◽  
Vol 16 (8) ◽  
pp. 4566-4572 ◽  
Author(s):  
S L Lee ◽  
Y Wang ◽  
J Milbrandt
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
Zinc Finger ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Hematopoietic Progenitor Cell ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Macrophage Differentiation ◽  
Marrow Cells

The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bone marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL-60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A-/- bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A-/- macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A-/- mice showed no increased mortality or bacteria] burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.

Colony formation by primitive haemopoietic progenitors in cocultures of bone marrow cells and stromal cells

1985 ◽  
Vol 60 (1) ◽  
pp. 129-136 ◽  
Author(s):  
M. Y. Gordon ◽  
J. A. Hibbin ◽  
L. U. Kearney ◽  
E. C. Gordon-Smith ◽  
J. M. Goldman
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Marrow Cells

scholarly journals In vivo modulation of myelopoiesis by prostaglandin E2. III. Induction of suppressor cells in marrow and spleen capable of mediating inhibition of CFU-GM proliferation

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1633-1640
Author(s):  
LM Pelus ◽  
PS Gentile
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Steady State ◽  
Prostaglandin E2 ◽  
Bone Marrow Cells ◽  
Suppressor Cells ◽  
Spleen Cells ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Intravenous (IV) injection of 0.1 to 10 micrograms of authentic prostaglandin E2 (PGE2) in intact steady-state mice induces a population of bone marrow and spleen cells having the capacity to suppress CFU-GM proliferation when admixed with normal bone marrow cells. Equivalent suppression of CFU-GM committed to monocytic as well as granulocytic differentiation was observed using colony-stimulating factors (CSFs) differing in their lineage specificities and by direct morphological analysis of proliferating clones. Kinetic analysis indicates that suppressive bone marrow cells appear within 2 hours after PGE2 injection, are maximal at 6 hours, and are no longer observed by 24 hours postinjection. Positive and negative selection studies using monoclonal antibodies indicate that the PGE2-induced suppressor cells react positively with anti-GMA 1.2, MAC1, and F4/80 monoclonal antibodies, suggesting a myeloid/monocytic origin. As few as 1,000 positively selected bone marrow or spleen cells were able to inhibit maximally normal CFU-GM proliferation by 50,000 control bone marrow cells. Suppression of normal CFU-GM can be substituted for by 24- hour cell-free supernates from unseparated bone marrow cells or GMA 1.2 or F4/80 positively selected marrow or spleen cells from PGE2-treated but not control mice. These supernates also inhibited BFU-E proliferation. Injection of as few as 2 million bone marrow cells from PGE2-treated mice into steady-state mice or animals hematopoietically rebounding following a sublethal injection of cyclophosphamide significantly suppressed total CFU-GM proliferation in recipient mice within 6 hours. In summary, these studies describe the detection of a novel hematopoietic control network induced by PGE2 in intact mice.

scholarly journals Effects of neuraminidase on the regulation of erythropoiesis

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 784-788 ◽  
Author(s):  
VF LaRussa ◽  
F Sieber ◽  
LL Sensenbrenner ◽  
SJ Sharkis
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Colony Growth ◽  
Mouse Bone Marrow ◽  
Continuous Exposure ◽  
Low Concentrations ◽  
High Concentrations ◽  
Marrow Cells

Abstract In this article, we present evidence that sialic acid-containing surface components play a role in the regulation of erythropoiesis. A 1- hr exposure of mouse bone marrow cells to high concentrations of neuraminidase reduced erythroid colony formation. Coculture of 10(6) untreated thymocytes with neuraminidase-treated bone marrow cells restored erythroid colony growth. Neuraminidase-treated thymocytes retained their ability to suppress erythroid colony formation by untreated marrow cells, but lost their ability to enhance erythroid colony formation. Continuous exposure to low concentrations of neuraminidase enhanced erythroid bone marrow cell colony growth in response to a suboptimal dose of erythropoietin.

scholarly journals In vivo modulation of myelopoiesis by prostaglandin E2. III. Induction of suppressor cells in marrow and spleen capable of mediating inhibition of CFU-GM proliferation

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1633-1640 ◽  
Author(s):  
LM Pelus ◽  
PS Gentile
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Steady State ◽  
Prostaglandin E2 ◽  
Bone Marrow Cells ◽  
Suppressor Cells ◽  
Spleen Cells ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Abstract Intravenous (IV) injection of 0.1 to 10 micrograms of authentic prostaglandin E2 (PGE2) in intact steady-state mice induces a population of bone marrow and spleen cells having the capacity to suppress CFU-GM proliferation when admixed with normal bone marrow cells. Equivalent suppression of CFU-GM committed to monocytic as well as granulocytic differentiation was observed using colony-stimulating factors (CSFs) differing in their lineage specificities and by direct morphological analysis of proliferating clones. Kinetic analysis indicates that suppressive bone marrow cells appear within 2 hours after PGE2 injection, are maximal at 6 hours, and are no longer observed by 24 hours postinjection. Positive and negative selection studies using monoclonal antibodies indicate that the PGE2-induced suppressor cells react positively with anti-GMA 1.2, MAC1, and F4/80 monoclonal antibodies, suggesting a myeloid/monocytic origin. As few as 1,000 positively selected bone marrow or spleen cells were able to inhibit maximally normal CFU-GM proliferation by 50,000 control bone marrow cells. Suppression of normal CFU-GM can be substituted for by 24- hour cell-free supernates from unseparated bone marrow cells or GMA 1.2 or F4/80 positively selected marrow or spleen cells from PGE2-treated but not control mice. These supernates also inhibited BFU-E proliferation. Injection of as few as 2 million bone marrow cells from PGE2-treated mice into steady-state mice or animals hematopoietically rebounding following a sublethal injection of cyclophosphamide significantly suppressed total CFU-GM proliferation in recipient mice within 6 hours. In summary, these studies describe the detection of a novel hematopoietic control network induced by PGE2 in intact mice.

Pharmacologic Restoration of PP2A Activity and Interference with the SET-PP2A Interplay by FTY720 and Its Non-Immunosuppressive Derivative as a Novel and Efficient Therapy for Ph-Negative Myeloproliferative Disorders

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 775-775
Author(s):  
Joshua J Oaks ◽  
A. Mukhopadhyay ◽  
Ramasamy Santhanam ◽  
S. A Saddoughi ◽  
Christopher Walker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Myeloproliferative Disorders ◽  
Transformed Cells ◽  
Wild Type ◽  
Pp2a Activity ◽  
C6 Ceramide ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Abstract 775 We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2). Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients. To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells. Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min). Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures: Verstovsek: Incyte Corporation: Research Funding.

Impaired Granulocytic Differentiation In Vitro in Hematopoietic Cells Lacking Retinoic Acid Receptors α1 and γ

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 607-615 ◽  
Author(s):  
Jean Labrecque ◽  
Deborah Allan ◽  
Pierre Chambon ◽  
Norman N. Iscove ◽  
David Lohnes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retinoic Acid ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Retinoic Acid Receptors ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Transcripts for the retinoic acid receptors (RARs) α1, α2, γ1, and γ2 were found in the granulocytic lineage (Gr-1+cells) through semiquantitative polymerase chain reaction (PCR) analysis. The screening of single cell cDNA libraries derived from hematopoietic progenitors also showed the presence of RARα and, to a lesser extent, RARγ transcripts in committed granulocyte (colony-forming unit-granulocyte [CFU-G]) or granulocyte-macrophage (CFU-GM) colony-forming cells. The contribution of RARα1 and γ to hematopoietic cell differentiation was therefore investigated in mice bearing targeted disruption of either one or both of these loci. Because RARγ and RARα1γ compound null mutants die shortly after birth, bone marrow cells were collected from fetuses at 18.5 days postcoitum (dpc) and evaluated for growth and differentiation in culture in the presence of Steel factor (SF), interleukin-3 (IL-3), and erythropoietin (Epo). The frequency of colony-forming cells from bone marrow populations derived from RARα1/γ double null mice was not significantly different from that of RARγ or RARα1 single nulls or from wild-type controls. In addition, the distribution of erythroid, granulocyte, and macrophage colonies was comparable between hematopoietic cells from all groups, suggesting that lineage commitment was not affected by the lack of RARα1 and/or RARγ. Colony cells were then harvested individually and evaluated by morphologic criteria. While terminal granulocyte differentiation was evident in wild-type cells and colonies from either single null mutant, colonies derived from RARα1−/−γ−/− bone marrow populations were blocked at the myelocyte and, to a lesser extent, at the metamyelocyte stages, whereas erythroid and macrophage differentiation was not affected. Together, these results indicate that both RARα1 and γ are required for terminal maturation in the granulocytic lineage in vitro, but appear to be dispensable for the early stages of hematopoietic cell development. Our results raise the possibility that in acute promyelocytic leukemia (APL), the different RARα fusion proteins cause differentiation arrest at a stage when further maturation requires not only RARα, but also RARγ. Finally, bone marrow cells appear to differentiate normally in vivo, suggesting an effective compensation mechanism in the RARα1/γ double null mice.

scholarly journals Thrombopoietin Does Not Induce Lineage-Restricted Commitment of Mpl-R Expressing Pluripotent Progenitors But Permits Their Complete Erythroid and Megakaryocytic Differentiation

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3544-3553 ◽  
Author(s):  
Frédérique Goncalves ◽  
Catherine Lacout ◽  
Jean-Luc Villeval ◽  
Françoise Wendling ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Constitutive Expression ◽  
Retroviral Vectors ◽  
Granulocytic Differentiation ◽  
Hematopoietic Stem ◽  
Infected Cells ◽  
Marrow Cells

In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU–treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R–infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R–transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R– or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R–infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R– or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R–infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.

scholarly journals Parvovirus B19-induced perturbation of human megakaryocytopoiesis in vitro

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 1997-2004 ◽  
Author(s):  
A Srivastava ◽  
E Bruno ◽  
R Briddell ◽  
R Cooper ◽  
C Srivastava ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Replication ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Northern Analysis ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Marrow Cells

Abstract Parvovirus B19 infection leads to transient aplastic crises in individuals with chronic hemolytic anemias or immunodeficiency states. An additional unexplained sequela of B19 infection is thrombocytopenia. Because B19 is known to have a remarkable tropism for human erythropoietic elements, and is not known to replicate in nonerythroid cells, the etiology of this thrombocytopenia is uncertain. We sought to define the pathobiology of B19-associated thrombocytopenia by examining the role of B19 on in vitro megakaryocytopoiesis. B19 infection of normal human bone marrow cells significantly suppressed megakaryocyte (MK) colony formation compared with mock-infected cells. No such inhibition was observed with a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The B19-MK cell interaction was also studied at the molecular level. Whereas low-density bone marrow cells containing erythroid precursor cells supported B19 DNA replication, no viral DNA replication was observed in B19-infected MK-enriched fractions as determined by the presence of viral DNA replicative intermediates on Southern blots. However, analysis of total cytoplasmic RNA isolated from B19-infected MK fractions showed a low-level expression of the B19 genome as detected by quantitative RNA dot blots as well as by Northern analysis. Furthermore, a frame-shift mutation in a recombinant AAV-B19 hybrid genome segment that encodes the viral nonstructural (NS1) protein significantly reduced the observed inhibition of MK colony formation. These studies indicate tissue- tropism of B19 beyond the erythroid progenitor cell, and lend support to the hypothesis that B19 genome expression may be toxic to cell populations that are nonpermissive for viral DNA replication.

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