scholarly journals 20-HETE Participates in Intracerebral Hemorrhage-Induced Acute Injury by Promoting Cell Ferroptosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Ranran Han ◽  
Jieru Wan ◽  
Xiaoning Han ◽  
Honglei Ren ◽  
John R. Falck ◽  
...  
Keyword(s):  
Cell Death ◽  
Intracerebral Hemorrhage ◽  
Ex Vivo ◽  
Neuroprotective Effect ◽  
Lipid Peroxide ◽  
Mitogen Activated Protein Kinase ◽  
Acute Injury ◽  
Arachidonic Acid Metabolite ◽  
Lesion Volume

Intracerebral hemorrhage (ICH) is a highly fatal type of stroke that leads to various types of neuronal death. Recently, ferroptosis, a form of cell death resulting from iron-dependent lipid peroxide accumulation, was observed in a mouse ICH model. N-hydroxy-N′-(4-n-butyl-2-methylphenyl)-formamidine (HET0016), which inhibits synthesis of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE), has shown a protective effect after ICH. However, the underlying mechanisms of the neuroprotective effect need further investigation. We explored whether 20-HETE participates in ICH-induced ferroptosis ex vivo by using hemoglobin-treated organotypic hippocampal slice cultures (OHSCs) and in vivo by using a collagenase-induced ICH mouse model. Ex vivo, we found that the 20-HETE synthesis inhibitor HET0016 and antagonist 20-6,15-HEDGE reduced hemoglobin-induced cell death, iron deposition, and lipid reactive oxygen species levels in OHSCs. Furthermore, 20-HETE inhibition in OHSCs increased the expression of glutathione peroxidase (GPX) 4, an antioxidant enzyme that serves as a main regulator of ferroptosis. In contrast, exposure of OHSCs to the 20-HETE stable mimetic 20-5,14-HEDGE induced cell death that was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. In vivo, HET0016 treatment ameliorated focal deficits, reduced lesion volume, and decreased iron accumulation around the lesion at day 3 and 7 after ICH. In addition, lipid peroxidation was decreased and expression of GPX4 was increased in the HET0016-treated ICH group. The mitogen-activated protein kinase pathway also was inhibited by HET0016 in vivo. These results indicate that 20-HETE contributes to ICH-induced acute brain injury in part by activating ferroptosis pathways, thereby providing an upstream target for inhibiting ferroptosis.

scholarly journals Endonuclease G mediates endothelial cell death induced by carbamylated LDL

2011 ◽  
Vol 300 (6) ◽  
pp. H1997-H2004 ◽  
Author(s):  
Eugene O. Apostolov ◽  
Debarti Ray ◽  
Wilson M. Alobuia ◽  
Marina V. Mikhailova ◽  
Xiaoying Wang ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Endothelial Cells ◽  
Cell Death ◽  
Kidney Disease ◽  
Dna Fragmentation ◽  
Ex Vivo ◽  
Mitotic Cell ◽  
Mitogen Activated Protein Kinase ◽  
Endonuclease G

End-stage kidney disease is a terminal stage of chronic kidney disease, which is associated with a high incidence of cardiovascular disease. Cardiovascular disease frequently results from endothelial injury caused by carbamylated LDL (cLDL), the product of LDL modification by urea-derived cyanate. Our previous data suggested that cLDL induces mitogen-activated protein kinase-dependent mitotic DNA fragmentation and cell death. However, the mechanism of this pathway is unknown. The current study demonstrated that cLDL-induced endothelial mitotic cell death is independent of caspase-3. The expression of endonuclease G (EndoG), the nuclease implicated in caspase-independent DNA fragmentation, was significantly increased in response to cLDL exposure to the cells. The inhibition of EndoG by RNAi protected cLDL-induced DNA fragmentation, whereas the overexpression of EndoG induced more DNA fragmentation in endothelial cells. Ex vivo experiments with primary endothelial cells isolated from wild-type (WT) and EndoG knockout (KO) mice demonstrated that EndoG KO cells are partially protected against cLDL toxicity compared with WT cells. To determine cLDL toxicity in vivo, we administered cLDL or native LDL (nLDL) intravenously to the WT and EndoG KO mice and then measured floating endothelial cells in blood using flow cytometry. The results showed an increased number of floating endothelial cells after cLDL versus nLDL injection in WT mice but not in EndoG KO mice. Finally, the inhibitors of MEK-ERK1/2 and JNK-c-jun pathways decreased cLDL-induced EndoG overexpression and DNA fragmentation. In summary, our data suggest that cLDL-induced endothelial toxicity is caspase independent and results from EndoG-dependent DNA fragmentation.

Abstract P419: A Systematic Characterization of Brain Endothelial Cell Death in Intracerebral Hemorrhage

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Maulana Ikhsan ◽  
Marietta Zille
Keyword(s):  
Cell Death ◽  
Intracerebral Hemorrhage ◽  
Brain Tissue ◽  
Vascular Integrity ◽  
Regulated Cell Death ◽  
Injury Mechanisms ◽  
Endothelial Cell Death ◽  
Cell Death Mechanisms ◽  
Underlying Mechanisms

Introduction: Intracerebral hemorrhage (ICH) is a type of stroke caused by the loss of vascular integrity leading to bleeding within the brain tissue. Hematoma-derived factors cause secondary injury mechanisms such as cell death days to weeks after the event and in regions distant from the primary insult. Increasing evidence suggests that hemoglobin released by the hematoma is one of the major contributors to neuronal injury in ICH. To date, it is unclear whether brain endothelial cells (EC) are similarly vulnerable to hemolysis products and undergo regulated cell death. Hypothesis: We hypothesized that brain EC undergo multiple, different modes of cell death after ICH and that the underlying mechanisms are different compared to neurons. Methods: We systematically investigated cell death mechanisms in brain EC after exposure to the hemolysis product hemin. We used chemical inhibitors of apoptosis, autophagy, ferroptosis, necroptosis, and parthanatos and assessed biochemical markers of these cell death modes. Results: Brain EC viability was concentration-dependently decreased, starting at higher hemin concentrations than neurons. Treatment of EC with ferroptosis inhibitors protective against hemin toxicity in neurons and against ICH in vivo showed that only N-acetylcysteine and deferoxamine protected brain EC, while ferrostatin-1 and U0126 did not abrogate EC death. The autophagy inhibitor bafilomycin A1 also reduced EC death and hemin increased the expression of the autophagy marker LC3. While inhibitors against apoptosis and parthanatos were not effective, the necroptosis inhibitor GSK872 demonstrated a partial protective effect. Conclusions: Our data suggest that ICH induces different mechanisms of death in EC (ferroptosis and autophagy) compared to neurons (ferroptosis and necroptosis) and may thus warrant a combinatorial therapeutic approach. Further investigations in human and ovine ICH brain tissue are ongoing.

scholarly journals APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

Haematologica ◽  
2021 ◽  
Author(s):  
Rudy Birsen ◽  
Clement Larrue ◽  
Justine Decroocq ◽  
Natacha Johnson ◽  
Nathan Guiraud ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Lipid Peroxides ◽  
Death Process ◽  
Glutathione Biosynthesis ◽  
Acute Myeloid

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia. APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Aloe Metabolites Prevent LPS-Induced Sepsis and Inflammatory Response by Inhibiting Mitogen-Activated Protein Kinase Activation

2017 ◽  
Vol 45 (04) ◽  
pp. 847-861 ◽  
Author(s):  
Chia-Yang Li ◽  
Katsuhiko Suzuki ◽  
Yung-Li Hung ◽  
Meng-Syuan Yang ◽  
Chung-Ping Yu ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Aloe Vera ◽  
Peritoneal Macrophages ◽  
Mitogen Activated Protein Kinase ◽  
Protective Effects ◽  
Raw264.7 Macrophages ◽  
Mitogen Activated Protein ◽  
Anti Inflammatory

Aloe, a polyphenolic anthranoid-containing Aloe vera leaves, is a Chinese medicine and a popular dietary supplement worldwide. In in vivo situations, polyphenolic anthranoids are extensively broken down into glucuronides and sulfate metabolites by the gut and the liver. The anti-inflammatory potential of aloe metabolites has not been examined. The aim of this study was to investigate the anti-inflammatory effects of aloe metabolites from in vitro (lipopolysaccharides (LPS)-activated RAW264.7 macrophages) and ex vivo (LPS-activated peritoneal macrophages) to in vivo (LPS-induced septic mice). The production of proinflammatory cytokines (TNF-[Formula: see text] and IL-12) and NO was determined by ELISA and Griess reagents, respectively. The expression levels of iNOS and MAPKs were analyzed by Western blot. Our results showed that aloe metabolites inhibited the expression of iNOS, decreased the production of TNF-[Formula: see text], IL-12, and NO, and suppressed the phosphorylation of MAPKs by LPS-activated RAW264.7 macrophages. In addition, aloe metabolites reduced the production of NO, TNF-[Formula: see text] and IL-12 by murine peritoneal macrophages. Furthermore, aloe administration significantly reduced the NO level and exhibited protective effects against sepsis-related death in LPS-induced septic mice. These results suggest that aloe metabolites exerted anti-inflammatory effects in vivo, and that these effects were associated with the inhibition of inflammatory mediators. Therefore, aloe could be considered an effective therapeutic agent for the treatment of sepsis.

scholarly journals OP32 Mincle signalling promotes intestinal mucosal inflammation through induction of macrophage pyroptosis and neutrophil chemotaxis in Crohn’s disease

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S031-S031
Author(s):  
W GONG ◽  
K Guo ◽  
J Ren
Keyword(s):  
Intestinal Inflammation ◽  
Ex Vivo ◽  
Experimental Colitis ◽  
Mitogen Activated Protein Kinase ◽  
Mucosal Inflammation ◽  
Neutrophil Chemotaxis ◽  
Knock Out ◽  
Mitogen Activated Protein ◽  
Proinflammatory Role

Abstract Background Macrophage-inducible C-type lectin (Mincle) signalling plays a proinflammatory role in different organs such as the brain and liver, but its role in intestinal inflammation remains unknown. Methods We studied the characteristics of Mincle signalling expression in CD patients and experimental colitis. The functional role of Mincle signalling in the intestine was addressed in experimental colitis models in vivo by using mice with Mincle knock out (Mincle−/−), neutralising anti-Mincle antibody, Mincle pharmacologic agonist and RNA-seq genome expression analysis. Bone marrow-derived macrophages were collected from mice and used to further verify the effect of Mincle signalling in macrophages. Results Mincle signalling was significantly elevated in active human CD and experimental colitis, and macrophages were the principal leukocyte subset that up-regulates Mincle signalling. Mincle deficiency ameliorated the colitis by reducing induced macrophage pyroptosis (Figure 1), whereas activation of Mincle with the pharmacologic agonist worsened the intestinal inflammation (Figure 2). Moreover, the ex vivo studies confirmed that Mincle signalling activation promoted and its absence restricted release of proinflammatory cytokines from pyroptosis of macrophage (Figure 3). Finally, Mincle/Syk signalling could promote the production of chemokines to recruit neutrophils by activating Mitogen-Activated Protein Kinase (MAPK) during inflammation (Figure 4). Conclusion Mincle signalling promotes intestinal mucosal inflammation through induction of macrophage pyroptosis and neutrophil chemotaxis. Modulation of the Mincle/Syk axis emerges as a potential therapeutic strategy to target inflammation and treat CD.

scholarly journals Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-induced acute lung injury in mice

2010 ◽  
Vol 299 (3) ◽  
pp. L312-L322 ◽  
Author(s):  
Isabelle Métrailler-Ruchonnet ◽  
Alessandra Pagano ◽  
Stéphanie Carnesecchi ◽  
Karim Khatib ◽  
Pedro Herrera ◽  
...  
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Lung Injury ◽  
Ex Vivo ◽  
Lung Damage ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lung Weight

Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.

Treatment with Bortezomib Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1426-1426
Author(s):  
Yachiyo Kuwatsuka ◽  
Yosuke Minami ◽  
Ryohei Tanizaki ◽  
Miho Minami ◽  
Akihiro Abe ◽  
...  
Keyword(s):  
Cell Death ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Leukemia Cells ◽  
Imatinib Treatment ◽  
Spleen Cells ◽  
Quiescent Cells ◽  
Nog Mice

Abstract Abstract 1426 Poster Board I-449 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. In order to examine mechanisms of drug resistance in LSCs and to seek strategies to overcome the resistance, we used Ph-positive acute lymphoblastic leukemia patient cells serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Recently several publications have suggested that proteasome inhibitors can induce selective cell death in LSCs. Spleen cells derived from leukemic NOG mice were treated ex vivo with imatinib and the proteasome inhibitor, bortezomib and cell viablility (PI-/Annexin-V-) was compared between treated and non-treated cells. After treatment with imatinib, significantly more residual cells were observed in the CD34+CD38- population compared to the CD34+CD38+ or CD34-CD38+ populations. With nM level of bortezomib, substantial cell death was induced in all populations with up-regulation of phospho-p53 (Ser15). Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with imatinib treatment, but not with bortezomib treatment. Regarding cell cycle states, a higher percentage of Hoechst-33342low/Pyronin-Ylow cells was observed in the CD34+CD38- population relative to the other populations, suggesting more cells in the G0 state among the CD34+CD38- population. In co-culturing with S17 stromal cells, quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib, while substantial cell death including CD34+ population was induced with nM level of bortezomib. We are also investigating more detailed biomarkers in the cell death and effects of these drugs both on the primitive leukemia cells and normal hematopoietic cells using the in vivo leukemic NOG mice systems. These results imply that resistance to imatinib in Ph-positive leukemia quiescent cells is independent of BCR-ABL phosphorylation and that treatment with bortezomib can overcome the resistance of Ph-positive LSCs. Disclosures Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe: Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.

Activity of MK1775, a selective Wee1 inhibitor, alone or in combination with gemcitabine, in sarcomas.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 10084-10084
Author(s):  
Jenny Kreahling ◽  
Damon R. Reed ◽  
Parastou Foroutan ◽  
Gary Martinez ◽  
Robert Gillies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Soft Tissue ◽  
Soft Tissue Sarcoma ◽  
Therapeutic Efficacy ◽  
Ex Vivo ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Primary Therapy

10084 Background: Sarcomas consist of more than 50 subtypes of mesenchymal tumors. Doxorubicin alone or in combination has been the primary therapy for treatment of sarcomas; however, the response rates are suboptimal in many of the more common adult subtypes of soft tissue sarcoma. Accordingly, new agents are needed for the treatment of this heterogeneous group of diseases. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Methods: MK1775 treatment of multiple sarcoma preclinical models at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death. In our current study we have investigated the therapeutic efficacy of MK1775 in sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo in a xenograft model of osteosarcoma both alone and in combination with gemcitabine. Results: In patient-derived bone and soft tissue sarcoma samples ex vivo treatments show MK1775 in combination with gemcitabine causes significant apoptotic cell death suggesting that this treatment may represent a novel approach in the treatment of sarcomas. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 mutational status. Furthermore, in a patient-derived osteosarcoma xenograft mouse model we show the therapeutic efficacy of MK1775 in vivo by utilizing magnetic resonance imaging (MRI) and diffusion MRI methods. Our data shows MK1775 in combination with gemcitabine dramatically slows tumor growth, increases apoptotic cell death and increases CDC2 activity. Cell viability, a clinically established prognostic indicator of survival, was lowest with the combination and very low in animals treated with MK1775 alone. This was mainly due to increased mineralization of the tumors. Caspase-3 was increased in MK1775 treated animals by immunohistochemistry as well. Conclusions: These results together with the promising safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent alone or in combination with gemcitabine in the treatment of both adult as well as pediatric sarcoma patients.

Factor XII: New life for an old protein

2010 ◽  
Vol 104 (11) ◽  
pp. 915-918 ◽  
Author(s):  
Gretchen LaRusch ◽  
Alvin Schmaier
Keyword(s):  
Cell Growth ◽  
Knockout Mice ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Antigen Expression ◽  
Mitogen Activated Protein Kinase ◽  
Factor Xii ◽  
Contact Activation ◽  
Urokinase Plasminogen Activator Receptor

SummaryRatnoff and his coworkers recognised that factor XII (XII) stimulates cell growth and activates mitogen-activated protein kinase. We determined the receptor(s) for this function and the consequence of this signalling pathway. Investigations show that the urokinase plasminogen activator receptor serves as the XII binding site on cultured umbilical vein endothelial cells. When XII binds, it stimulates ERK1/2 and Akt S473 phosphorylation. These events are distinct because when cell mTORC2 is absent, XII phosphorylates ERK1/2 but not Akt S473. Zymogen XII is an equal stimulator of signalling as XIIa or inhibitor-treated XIIa. Peptides from uPAR domain 2 block XII binding and ERK1/2 and Akt phosphorylation. Furthermore, antibodies to the integrins β1 and α5 block XII signalling. Likewise, inhibitors to the EGFR block XII-induced phosphorylation events. XII stimulates cell growth and proliferation. XII induces angiogenesis ex vivo in normal aortic sprouts and in vivo in matrigel plugs in normal mice, but not in aorta from uPAR knockout mice or matrigel plugs placed into uPAR-deleted mice. Skin biopsies constitutively or in a wound nine days after injury show reduced CD31 antigen expression in specimens from XII knockout mice compared to wild-type mice. These studies indicate that XII stimulates angiogenesis, a physiologic function independent of contact activation.

scholarly journals Activation of Simultaneous Apoptosis and Necroptosis to Eradicate Drug Resistant Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1283-1283
Author(s):  
Scott McComb ◽  
Julia Aguadé-Gorgorió ◽  
Blerim Marovca ◽  
Lena Harder ◽  
Gunnar Cario ◽  
...  
Keyword(s):  
Cell Death ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Short Circuit ◽  
Large Set ◽  
Ex Vivo Model ◽  
Resistant Disease

Abstract Dysregulation of apoptotic pathways provides an indiscriminate mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death induced by many chemotherapeutic compounds. Here we have assessed the potential of SMAC mimetic (SM) compounds to short circuit cell death resistance by blocking the pro-survival cellular inhibitor of apoptosis (cIAP) proteins. By screening a large set of patient-derived precursor B-cell ALL samples in an ex vivo model of the leukemia microenvironment we detect exquisite sensitivity to two different SM compounds, Birinapant and LCL161, in about one third of ALL samples. Strong ex vivo SM activity correlated with potent in vivo anti-leukemic efficacy against de novo refractory and relapsed ALL xenografts. Intriguingly, we find that although SM-sensitivity is independent of TNF and TNFR1 levels, expression of TNFR2 is highly predictive of response to SM in two separate cohorts of ALL samples, suggesting that TNFR2 expression may represent a promising biomarker for identifying SM-sensitive cells. Downstream, we employ a novel and powerful multi-colour Lenti-CRISPR approach to show that simultaneous disruption of both apoptotic and necroptotic genes is necessary to block SM-induced death. In contrast, disruption of RIP1 alone was adequate to block SM-induced apoptosis and necroptosis. Surprisingly, RIP1 loss had no significant impact on response to standard anti-leukemic therapies, supporting a view that the RIP1-dependent death pathway is not likely to be selected against in leukemia cells that have failed to respond to front line therapy. These results provide the first evidence that SM compounds can circumvent apoptotic escape in drug-refractory ALL through parallel activation of both RIP1-dependent apoptosis and necroptosis. Furthermore, our data strongly support further development of SM as anti-leukemic agents for treatment in resistant disease. Disclosures No relevant conflicts of interest to declare.

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