scholarly journals Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Nitin Puri ◽  
Yevgeniy Arefiev ◽  
Robert Chao ◽  
David Sacerdoti ◽  
Hibba Chaudry ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heme Oxygenase ◽  
Iron Homeostasis ◽  
Transmembrane Protein ◽  
Low Grade ◽  
Obese Mice ◽  
Cellular Iron ◽  
Cobalt Protoporphyrin ◽  
Stress Response System ◽  
Metabolic Regulators

Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin-a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO) is a stress response system which reduces oxidative stress. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in hepatic tissues from lean and obese mice. Obese mice exhibited hyperglycemia (p<0.05); increased levels of proinflammatory cytokines (MCP-1, IL-6,p<0.05); oxidative stress (p<0.05); and increased hepatic hepcidin levels (p<0.05). Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p<0.05). However, this effect is accompanied by a significant decline in ferritin expression. Additionally, there are reduced insulin receptor phosphorylation and attenuation of metabolic regulators pAMPK, pAKT, and pLKB1. Cobalt protoporphyrin- (CoPP-) induced HO-1 upregulation in obese mice reversed these alterations (p<0.05), while attenuating hepatic hepcidin levels. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP) (p<0.05). Our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin.

Abstract P338: Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Hibba Chaudhry ◽  
Alexandra Nichols ◽  
Komal Sodhi ◽  
Krithika Srikanthan ◽  
Athar Nawab
Keyword(s):  
Oxidative Stress ◽  
Heme Oxygenase ◽  
Iron Homeostasis ◽  
Transmembrane Protein ◽  
Low Grade ◽  
Obese Mice ◽  
Liver Iron ◽  
Cellular Iron ◽  
Cobalt Protoporphyrin ◽  
Stress Response System

Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin- a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO) is a stress response system, the induction of which reduces oxidative stress thereby abating patho-physiological conditions such as obesity and metabolic syndrome.8 week old male obese (ob) mice and their age- and sex-matched lean mice were used as controls. CoPP was administered intraperitoneally once a week (3 mg/kg) for 6 weeks to obese mice. CoPP plus stannous mesoporphyrin (SnMP) was administered intraperitoneally three times a week (20 mg/kg) for 6 weeks. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in tissues from lean and obese mice. Obese mice exhibited hyperglycemia along with increased levels of pro-inflammatory cytokines (MCP-1, IL-6, p<0.05), oxidative stress and increased hepatic hepcidin levels (p<0.05). Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p<0.05). Further, our results showed attenuation of insulin receptor phosphorylation and attenuation of metabolic regulators including pAMPK, pAKT and pLKB1. Cobalt protoporphyrin (CoPP)-induced HO-1 up-regulation in obese mice and reversed these pathophysiological alterations (p<0.05) while attenuating hepatic hepcidin levels and enhancing ferritin expression. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP) (p<0.05). Taken together, our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin in conjunction with the rescue of cellular ferritin levels. Therefore, these findings may prove an effective strategy in treating the metabolic consequences of obesity including alteration of liver iron homeostasis.

scholarly journals Heme Oxygenase-1 Induction by Cobalt Protoporphyrin Ameliorates Cholestatic Liver Disease in a Xenobiotic-Induced Murine Model

2021 ◽  
Vol 22 (15) ◽  
pp. 8253
Author(s):  
Jung-Yeon Kim ◽  
Yongmin Choi ◽  
Jaechan Leem ◽  
Jeong Eun Song
Keyword(s):  
Oxidative Stress ◽  
Liver Disease ◽  
Heme Oxygenase ◽  
Murine Model ◽  
Liver Diseases ◽  
Transforming Growth Factor ◽  
Heme Oxygenase 1 ◽  
Cholestatic Liver Disease ◽  
Hepatocyte Apoptosis ◽  
Cobalt Protoporphyrin

Cholestatic liver diseases can progress to end-stage liver disease and reduce patients’ quality of life. Although their underlying mechanisms are still incompletely elucidated, oxidative stress is considered to be a key contributor to these diseases. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that displays antioxidant action. It has been found that this enzyme plays a protective role against various inflammatory diseases. However, the role of HO-1 in cholestatic liver diseases has not yet been investigated. Here, we examined whether pharmacological induction of HO-1 by cobalt protoporphyrin (CoPP) ameliorates cholestatic liver injury. To this end, a murine model of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet feeding was used. Administration of CoPP ameliorated liver damage and cholestasis with HO-1 upregulation in DDC diet-fed mice. Induction of HO-1 by CoPP suppressed the DDC diet-induced oxidative stress and hepatocyte apoptosis. In addition, CoPP attenuated cytokine production and inflammatory cell infiltration. Furthermore, deposition of the extracellular matrix and expression of fibrosis-related genes after DDC feeding were also decreased by CoPP. HO-1 induction decreased the number of myofibroblasts and inhibited the transforming growth factor-β pathway. Altogether, these data suggest that the pharmacological induction of HO-1 ameliorates cholestatic liver disease by suppressing oxidative stress, hepatocyte apoptosis, and inflammation.

scholarly journals Antioxidant Roles of Heme Oxygenase, Carbon Monoxide, and Bilirubin in Cerebral Circulation during Seizures

2012 ◽  
Vol 32 (6) ◽  
pp. 1024-1034 ◽  
Author(s):  
Helena Parfenova ◽  
Charles W Leffler ◽  
Shyamali Basuroy ◽  
Jianxiong Liu ◽  
Alexander L Fedinec
Keyword(s):  
Oxidative Stress ◽  
Carbon Monoxide ◽  
Heme Oxygenase ◽  
Vascular Dysfunction ◽  
Antioxidant Properties ◽  
Cerebrovascular Function ◽  
Cerebrovascular Dysfunction ◽  
Cobalt Protoporphyrin ◽  
Endogenous Antioxidant ◽  
The Brain

Postictal cerebrovascular dysfunction is an adverse effect of seizures in newborn piglets. The brain heme oxygenase (HO) provides protection against cerebrovascular dysfunction. We investigated the contribution of reactive oxygen species (ROS) to seizure-induced vascular damage and the mechanism of HO vasoprotection. In a bicuculline model of seizures, we addressed the hypotheses: (1) seizures increase brain ROS; (2) ROS contribute to cerebral vascular dysfunction; (3) ROS initiate a vasoprotective mechanisms by activating endogenous HO; and (4) HO products have antioxidant properties. As assessed by dihydroethidium oxidation (ox-DHE), seizures increased ROS in cerebral vessels and cortical astrocytes; ox-DHE elevation was prevented by tiron and apocynin. An HO inhibitor, tin protoporphyrin, potentiated, whereas an HO-1 inducer, cobalt protoporphyrin, blocked seizure-induced increase in DHE oxidation. Heme oxygenase products carbon monoxide (CO) (CORM-A1) and bilirubin attenuated ox-DHE elevation during seizures. Antioxidants tiron and bilirubin prevented the loss of postictal cerebrovascular dilations to bradykinin, glutamate, and sodium nitroprusside. Tiron and apocynin abrogated activation of the brain HO during seizures. Overall, these data suggest that long-term adverse cerebrovascular effects of seizures are attributed to oxidative stress. On the other hand, seizure-induced ROS are required for activation of the endogenous antioxidant HO/CO/bilirubin system that alleviates oxidative stress-induced loss of postictal cerebrovascular function in piglets.

scholarly journals Antidiabetic Potential of the Heme Oxygenase-1 Inducer Curcumin Analogues

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Yong Son ◽  
Ju Hwan Lee ◽  
Yong-Kwan Cheong ◽  
Hun-Taeg Chung ◽  
Hyun-Ock Pae
Keyword(s):  
Oxidative Stress ◽  
Er Stress ◽  
Heme Oxygenase ◽  
Stress Responses ◽  
Therapeutic Potential ◽  
Heme Oxygenase 1 ◽  
Low Grade ◽  
Cellular Protection ◽  
Inflammatory Stress ◽  
Curcumin Analogues

Although there is a therapeutic treatment to combat diabetes, the identification of agents that may deal with its more serious aspects is an important medical field for research. Diabetes, which contributes to the risk of cardiovascular disease, is associated with a low-grade chronic inflammation (inflammatory stress), oxidative stress, and endoplasmic reticulum (ER) stress. Because the integration of these stresses is critical to the pathogenesis of diabetes, agents and cellular molecules that can modulate these stress responses are emerging as potential targets for intervention and treatment of diabetic diseases. It has been recognized that heme oxygenase-1 (HO-1) plays an important role in cellular protection. Because HO-1 can reduce oxidative stress, inflammatory stress, and ER stress, in part by exerting antioxidant, anti-inflammatory, and antiapoptotic effects, HO-1 has been suggested to play important roles in pathogenesis of diabetes. In the present review, we will explore our current understanding of the protective mechanisms of HO-1 in diabetes and present some emerging therapeutic options for HO-1 expression in treating diabetic diseases, together with the therapeutic potential of curcumin analogues that have their ability to induce HO-1 expression.

scholarly journals Reciprocal Effects of Oxidative Stress on Heme Oxygenase Expression and Activity Contributes to Reno-Vascular Abnormalities in EC-SOD Knockout Mice

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Tomoko Kawakami ◽  
Nitin Puri ◽  
Komal Sodhi ◽  
Lars Bellner ◽  
Toru Takahashi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heme Oxygenase ◽  
Redox Homeostasis ◽  
Inhibitory Effect ◽  
Serum Adiponectin ◽  
Causative Role ◽  
Vascular Abnormalities ◽  
Chronic Oxidative Stress ◽  
Cobalt Protoporphyrin ◽  
Physiological Abnormalities

Heme oxygenase (HO) system is one of the key regulators of cellular redox homeostasis which responds to oxidative stress (ROS) via HO-1 induction. However, recent reports have suggested an inhibitory effect of ROS on HO activity. In light of these conflicting reports, this study was designed to evaluate effects of chronic oxidative stress on HO system and its role in contributing towards patho-physiological abnormalities observed in extracellular superoxide dismutase (EC-SOD, SOD3) KO animals. Experiments were performed in WT and EC-SOD(−/−)mice treated with and without HO inducer, cobalt protoporphyrin (CoPP). EC-SOD(−/−)mice exhibited oxidative stress, renal histopathological abnormalities, elevated blood pressure, impaired endothelial function, reduced p-eNOS, p-AKT and increased HO-1 expression; although, HO activity was significantly (P<0.05) attenuated along with attenuation of serum adiponectin and vascular epoxide levels (P<0.05). CoPP, in EC-SOD(−/−)mice, enhanced HO activity (P<0.05) and reversed aforementioned pathophysiological abnormalities along with restoration of vascular EET, p-eNOS, p-AKT and serum adiponectin levels in these animals. Taken together our results implicate a causative role of insufficient activation of heme-HO-adiponectin system in pathophysiological abnormalities observed in animal models of chronic oxidative stress such as EC-SOD(−/−)mice.

scholarly journals Ferroptosis Signaling and Regulators in Atherosclerosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuqin Wang ◽  
Yajie Zhao ◽  
Ting Ye ◽  
Liming Yang ◽  
Yanna Shen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Iron Homeostasis ◽  
Hippo Pathway ◽  
Signal Pathways ◽  
Independent Manner ◽  
Cellular Iron ◽  
New Ideas ◽  
Abnormal Lipid Metabolism ◽  
The Hippo Pathway ◽  
Cellular Iron Homeostasis

Atherosclerosis (AS) is a major cause of cardiovascular diseases such as coronary heart disease, heart failure and stroke. Abnormal lipid metabolism, oxidative stress and inflammation are the main features of AS. Ferroptosis is an iron-driven programmed cell death characterized by lipid peroxidation, which have been proved to participate in the development and progression of AS by different signal pathways. NRF2-Keap1 pathway decreases ferroptosis associated with AS by maintaining cellular iron homeostasis, increasing the production glutathione, GPX4 and NADPH. The p53 plays different roles in ferroptosis at different stages of AS in a transcription-dependent and transcription- independent manner. The Hippo pathway is involved in progression of AS, which has been proved the activation of ferroptosis. Other transcription factors, such as ATF3, ATF4, STAT3, also involved in the occurrence of ferroptosis and AS. Certain proteins or enzymes also have a regulatory role in AS and ferroptosis. In this paper, we review the mechanism of ferroptosis and its important role in AS in an attempt to find a new relationship between ferroptosis and AS and provide new ideas for the future treatment of AS.

scholarly journals Vitamin D-Mediated Anti-cancer Activity Involves Iron Homeostatic Balance Disruption and Oxidative Stress Induction in Breast Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Khuloud Bajbouj ◽  
Lina Sahnoon ◽  
Jasmin Shafarin ◽  
Abeer Al-Ali ◽  
Jibran Sualeh Muhammad ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Oxidative Stress ◽  
Vitamin D ◽  
Breast Cancer Cells ◽  
Iron Homeostasis ◽  
Stress Induction ◽  
Protein Levels ◽  
Cellular Iron ◽  
Anti Cancer ◽  
Cellular Iron Homeostasis

Background: Vitamin D deficiency associates with high risk of breast cancer (BRCA) and increased cellular iron. Vitamin D exerts some of its anti-cancer effects by regulating the expression of key iron regulatory genes (IRGs). The association between vitamin D and cellular iron content in BRCA remains ambiguous. Herein, we addressed whether vitamin D signaling exerts a role in cellular iron homeostasis thereby affecting survival of breast cancer cells.Methods: Expression profile of IRGs in vitamin D-treated breast cancer cells was analyzed using publicly available transcriptomic datasets. After treatment of BRCA cell lines MCF-7 and MDA-MB-231 with the active form of vitamin D, labile iron content, IRGs protein levels, oxidative stress, and cell survival were evaluated.Results: Bioinformatics analysis revealed several IRGs as well as cellular stress relates genes were differentially expressed in BRCA cells. Vitamin D treatment resulted in cellular iron depletion and differentially affected the expression of key IRGs protein levels. Vitamin D treatment exerted oxidative stress induction and alteration in the cellular redox balance by increasing the synthesis of key stress-related markers. Collectively, these effects resulted in a significant decrease in BRCA cell survival.Conclusion: These findings suggest that vitamin D disrupts cellular iron homeostasis leading to oxidative stress induction and cell death.

scholarly journals A novel mechanism of heme degradation to biliverdin studied by QM/MM and QM calculations

2018 ◽  
Vol 47 (25) ◽  
pp. 8283-8291 ◽  
Author(s):  
Fatemeh Sadat Alavi ◽  
Mahin Gheidi ◽  
Mansour Zahedi ◽  
Nasser Safari ◽  
Ulf Ryde
Keyword(s):  
Oxidative Stress ◽  
Heme Oxygenase ◽  
Iron Homeostasis ◽  
Heme Degradation

Heme degradation by heme oxygenase enzymes is important for maintaining iron homeostasis and prevention of oxidative stress.

scholarly journals Regulation of Iron Storage by CsrA Supports Exponential Growth of Escherichia coli

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Christine Pourciau ◽  
Archana Pannuri ◽  
Anastasia Potts ◽  
Helen Yakhnin ◽  
Paul Babitzke ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Escherichia Coli ◽  
Exponential Growth ◽  
Iron Homeostasis ◽  
Iron Storage ◽  
Content Type ◽  
Mrna Targets ◽  
Cellular Iron ◽  
The Impact

ABSTRACT The global regulatory protein CsrA coordinates gene expression in response to physiological cues reflecting cellular stress and nutrition. CsrA binding to the 5′ segments of mRNA targets affects their translation, RNA stability, and/or transcript elongation. Recent studies identified probable mRNA targets of CsrA that are involved in iron uptake and storage in Escherichia coli, suggesting an unexplored role for CsrA in regulating iron homeostasis. Here, we assessed the impact of CsrA on iron-related gene expression, cellular iron, and growth under various iron levels. We investigated five new targets of CsrA regulation, including the genes for 4 ferritin or ferritin-like iron storage proteins (ISPs) and the stress-inducible Fe-S repair protein, SufA. CsrA bound with high affinity and specificity to ftnB, bfr, and dps mRNAs and inhibited their translation, while it modestly activated ftnA expression. Furthermore, CsrA was found to regulate cellular iron levels and support growth by repressing the expression of genes for ISPs, most importantly, ferritin B (FtnB) and bacterioferritin (Bfr). Iron starvation did not substantially affect cellular levels of CsrA or its small RNA (sRNA) antagonists, CsrB and CsrC. csrA disruption led to increased resistance to the lethal effects of H2O2 during exponential growth, consistent with a regulatory role in oxidative stress resistance. We propose that during exponential growth and under minimal stress, CsrA represses the deleterious expression of the ISPs that function under oxidative stress and stationary-phase conditions (FtnB, Bfr, and Dps), thus ensuring that cellular iron is available to processes that are required for growth. IMPORTANCE Iron is an essential micronutrient for nearly all living organisms but is toxic in excess. Consequently, the maintenance of iron homeostasis is a critical biological process, and the genes involved in this function are tightly regulated. Here, we explored a new role for the bacterial RNA binding protein CsrA in the regulation of iron homeostasis. CsrA was shown to be a key regulator of iron storage genes in Escherichia coli, with consequential effects on cellular iron levels and growth. Our findings establish a model in which robust CsrA activity during the exponential phase of growth leads to repression of genes whose products sequester iron or divert it to unnecessary stress response processes. In so doing, CsrA supports E. coli growth under iron-limiting laboratory conditions and may promote fitness in the competitive iron-limited environment of the host large intestine.

An Anti-Apoptotic Molecule, Anamorsin, Functions in Both Iron-Sulfur Protein Assembly and Cellular Iron Homeostasis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2106-2106 ◽  
Author(s):  
Akira Tanimura ◽  
Yuri Kondo ◽  
Hirokazu Tanaka ◽  
Itaru Matsumura ◽  
Tomohiko Ishibashi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Lines ◽  
Iron Homeostasis ◽  
Binding Activity ◽  
Wild Type ◽  
S Protein ◽  
Aconitase Activity ◽  
Cellular Iron ◽  
Iron Sulfur ◽  
Retrovirus Vector

Abstract Abstract 2106 Introduction: Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells (J.Exp.Med. 199:581–592, 2004). AM has no structural homology to any known anti-apoptosis molecules such as Bcl-2 and IAP family members. AM-deficient (AM−/−) mice are embryonic lethal at late gestation. Its embryos are anemic and the size of embryos is very small. It is thought that AM plays a crucial role in hematopoiesis, however the precise biological mechanisms of AM remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569-5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the cellular iron regulation. Methods and Results: At first, in order to analyze molecular and cellular events, we established cell lines from wild-type or AM-deficient embryonic fetal liver (14.5dpc) by using SV40 large T antigen. We isolated 5 wild-type cell lines (AM WTs) and 2 AM-deficient cell lines (AM KOs) respectively. Next, we compared the cell growth and apoptosis in both cell lines and found that the growth rate of AM KOs were slightly lower than that of AM WTs although these cell lines were immortalized. AM KOs showed more significant apoptosis induced by oxidative stress; the percents of Annexin V positive fraction were 12 ± 4 and 36 ± 6 in AM WTs and AM KOs respectively under the condition of 0.1mM H2O2 for 16hr. In addition to oxidative stress, AM KOs were more sensitive to UV irradiation. These differences were cancelled by transduction of AM-expression retrovirus vector in AM KOs. It was reported that Dre2 functions in cytosolic Fe/S protein biogenesis. We examined whether AM might be involved in the maturation of cytosolic Fe/S proteins. Iron regulatory protein 1 (IRP1) is a well-known cytosolic Fe/S protein with dual functions; in the presence of an [4Fe-4S] cluster it functions as a cytosolic aconitase, while IRP1 binds to mRNA stem-loop structures called iron responsive elements (IREs) and confer the mRNA stability when the [4Fe-4S] cluster is missing. In the iron-deficient cells, IRP1 binds to IREs located at the mRNA of iron transferrin receptor (TfR), ferritin and other iron metabolism transcripts, thereby enhancing iron uptake. In this way, it is thought that IRP1 plays important roles in iron homeostasis. We therefore compared the aconitase activity and IRE binding activities of IRP1 between AM WTs and AM KOs and found that AM deficiency resulted in the decrease of cytosolic aconitase activity (approximately 30% compared to AM WTs). In contrast to cytosolic aconitase activity, the mitochondrial aconitase activity showed little change regardless of AM deficiency. In order to analyze whether AM deficiency might increase IRE binding activity of IRP1, cytoplasmic extracts of AM WTs and AM KOs were compared by RNA precipitation assay. In AM KOs, the expression level of IRP1 decreased approximately one third compared to AM WTs. However, the binding activity of IRP1 to biotin-labeled IRE increased in the extract of AM KOs approximately three-fold in comparison to AM WTs. These differences was cancelled by transduction of AM-expression retrovirus vector to AM KOs. All these findings demonstrated the involvement of AM in the maturation of the cytosolic Fe/S protein, IRP1. Furthermore, we examined the expression of TfR, which is known to be modulated by IRP1-mediated posttranscriptional regulation. In the presence of iron chelator, desferrioxamine, the expression of TfR in AM WTs was markedly elevated. On the other hand, in AM KOs, the expression of TfR was hardly elevated. Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration due to the decreased function of IRP1. Conclusion: Our current findings indicate that AM is essential for cytosolic Fe/S cluster biogenesis and iron homeostasis. Now the influence of the AM-mediated iron homeostasis on hematopoiesis is under investigation. Disclosures: No relevant conflicts of interest to declare.

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