Abstract 466: Stigmasterol Accumulation in ABCG8 Knockout Mice Does Not Account for Xenosterol Toxicity

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Shailendra B Patel
Keyword(s):  
Body Fat ◽  
Knockout Mice ◽  
Biological Effect ◽  
Target Genes ◽  
Cardiac Fibrosis ◽  
Plasma Cholesterol ◽  
Biological Effects ◽  
Cholesterol Homeostasis ◽  
Significant Toxicity ◽  
Glucose Plasma

Xenosterol accumulation mice deficient in sterolin function leads to significant toxicity, with infertility, decreased body fat accumulation, macrothrombocytopenia, cardiac fibrosis and premature death1, 2. The predominant xenosterols are phytosterols derived from the diet and are a mixture of sitosterol (typically 70%), campesterol (~20%) and stigmasterol (~5-10%). Although sitosterol has been shown to have some biological effect in tissue culture, only stigmasterol has been shown to have a potent biological effect, by activating the transcriptional factor, Lxr3. To delineate whether all of the toxic biological effects were mediated by stigmasterol accumulation, we designed a diet that was supplemented with highly enriched stigmasterol (>80% purity) and fed this to Abcg8 knockout mice. Stigmasterol accumulation in the plasma and tissues was comparable to the levels observed in mice fed a mixed phytosterol-enriched diet that had been shown to result in toxicity. Over a 12-week period, both male and female Abcg8 knockout mice gained normal amounts of weight, body fat, showed no disturbances in tail-cuff measured blood pressure, and plasma analyses showed no abnormalities of platelet counts or volumes, blood glucose, plasma cholesterol, despite accumulation of stigmasterol in the plasma and tissues. Fertility testing showed no abnormalities. Gene expression analyses of livers did not show any consistent patterns, although Lxr target genes were not up-regulated. These data do not support the concept that stigmasterol accumulation, at levels of 5-10mg/dL in plasma, account for the xenosterol-mediated toxicity observed. 1. McDaniel, A.L., H.M. Alger, J.K. Sawyer, K.L. Kelley, N.D. Kock, J.M. Brown, R.E. Temel, and L.L. Rudel, Phytosterol feeding causes toxicity in ABCG5/G8 knockout mice. Am J Pathol, 2013. 182(4): p. 1131-8. 2. Solca, C., G.S. Tint, and S.B. Patel, Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice. J Lipid Res, 2013. 54(2): p. 397-409. 3. Yang, C., L. Yu, W. Li, F. Xu, J.C. Cohen, and H.H. Hobbs, Disruption of cholesterol homeostasis by plant sterols. J. Clin. Invest., 2004. 114(6): p. 813-22.

Abstract 667: Ergosterol or Brassicasterol Accumulations in ABCG8 Knockout Mice Do Not Account for Xenosterol Toxicity

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Shailendra B Patel
Keyword(s):  
Body Fat ◽  
Knockout Mice ◽  
Biological Effect ◽  
Target Genes ◽  
Cardiac Fibrosis ◽  
Plasma Cholesterol ◽  
Transcriptional Factor ◽  
Significant Toxicity ◽  
Metabolism Enzymes ◽  
Gene Expression Analyses

Xenosterol accumulation mice deficient in sterolin function leads to significant toxicity, with infertility, decreased body fat accumulation, macrothrombocytopenia, cardiac fibrosis and premature death1,2. Although sitosterol has been shown to have some biological effect in tissue culture, only stigmasterol has been shown to have a potent biological effect, by activating the transcriptional factor, Lxr3. In patients with sitosterolemia, feeding shell-fish sterols led to accumulation of shell-fish sterols in their plasma4, suggesting any xenosterol could accumulate, if fed, to mammals deficient in Abcg5 or Abcg8. Fungi do not utilize cholesterol, but instead use ergosterol as the primary membrane sterol. To delineate whether ergosterol could accumulate in Abcg8 knockout mice, and lead to toxicity, we designed a diet that was supplemented with highly enriched ergosterol (>98% purity) and fed this to Abcg8 knockout mice. Over a 12-week period, both male and female Abcg8 knockout mice fed an ergosterol-enriched diet gained normal amounts of weight, body fat, showed no disturbances in tail-cuff measured blood pressure, and showed no abnormalities of platelet counts or volumes, blood glucose, or plasma cholesterol. Fertility testing showed no abnormalities. Dramatically, analyses of plasma from these mice showed no accumulation of ergosterol, but a dramatic increase in plasma brassicasterol, with levels reaching 80mg/dL in plasma. Gene expression analyses of livers did not show any consistent patterns, although Lxr target genes were not up-regulated. These data do not support the concept that brassicasterol accumulation, at levels of 80mg/dL in plasma, account for the xenosterol-mediated toxicity observed. Additionally, the intestine shows powerful 1st pass detoxification of ergosterol by converting it to brassicaterol by using (presumably) dehydrocholesterol Δ7 reductase enzyme and highlights the importance of intermediary metabolism enzymes as part of the defense against xenosterols. 1. McDaniel, A.L.et al Am J Pathol, 2013. 182(4): p. 1131-8. 2. Solca, C. et al J Lipid Res, 2013. 54(2): p. 397-409. 3. Yang, C.et al J. Clin. Invest., 2004. 114(6): p. 813-22. 4. Gregg, R.E.et al J. Clin. Invest., 1986. 77(6): p. 1864-72.

scholarly journals SREBP2 mediates the modulation of intestinal NPC1L1 expression by curcumin

2011 ◽  
Vol 301 (1) ◽  
pp. G148-G155 ◽  
Author(s):  
Pradeep Kumar ◽  
Pooja Malhotra ◽  
Ke Ma ◽  
Amika Singla ◽  
Omar Hedroug ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Plasma Cholesterol ◽  
Biological Effects ◽  
Cholesterol Absorption ◽  
Inhibitory Effect ◽  
Cholesterol Homeostasis ◽  
Cholesterol Esterification ◽  
Dependent Manner ◽  
Intestinal Cholesterol Absorption

Curcumin, the major phenolic compound in the spice turmeric, exhibits numerous biological effects, including lowering plasma cholesterol and preventing diet-induced hypercholesterolemia. The mechanisms underlying the hypocholesterolemic effect of curcumin are not fully understood. In this regard, intestinal Niemann-Pick C1-like 1 (NPC1L1) cholesterol transporter, the molecular target of intestinal cholesterol absorption inhibitor ezetimibe, plays an essential role in the maintenance of cholesterol homeostasis. The current studies were designed to investigate the effect of curcumin on NPC1L1 function, expression, and promoter activity in intestinal Caco-2 monolayers. NPC1L1 function was evaluated by the measurement of ezetimibe-sensitive [3H]cholesterol esterification. Relative abundance of NPC1L1 mRNA and protein was evaluated by real-time PCR and Western blotting, respectively. Luciferase assays were used to measure NPC1L1 promoter activity. Our results showed that curcumin significantly inhibited ezetimibe-sensitive cholesterol esterification in a dose-dependent manner with a maximum decrease (by 52% compared with control) occurring at 50 μM concentration. Curcumin treatment of Caco-2 monolayers also significantly decreased NPC1L1 mRNA and protein expression. Similarly, the promoter activity of the NPC1L1 gene was inhibited significantly (55%) by 50 μM curcumin. The decrease in NPC1L1 promoter activity by curcumin was associated with a reduction in the expression and the DNA-binding activity of the sterol response element-binding protein 2 (SREBP2) transcription factor. Furthermore, the overexpression of active SREBP2 protected NPC1L1 from the inhibitory effect of curcumin. Our studies demonstrate that curcumin directly modulates intestinal NPC1L1 expression via transcriptional regulation and the involvement of SREBP2 transcription factor.

Abstract 129: Liver-Specific and Intestine-Specific ACAT2 Knockout Mice Are Equally Protected from Diet-Induced Hepatic Cholesterol Accumulation

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jun Zhang ◽  
Kathryn Kelley ◽  
Stephanie Marshall ◽  
Matthew Davis ◽  
Martha Wilson ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Plasma Cholesterol ◽  
Dietary Cholesterol ◽  
Cholesterol Absorption ◽  
Cholesterol Homeostasis ◽  
Cholesterol Accumulation ◽  
Very Low Density Lipoproteins ◽  
Plasma Triglycerides ◽  
Hepatic Lipid Accumulation ◽  
Vldl Cholesterol

Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) is exclusively expressed in the small intestine and liver. ACAT2 facilitates the movement of cholesterol among tissues by generating cholesteryl ester (CE) for packaging into newly synthesized chylomicrons and very low-density lipoproteins (VLDL). In these studies we investigated whether CE derived from either the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. For this purpose, we generated both liver-specific (ACAT2L-/L-) and intestine-specific (ACAT2SI-/SI-) ACAT2 knockout mice, and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. Interestingly, diet-induced accumulation of hepatic CE was similarly decreased in both ACAT2L-/L- and ACAT2SI-/SI- mice, and free cholesterol did not build up in the liver. Compared with control mice, both ACAT2L-/L- and ACAT2SI-/SI- mice had lower levels of plasma VLDL-cholesterol but higher plasma triglycerides. ACAT2SI-/SI- but not ACAT2L-/L- mice had blunted cholesterol absorption. Collectively, both ACAT2L-/L- and ACAT2SI-/SI- mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of either intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice, indicating that inhibition of ACAT2 expression in either tissue likely would be beneficial for atheroprotection.

Abstract 692: Therapeutic Inhibition of miR-33 Does Not Promote Obesity, Insulin Resistance or Hepatic Lipid Accumulation

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Denuja Karunakaran ◽  
Michéle Geoffrion ◽  
Danyk Barrett ◽  
Mary-Ellen Harper ◽  
Christine C Esau ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Protein Expression ◽  
Hepatic Steatosis ◽  
Target Gene ◽  
Target Genes ◽  
Plasma Cholesterol ◽  
Cholesterol Homeostasis ◽  
Serum Triglycerides ◽  
Carbohydrate Utilization ◽  
Pharmacological Inhibition

Inhibition of miR-33, a critical post-transcriptional regulator of cholesterol homeostasis, prevents atherosclerosis progression both dependently and independently of its effects on circulating HDL levels. However, recent studies suggest that genetic deletion or inhibition of miR-33 may adversely affect metrics of insulin resistance, lipid metabolism and hepatic steatosis. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to determine the effects of long-term pharmacological inhibition of miR33 in a mouse model of diet-induced obesity (DIO). Results: C57BL6/J mice were fed a high-fat diet in conjunction with anti-miR therapy (10mg/kg of control anti-miR or anti-miR33) for 20 weeks. There was significant but equivalent weight gain in all 3 groups. While anti-miR33 therapy increased total plasma cholesterol compared to control anti-miR treated mice (p≤0.01), there was no differences relative to PBS treated mice. Anti-miR33 treatment also resulted in a marked decrease in serum triglycerides relative to control anti-miR (29% decrease, p≤0.05) but once again not compared to PBS treated mice. Metrics of insulin resistance (GTT, ITT) were not altered in anti-miR33 treated mice relative to controls. Interestingly, indirect calorimetric measurements demonstrated that anti-miR33 treated mice had reduced respiratory exchange ratios (RER) compared to both control anti-miR and PBS treated mice (18% decrease, p≤0.05), suggesting an increase in fatty acid versus carbohydrate utilization in anti-miR33 treated mice. In addition, hepatic protein expression of known miR-33 target gene ABCA1 was de-repressed upon miR-33 inhibition, indicating that miR-33 was efficiently inhibited in this model. In contrast, putative miR-33 target gene SREBP-1 protein expression was not altered, nor were SREBP-1 downstream target genes FASN and ACC. Finally, anti-miR33 treated mice did not accumulate more lipids in their livers relative to control. In conclusion, we show in a DIO model that therapeutic silencing of miR-33 does not promote hepatic steatosis nor does it increase metrics of insulin resistance, suggesting that pharmacological inhibition of miR-33 may be a safe therapeutic for the treatment of atherosclerosis.

scholarly journals Human adventitial pericytes secrete bioactive factors exerting distinct biological effects on cardiac cells: hints for cardiac repair

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
E Avolio ◽  
G Mangialardi ◽  
S Slater ◽  
V.V Alvino ◽  
K Heesom ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Stromal Cells ◽  
Synthetic Peptides ◽  
Biological Effect ◽  
Biological Effects ◽  
Biological Properties ◽  
Cardiac Cells ◽  
Mass Spectrometry Analysis ◽  
Funding Source ◽  
Paracrine Factors

Abstract Background Pericytes are attracting much attention as potential candidates for successful cell therapy of myocardial ischaemia. Intramyocardially delivered adventitial pericytes (APCs) secrete paracrine factors which stimulate angiogenesis and recruitment of cardiac stromal cells, reduce fibrosis and promote cardiomyocyte proliferation and viability. However, factors responsible for these biological effects have not been elucidated yet. Purpose To exploit the components of APC secretome exerting a biological effect on cardiac cells with the aim to discover new druggable targets with potential therapeutic activity. Methods and results APCs were derived from saphenous veins of adult patients (n=13, 68±11 yrs, all with coronary artery disease - CAD). The APC-conditioned medium (CM) stimulated the proliferation of human iPS-derived cardiomyocytes compared with unconditioned medium (UCM) (EdU incorporation, 1.3-fold increases, P=0.004). Stimulation with APC-CM increased the number of mitotic figures in cardiomyocytes (Aurora B, 1.5-fold increases compared to UCM, P=0.002). Furthermore, APC-CM abrogated the hypoxia-induced apoptosis in cardiomyocytes (2-fold increase in Caspase 3/7 activity in hypoxic cells exposed to UCM compared to normoxic cells, P=0.002). We also found that APC-CM stimulates the migration of human cardiac stromal cells (CSCs) obtained from healthy donors (n=6, 54±11 yrs) in both a transwell and scratch migration assays (n=6, P<0.01 and P<0.05 vs UCM respectively). Interestingly, APC-CM activated also the migration of HUVECs (n=6, P<0.01 vs UCM) but did not attract fibroblasts. Next, we aimed to identify the biologically active components of the APC-CM. Depletion of exosomes and heat and RNase treatments did not abolish the pro-migratory action of the APC-CM, while this was abrogated by Proteinase K. Fractionation of the APC-CM based on the MW indicated that the bioactive peptides have MW >30KDa. The pro-migratory fractions of the APC-CM obtained from size exclusion chromatography underwent mass spectrometry analysis (n=3 APCs). This identified 14 proteins uniquely present in the pro-migratory fractions. The two most relevant candidates were SPARC and TGFBI, both confirmed by ELISA. Intriguingly, the recombinant SPARC and TGFBI failed to reproduce the biological effect of APC-CM on CSC migration, suggesting that the secreted proteins may carry unique post-translational modifications not found in synthetic peptides. Further analyses are being carried out to reveal the biological properties of the endogenous SPARC and TGFBI. Conclusions This study suggests a fascinating approach based on the use of the active component of the APC-CM as a surrogate of APC therapy. If the biological properties of the cellular proteins will be successfully reproduced in synthetic peptides in vitro, this innovative approach may extend the benefits of APC therapy to all those patients with CAD for whom cell therapy is not an available option. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): British Heart Foundation programme grant “Unravelling mechanism of stem cell depletion for the preservation of regenerative fitness in patients with diabetes”

scholarly journals Liver-Specific β-Catenin Knockout Mice Exhibit Defective Bile Acid and Cholesterol Homeostasis and Increased Susceptibility to Diet-Induced Steatohepatitis

2010 ◽  
Vol 176 (2) ◽  
pp. 744-753 ◽  
Author(s):  
Jaideep Behari ◽  
Tzu-Hsuan Yeh ◽  
Lindsay Krauland ◽  
Wade Otruba ◽  
Benjamin Cieply ◽  
...  
Keyword(s):  
Bile Acid ◽  
Knockout Mice ◽  
Cholesterol Homeostasis ◽  
Increased Susceptibility

scholarly journals Regulation of Cholesterol Homeostasis by the Liver X Receptors in the Central Nervous System

2002 ◽  
Vol 16 (6) ◽  
pp. 1378-1385 ◽  
Author(s):  
Karl D. Whitney ◽  
Michael A. Watson ◽  
Jon L. Collins ◽  
William G. Benson ◽  
Tammy M. Stone ◽  
...  
Keyword(s):  
Gene Expression ◽  
Central Nervous System ◽  
Nervous System ◽  
Cholesterol Efflux ◽  
Target Genes ◽  
Cholesterol Homeostasis ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
The Central Nervous System ◽  
Lxr Agonists

Abstract The nuclear oxysterol receptors liver X receptor-α [LXRα (NR1H3)] and LXRβ (NR1H2) coordinately regulate genes involved in cholesterol homeostasis. Although both LXR subtypes are expressed in the brain, their roles in this tissue remain largely unexplored. In this report, we show that LXR agonists have marked effects on gene expression in murine brain tissue both in vitro and in vivo. In primary astrocyte cultures, LXR agonists regulated several established LXR target genes, including ATP binding cassette transporter A1, and enhanced cholesterol efflux. In contrast, little or no effect on gene expression or cholesterol efflux was detected in primary neuronal cultures. Treatment of mice with a selective LXR agonist resulted in the induction of several LXR target genes related to cholesterol homeostasis in the cerebellum and hippocampus. These data provide the first evidence that the LXRs regulate cholesterol homeostasis in the central nervous system. Because dysregulation of cholesterol balance is implicated in central nervous system diseases such as Alzheimer’s and Niemann-Pick disease, pharmacological manipulation of the LXRs may prove beneficial in the treatment of these disorders.

scholarly journals SMADS-Mediate Molecular Mechanisms in Sjögren’s Syndrome

2021 ◽  
Vol 22 (6) ◽  
pp. 3203
Author(s):  
Margherita Sisto ◽  
Domenico Ribatti ◽  
Sabrina Lisi
Keyword(s):  
Sjögren’S Syndrome ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Sjögren's Syndrome ◽  
Biological Effects ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
Mesenchymal Transition ◽  
Sjögren‘S Syndrome

There is considerable interest in delineating the molecular mechanisms of action of transforming growth factor-β (TGF-β), considered as central player in a plethora of human conditions, including cancer, fibrosis and autoimmune disease. TGF-β elicits its biological effects through membrane bound serine/threonine kinase receptors which transmit their signals via downstream signalling molecules, SMADs, which regulate the transcription of target genes in collaboration with various co-activators and co-repressors. Until now, therapeutic strategy for primary Sjögren’s syndrome (pSS) has been focused on inflammation, but, recently, the involvement of TGF-β/SMADs signalling has been demonstrated in pSS salivary glands (SGs) as mediator of the epithelial-mesenchymal transition (EMT) activation. Although EMT seems to cause pSS SG fibrosis, TGF-β family members have ambiguous effects on the function of pSS SGs. Based on these premises, this review highlights recent advances in unravelling the molecular basis for the multi-faceted functions of TGF-β in pSS that are dictated by orchestrations of SMADs, and describe TGF-β/SMADs value as both disease markers and/or therapeutic target for pSS.

Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_2) ◽  
Author(s):  
Shuang Li ◽  
Dong Han ◽  
Dachun Yang
Keyword(s):  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Cardiac Fibrosis ◽  
Transient Receptor Potential ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Fibroblasts ◽  
Gene Transfection ◽  
Pressure Overload

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

scholarly journals Dietary Isoliquiritigenin at a Low Dose Ameliorates Insulin Resistance and NAFLD in Diet-Induced Obesity in C57BL/6J Mice

2018 ◽  
Vol 19 (10) ◽  
pp. 3281 ◽  
Author(s):  
Youngmi Lee ◽  
Eun-Young Kwon ◽  
Myung-Sook Choi
Keyword(s):  
Insulin Resistance ◽  
Energy Expenditure ◽  
Body Fat ◽  
Fat Mass ◽  
High Fat Diet ◽  
Plasma Cholesterol ◽  
Cellular Respiration ◽  
Body Fat Mass ◽  
High Fat ◽  
Diet Induced Obesity

Isoliquiritigenin (ILG) is a flavonoid constituent of Glycyrrhizae plants. The current study investigated the effects of ILG on diet-induced obesity and metabolic diseases. C57BL/6J mice were fed a normal diet (AIN-76 purified diet), high-fat diet (40 kcal% fat), and high-fat diet +0.02% (w/w) ILG for 16 weeks. Supplementation of ILG resulted in decreased body fat mass and plasma cholesterol level. ILG ameliorated hepatic steatosis by suppressing the expression of hepatic lipogenesis genes and hepatic triglyceride and fatty acid contents, while enhancing β-oxidation in the liver. ILG improved insulin resistance by lowering plasma glucose and insulin levels. This was also demonstrated by the intraperitoneal glucose tolerance test (IPGTT). Additionally, ILG upregulated the expression of insulin signaling-related genes in the liver and muscle. Interestingly, ILG elevated energy expenditure by increasing the expression of thermogenesis genes, which is linked to stimulated mitochondrial biogenesis and uncoupled cellular respiration in brown adipose tissue. ILG also suppressed proinflammatory cytokine levels in the plasma. These results suggest that ILG supplemented at 0.02% in the diet can ameliorate body fat mass, plasma cholesterol, non-alcoholic fatty liver disease, and insulin resistance; these effects were partly mediated by increasing energy expenditure in high-fat fed mice.

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