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A Model of Type 2 Diabetes: BBZDR/Wor rat

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BRM is a successful biopharmaceutical company formed in 1996 by Dennis Guberski and Dr. Arthur Like of the University of Massachusetts Medical School (UMass). Over the course of 20 years the founders developed proprietary diabetes research models under the sponsorship of the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK). BRM licensed this intellectual property portfolio from UMass in 1998 and since that time has used these proprietary tools to become one of the leading sources of customized preclinical contract research specializing in type 1 and type 2 diabetes.

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A Model of Type 2 Diabetes: BBZDR/Wor rat

  1. 1. Biomedical Research Models, Inc Contract Discovery Research
  2. 2. BBZDR/Wor Rat • Obese (dyslipidemic) • Hyperleptinemic • Insulin Resistant (hyperinsulinemic) • Hyperglycemic • Hypertensive
  3. 3. BBZDR Rats Clinical Characteristics Genetic Predisposition (fa/fa homozygous) Obesity (Hyperleptinemia) Insulin Resistance (Hyperinsulinemia) Impaired Glucose Tolerance (IGT) Type 2 Diabetes (Hyperglycemia) Macrovascular Diabetic Complications: Hypertension, Accelerated Stenosis (Inducible), Atherosclerosis (Duration Dependent Microvascular Diabetic Complications: Retinopathy, Neuropathy, and Nephropathy
  4. 4. Features of BBZDR/Wor Rats OBESE LEAN MALES FEMALES MALES FEMALES Incidence of Diabetes 98% (N=225) 3% (N=217) 0% (N=729) 0% (N=783) Age at Onset 66.8± 2.7 72.1 ±4.6 N/A N/A Hypertension + ++ – – Insulin Resistance + + – – GLUT-2 – + + +
  5. 5. Normal Islets Lean Rat InsulinGlucagon
  6. 6. Islets from an Obese Rat with IGT InsulinGlucagon Glut 2 Receptor
  7. 7. 0 100 200 300 400 500 600 0 10 20 30 40 50 Time (min) OBESE > 16 LEAN > 6 3.4 mmol/kg glucose i.v. 1.7 mmol/kg arginine i.v. BloodGlucose(mg/dl) Blood Glucose: Fasted BBZDR Females
  8. 8. Insulin Release: Fasted Female BBZDR Rats 0 20 40 60 80 100 0 10 20 30 40 50 Time (min) OBESE > 16 LEAN > 6 3.4 mmol/kg glucose i.v. 1.7 mmol/kg arginine i.v. Insulin(ng/ml)
  9. 9. BBZDR/Wor Obese Rat Islet: Glut-2 Receptor(400x)
  10. 10. Islets from Type 2 Diabetic Rat Glucagon Insulin Glut-2 Receptor Glut-2 Control
  11. 11. Blood Glucose: Fasted Male BBZDR rats 0 100 200 300 400 500 600 700 0 10 20 30 40 50 Time (min) OBESE LEAN 3.4 mmol/kg glucose i.v. 1.7 mmol/kg arginine i.v. BloodGlucose(mg/dl)
  12. 12. Insulin Release: Fasted BBZDR Males 0 20 40 60 80 100 0 10 20 30 40 50 Time (min) Obese Lean 3.4 mmol/kg glucose i.v. 1.7 mmol/kg arginine i.v. Insulin(ng/ml)
  13. 13. Atherosclerosis in BBZDR rats
  14. 14. Blood Chemistry Profile of BBZDR Rats 4 Months of age 0 100 200 300 400 500 600 700 800 Glucose Cholesterol Triglyceride HDL mg/dl Lean Male Obese Female * * * * * * P<0.05 * * * *
  15. 15. Atherosclerosis in Renal Artery BBZDR Diabetic Rat (4mo duration)
  16. 16. Severe atherosclerosis: 10 months ~8 months duration of diabetes Complete occlusion of renal artery, x100 (Left); Arrows identify plaque, x400 (Both panels).
  17. 17. Measurement and Calculation of Neointima Thickness The % stenosis was calculated as the Internal Elastic Area (IEA) minus the Residual Area (RA) divided by the IEA ( x 100)
  18. 18. Neointima Thickness in Lean and Obese Male BBZDR Rats One Week Post Balloon Injury BBZDR Lean Rats Type 2 Diabetic Rats
  19. 19. Neointima Thickness in Lean and Obese Male BBZDR Rats 4 Weeks Post Balloon Injury BBZDR Lean Rats Type 2 Diabetes Rats
  20. 20. Atherosclerosis Plaque in BBZDR/Wor Obese Female Rats
  21. 21. Fatty Deposits and Inflammatory Cells in Occluded Obese Female Carotid Artery
  22. 22. % of Stenosis In Balloon Injured Rat Carotid Artery 0 5 10 15 20 25 30 35 40 45 50 7 14 21 28 Days Post Balloon Injury %Stenosis Lean Rat Obese Female Obese Male * * **
  23. 23. Nephropathy in the BBZDR Rat
  24. 24. Clinical Phases Microalbuminuria Clinical proteinuria Progression to ESRD
  25. 25. LEAN BBZDR Kidney at 5 Months of age Obese Female Obese Diabetic
  26. 26. Lean rat: 5 months old Normal Glomeruli: x100 (left); insert x400 (right).
  27. 27. Obese female: non-diabetic, 5 month old Evidence of mild glomeruli enlargement and mesangial expansion (arrows), x100 (Right); Insert: x400 (Right)
  28. 28. Aldose Reductase Lean BBZDR Obese BBZDR 6 months 4months duration
  29. 29. Sorbitol Dehydrogenase in BBZDR Kidneys Lean ControlObese T2 Diabetic
  30. 30. RAGE in BBZDR Rat Kidneys Obese T2 Diabetic Lean Control
  31. 31. Summary • The diabetic BBZDR male rat is a good model of diabetic nephropathy. • Supported by Clinical blood chemistry and Histopathology – Evidence of hyperfiltration and glomeruli hypertrophy at 3months age – Microalbumuria progressing to proteinuria from 3month through 12month – Mesangial expansion, basement membrane thicking and interstitial fibrosis – Tubule expansion, aschemia and diffuse necrosis after 12months of age (10 months duration)
  32. 32. Diabetic Polyneuropathy
  33. 33. Neuropathy in BBZDR • Experimental rat models of types 1 and 2 diabetes differ in sympathetic neuroaxonal dystrophy. J Neuropathol Exp Neurol. 2004 May;63(5):450-60 Schmidt RE, Dorsey DA, Beaudet LN, Parvin CA, Zhang W, Sima AA. • C-peptide corrects endoneurial blood flow but not oxidative stress in type 1 BB/Wor rats. Am J Physiol Endocrinol Metab. 2004 Sep;287(3):E497-505. Stevens MJ, Zhang W, Li F, Sima AA. • A comparison of diabetic polyneuropathy in type II diabetic BBZDR/Wor rats and in type I diabetic BB/Wor rats. Diabetologia, 2000 Jun;43(6):786-93 Sima AA, Zhang W, Xu G, Sugimoto K, Guberski D, Yorek MA.
  34. 34. Type 1 and 2 Diabetes Type 1 Diabetes ↑ Hyperglycemia Insulin / C-peptide ↓ ↑ Polyol Pathway ↓ NO ↓ Na+/K+- ATPase ↑ Oxidative Stress ↑ Nonenzymatic Glycation ↓ Neurotrophism Apoptosis ? Axonal Degeneration /Loss Impaired Regeneration Nodal / Paranodal Degeneration Acute Reversible NCV-Slowing Chronic Irreversible NCV-Slowing Glucose Sorbitol Fructose NADPH NADP Arginine Citrullin + NO ↓ GSSG GSH ↓ Pathogenetic Scheme of DPN Sima, Cell Mol Life Sci 2003.
  35. 35. Diabetic Polyneuropathy (DPN) Type 2 vs Type 1 Type 2 Type 1 • 35% /25 years • Slow progression • Mild axonal degeneration • No nodal changes • Close to 100% /25years • More rapid progression • Severe axonal degeneration and loss • Progressive nodal/paranodal • degeneration
  36. 36. Progression of Axo-glial Dysjunction Axo-glialdysjunction(%) A. BB/Wor – BBZDR/Wor- rats Duration Of Diabetes Baseline 4mo 6mo 8mo 12mo 14mo 0 10 20 30 40 50 Control BB/Wor BBZDR/ Wor Axo-glialdysjunction(%) 20 40 60 20 30 40 50 60 70 Age (years) Common slope: df =39, f=13.91, p< 0.001 Control: a= 0.24 b= 0.26 NIDM: a= 3.44 b= 0.26 IDDM: a= 25.43 b= 0.26 B. Human Sima et al., J Clin Invest 1984 Sima et al., N Engl J Med, 1986
  37. 37. 0 20 40 60 80 100 0 5 10 15 20 25 30 35 Teased Fiber Pathology in Type 1 and Type 2 Diabetic Patients Control Type 1 Type 2 n=19 n=11 n=17 Control Type 1 Type 2 n=19 n=11 n=17 p<0.001 p<0.005 p<0.001 p<0.001 Sima et al, JCI,1988
  38. 38. Structural Abnormalities In Sural Nerve Axonal Degeneration Fiber Number Nerve Regeneration P<0.001 P<0.001 P<0.01 P<0.001 PercentageofAxonal Degeneration(%) 0 5 10 15 20 25 C8 D1-8 D1CP D2-8 P<0.05 P=0.07 P<0.051000 800 600 400 200 0 NumberofMyelinated Fibers C8 D1-8 D1CP D2-8 P<0.001 P<0.001 P<0.05 C8 D1-8 D1CP D2-8 Fibers(%) 0 1 2 3 4 5 6 7 8 Sima et al., Diabetologia,2002
  39. 39. *: p<0.01, **: p<0.001 vs control. Blood Glucose Serum Insulin Serum C-peptide 0 100 200 300 400 500 600 0 200 400 600 800 1000 0 100 200 300 400 500 600 700 C D1 D1+C D2 C D1 D1+C D2 C D1 D1+C D2 ** ** ** * ** ** ** BloodGlucose(mmol/L) Insulin(pmol/L) C-peptide(pmol/L) Glucose, insulin and C-peptide plasma levels in BB/Wor- and BBZDR/Wor-rats Sima et al: Diabetologia, 2001
  40. 40. Na+/K+-ATPaseActivity (mmol/ADP/mg/wetwt./hr) Control Sham 10µg 100µg 500µ 1000µg hrC-peptide 0 200 400 600 800 1000 * † † *: p<0.01, vs age-matched controls; †: p<0.05 vs duration-matched sham-treated BB/W-rats. The Effect of C-peptide on Neural Na+/K+- ATPase Zhang et al: Exp. Diab.Res. 2001
  41. 41. 35 40 45 50 55 60 65 70 75 Control BB/Wor BB/Wor+C BBZDR/Wor Onset 1 Week 2 Week 3 Week 4 Week 5 Week Hyperglycemic Component Insulin/C-Peptide Deficiency Component Acute Metabolic NCV Defects
  42. 42. Diabetic Retinopathy (DR)
  43. 43. Retinopathy in BBZDR • N-3 polyunstaurated Fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function. Plos One. 2013;8(1):e55177. Epub 2013 Jan29. Tikhonenko M, Lydic TA, Opreanu M, Li Calzi S, Bozack S, McSorley KM, Sochacki AL, Faber MS, Hazra S, Duclos S, Guberski D, Reid GE, Grant MB, Busik JV • Diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock. Journal of Experimental Medicine. 2009 Dec21;206(13):2897-906 Busik JV, Tikhonenko M, Bhatwadekar A, Opreanu M, Yakubova N, Caballero S, Player D, Nakagawa T, Afzal A, Kielczewski J, Sochacki A, Hasty S, Li Calzi S, Kim S, Duclos S, Segal MS, Guberski DL, Esselman WJ, Boulton ME, Grant MB. • Time course of NADH oxidase,inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in the BBZ/Wor rat. Nitric Oxide. 2002 May;6(3): 295-304. Ellis,E.A., Guberski, D.L., Huston, B. and Grant, M.B.
  44. 44. Small Pressurized Artery Myography: As close as it gets to true organ perfusion or function 0 50 100 150 200 250 50 75 100 125 150 175 Diameter with tone Passive diameter WKY Pressure (mmHg) Diameter(µM)
  45. 45. Endothelial Dysfunction in BBZDR Rats  Obese-Diabetic; O Lean control (age-matched) -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone Pre-diabetic age 3-4 Weeks of Diabetes 5 Months of Diabetes 10 Months of Diabetes
  46. 46. Levels of Blood Sugars and Glycosylated Hemoglobin Blood Sugars (mg/dl) Total Glycosylated Hemoglobin (%) Diabetic (BBZ/Wor) 496 ± 15 13.0 ± 0.4 Non-Diabetic (BBDR /Wor) 123 ± 3 5.3 ± 0.5
  47. 47. VEGF and H2O2 Correlation H2O2% VEGF CORRELATION COEFFICIENT DIABETIC 78.7 ± 4.84 24.67 ± 0.33 0.82* NON-DIABETIC 39.0 ± 4.47 21.52 ± 0.43 0.83** *p = 0.001 **p = 0.0001
  48. 48. Percentage of Blood Vessels Positive for Peroxide Central Retina Peripheral Retina Diabetic (BBZ/Wor) 80.00 ± 10.52 44.24 ± 12.79 Non-Diabetic (BB DR /Wor) 38.28 ± 9.68 26.47 ± 8.62
  49. 49. Basement Membrane Thickness (nm) in Type 2 BBZDR Rats Diabetic (BBZ/Wor) 96.0 ± 13.32nm Non-Diabetic (BBDR /Wor) 60.4 ± 5.62nm
  50. 50. Immunocytochemical Localization of Fibronectin Colloidal Gold Particles/um2 Diabetic (BBZ/Wor) 4.45 ± 1.09 Non-Diabetic (BBDR /Wor) 1.56 ± 0.79
  51. 51. Animal Characteristics for iNOS and Nitrotyrosine Study ANIMAL GROUP AGE LENGTH OF DIABETES BLOOD GLUCOSE PRE-DIABETIC (BBZ/Wor) 1.5 – 2 months ND 142 ± 29 md/dl NEW ONSET DIABETES (BBZ/Wor) 2 months 2 – 6 days 383 ± 79 mg/dl CHRONIC DIABETES (BBZ/Wor) 7 – 20 months 4.8 – 18 months 387 ± 96 mg/dl NON-DIABETIC CONTROLS (BB DR /Wor) 7 – 20 months ND 130 ± 28 mg/dl
  52. 52. Colloidal gold localization of nitrotyrosine (arrows) in retina of BBZ/Wor rat with diabetes for two days
  53. 53. eNOS and iNOS Immunoreactivity in Retina eNOS iNOS BBZ/Wor 13.9 ± 2.3 part/50 µm 2 33.9 ± 10.0 part/50 µm 2 BBDR /Wor 78.8 ± 6.0 part/50 µm 2 3.5 ± 2.8 part/50 µm 2
  54. 54. iNOS and Nitrotyrosine Immunoreactivity ANIMAL GROUP iNOS NITROTYROSINE PRE-DIABETIC (BBZ/Wor) 3.7 ± 0.81 % C 8.2 ± 1.70 part/50 µm 2 C NEW ONSET DIABETES (BBZ/Wor) 69.6 ± 5.88 % A 60.8 ± 16.91 part./50 µm 2 A CHRONIC DIABETES (BBZ/Wor) 49.9 ± 9.75 % B 29.5 ± 4.31 part./50 µm 2 B NON-DIABETIC CONTROLS (BB DR /Wor) 8.7 ± 4.66 % C 9.0 ± 1.87 part./50 µm 2 C A: p = 0.0001 B: p = 0.0001 Means with the same letter (A, B, C) are not significantly different.
  55. 55. Time Course of Vascular Changes in BBZDR/Wor Rat With Duration of Hyperglycemia Length of hyperglycemia 1 week 4-6 months 18-24 months NADH oxidase ­ ­ ­ Free Radicals ­ ­ ­ iNOS ­ ­ ­ Nitrotyrosine ­ ­ ­ eNOS ¯ ¯ ¯ NOS cofactors - ¯ ¯ Vessel Diameter ­ ¯ ¯
  56. 56. Small Pressurized Artery Myography: As close as it gets to true organ perfusion or function 0 50 100 150 200 250 50 75 100 125 150 175 Diameter with tone Passive diameter WKY Pressure (mmHg) Diameter(µM)
  57. 57. Isolated Intact Pressurized Resistance Arteries: The heart of microvascular (patho) physiology • Cerebral • Coronary • Renal • Retinal, opthalmic
  58. 58. Inflow T=37.4 outflow The Arteriograph System: Total control over the micro vascular environment Pressure / flow control Open / close micromanipulator
  59. 59. The Pressure Arteriograph System: A controlled two cell system in native interaction
  60. 60. CONTROL 116 MICRONS U73122 185 MICRONS U73122+KCL 77 MICRONS 1.305 1.058 1.595 1.6 0.7 Quantitative Calcium Imaging (FURA-2)
  61. 61. 10 mmHg 122 MICRONS 70 mmHg 108 MICRONS 130 mmHg 112 MICRONS 0.2 3.0 1.095 1.408 1.564 Quantitative Membrane Potential Imaging (Di-8-ANNEPS)
  62. 62. 0 0 -40 -47 -39 -40 -60 V -60 -20 -20Arterial membrane potential, mV: Arterial diameter, µm: video edge detection Arterial wall [Ca ], nM:2+ 60 mm Hg 105 189 245 68 205103 100 mm Hg + 10 nM Nisoldipine 100 mm Hg 8 1416 2012 18 Time (min) Time (min)V Simultaneous Electrophysiology, diameter and calcium Imaging
  63. 63. Long-term artery culture: a physiological genomics platform • Viral transduction • Protein transduction • Ribozyme, siRNA • To VSM and/or ENDO
  64. 64. 3-4 Weeks of DiabetesPre-diabetic age 5 Months of Diabetes 10 Months of Diabetes 0 25 50 75 100 125 150 175 200 225 0 20 40 60 Intraluminal pressure (mm Hg) Myogenictone(%) 0 25 50 75 100 125 150 175 200 225 0 20 40 60 Intraluminal pressure (mm Hg) Myogenictone(%) 0 25 50 75 100 125 150 175 200 225 0 20 40 60 Intraluminal pressure (mmHg) Myogenictone(%)  Obese-Diabetic; O Lean control (age-matched) 0 25 50 75 100 125 150 175 200 225 0 20 40 60 Intraluminal Pressure (mmHg) Myogenictone(%) Characterization of Cerebral-arterial Dysfunction in BBZ/Wor Rats
  65. 65. Endothelial Dysfunction  Obese-Diabetic; O Lean control (age-matched) -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 log[Histamine] M %Myogenictone Pre-diabetic age 3-4 Weeks of Diabetes 5 Months of Diabetes 10 Months of Diabetes
  66. 66. Arterial smooth muscle hyper-reactivity Pre-diabetic age 3-4 Weeks of Diabetes 5 Months of Diabetes 10 Months of Diabetes  Obese-Diabetic; O Lean control (age-matched) -12 -11 -10 -9 -8 -7 -6 -5 0 25 50 75 100 log[5-Hydroxy tryptamine] M %KClConstriction -12 -11 -10 -9 -8 -7 -6 -5 0 25 50 75 100 log[5-Hydroxy tryptamine] M %KClConstriction -12 -11 -10 -9 -8 -7 -6 -5 0 25 50 75 100 log[5-Hydroxy tryptamine] M %KClConstriction -12 -11 -10 -9 -8 -7 -6 -5 0 25 50 75 100 log[5-Hydroxy tryptamine] M %KClConstriction
  67. 67. Pre-diabetic age 3-4 Weeks of Diabetes 5 Months of Diabetes 10 Months of Diabetes  Obese-Diabetic; O Lean control (age-matched) -9 -8 -7 -6 -5 -4 0 25 50 75 100 125 log[U-73122] M %Myogenictone -9 -8 -7 -6 -5 -4 0 25 50 75 100 125 log[U-73122] M %Myogenictone -9 -8 -7 -6 -5 -4 0 25 50 75 100 125 log[U-73122] M %Myogenictone -9 -8 -7 -6 -5 -4 0 25 50 75 100 125 log[U-73122] M %Myogenictone Arterial smooth muscle hyper-reactivity PLC activation
  68. 68. Autoregulation in rat (Sprague Dawley) ophthalmic artery 0 30 60 90 120 150 180 210 100 150 200 250 300 Diameter with tone Passive diameter Myogenic tone 0 25 50 Intraluminal Pressure (mm Hg) Lumendiameter(microns) Myogenictone(%)
  69. 69. -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 Serotonin Phenylephrine Vasopressin UK-14304 U-46619 log M [Vasoconstrictor] %KCl(60mM)response -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0 50 100 150 log M [Endothelin-1] %KCl(60mM)response Effect of different vasoconstrictors in ophthalmic artery Order of efficacy: Endothelin-1 > Vasopressin = Serotonin = Phenylephrine > U-46619 > UK 14304 Order of potency: Endothelin-1 > Vasopressin = Serotonin > U-46619 > UK 14304 > Phenylephrine
  70. 70. -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125 Histamine Carbachol Adenosine CGRP Isoprenaline VIP log M [Vasodilator] Myogenictone(%) Order of efficacy: Carbachol > Isoprenaline > Histamine = CGRP = VIP = Adenosine Order of potency: CGRP > VIP > Carbachol = Isoprenaline > Adenosine = Histamine Bradykinin, substance P and octreotide did not produce sustained dilatory responses Effect of different vasodilators in ophthalmic artery
  71. 71. -9 -8 -7 -6 -5 -4 -3 20 60 100 140 180 L-NAME Indomethacin log M [Inhibitor] Myogenictone(%) Effect of the inhibition of NOS and prostaglandin synthesis on myogenic tone in rat ophthalmic artery
  72. 72. CNS Controls Release of EPC’s • Bone marrow derived epithelial progenitor cells (EPCs) home to sites of vascular injury and help to maintain capillary integrity or re-perfuse acellular capillaries • The release of EPCs from bone marrow follows a circadian rhythmicity in response to local adrenergic signals (Mendez-Ferrer et al. 2008). Circadian rhythms are driven by a central clock (suprachiasmaticnucleus, SCN) and peripheral clocks (tissues) • Clock genes induce circadian rhythm for EPC release by synchronous interplay of positive (Clock, Bmal1) and negative (Per1, Per2, Cry1, Cry2) loops of the peripheral circadian clock J Busik et. al,. (2009) J. Exp. Med
  73. 73. Diabetes Decreases Circadian Release of EPCs.
  74. 74. qPCR mRNA Expression Analysis for Clock Genes
  75. 75. EPC Dysfunction in T2D
  76. 76. Decreased Adrenergic Function in T2D
  77. 77. Conclusions • BBZ has most of the features of diabetic retinopathy as observed in humans • Evaluation of drug therapies can be performed using – Histological studies – Immunological studies – Functional studies using small vessels

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