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1.
Diabetes Res Clin Pract ; 105(3): 373-81, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24956965

ABSTRACT

AIM: Advanced glycation endproducts (AGEs) have been shown to contribute to alteration of glomerular permselectivity to proteins in diabetes. Oxidative stress is required for AGE formation. Therefore we studied the effect of an antioxidant micronized purified flavonoid fraction (MPFF, Daflon(R) 500 mg), on urinary albumin clearance in diabetic rats. METHODS: Hyperglycaemia was induced by streptozotocin 55 mg/kg IM at days 0 and 7 in normotensive Wistar rats (NWR, diabetes duration 5 months) or hypertensive Wistar Kyoto rats (SHR, diabetes duration 2 months). MPFF was administered at 300 mg/kg/day, from day -2 until sacrifice. RESULTS: After 5 months of diabetes in NWR, MPFF reduced albumin clearance from 729±92 to 392±60 nl/min/kg, p<0.01, and restored albuminemia from 20.4±0.9 to 24.0±1 g/l, p<0.05; albumin fractional clearance was significantly diminished in the flavonoid-treated diabetic rats (0.360±0.037‰ versus 1.335±0.430‰ in the diabetic controls, p<0.001); MPFF did not significantly modify blood glucose and plasma fructosamine levels. After 2 months of diabetes in SHR, MPFF reduced albumin clearance from 243±121 to 101±47 nl/min/kg, p<0.05, and restored albuminemia from 21.1±1.6 to 26.7±2.2 g/l (p<0.05); MPFF also decreased plasma fluorescence characteristic of AGEs (p<0.02). Besides hesperetin, a main metabolite of MPFF recovered in plasma, inhibited in vitro the formation of the crosslinking AGE pentosidine in collagen incubated with high glucose (p<0.001). CONCLUSION: Our results confirm the role of glycoxidative stress in diabetic nephropathy. MPFF might be useful as complementary treatment for preventing diabetic microangiopathy.


Subject(s)
Albuminuria/drug therapy , Antioxidants/therapeutic use , Diabetes Mellitus, Experimental/metabolism , Diosmin/therapeutic use , Glycation End Products, Advanced/metabolism , Hypoalbuminemia/drug therapy , Phytotherapy , Rutaceae/chemistry , Animals , Blood Glucose/metabolism , Blood Pressure/drug effects , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/prevention & control , Flavonoids/therapeutic use , Fructosamine/blood , Glomerular Basement Membrane/drug effects , Glomerular Basement Membrane/pathology , Glycation End Products, Advanced/analysis , Hesperidin/therapeutic use , Hyperglycemia/chemically induced , Hyperglycemia/drug therapy , Kidney Glomerulus/metabolism , Male , Oxidative Stress/drug effects , Plant Extracts , Rats, Inbred SHR , Rats, Wistar
2.
Amino Acids ; 43(2): 697-707, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22002796

ABSTRACT

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.


Subject(s)
Collagen Type IV/biosynthesis , Glucose/physiology , Human Growth Hormone/physiology , Human Umbilical Vein Endothelial Cells/metabolism , Cell Proliferation , Cells, Cultured , Collagen Type IV/metabolism , Culture Media , Gelatinases/metabolism , Glucose/metabolism , Human Growth Hormone/pharmacology , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Indoles/pharmacology , Maleimides/pharmacology , Proline/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Serum
3.
Diabetes Res Clin Pract ; 77(2): 337-40, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17383766

ABSTRACT

Aspirin showed an inhibitory effect on the formation of pentosidine, a cross-linking advanced glycation endproduct, in collagen incubated with glucose in vitro. IC(50) was evaluated at 10mmol/l. Aspirin might act by metallic ion chelating (as did EDTA and DTPA) and by oxygen radical scavenging. Since aspirin was reported to inhibit retinopathy in diabetic dogs, it could act partly by inhibiting advanced glycation endproduct accumulation in long-lived proteins like collagens.


Subject(s)
Arginine/analogs & derivatives , Aspirin/pharmacology , Collagen/metabolism , Glycation End Products, Advanced/metabolism , Lysine/analogs & derivatives , Arginine/antagonists & inhibitors , Collagen/drug effects , Cross-Linking Reagents , Glucose/pharmacology , Glycosylation/drug effects , Lysine/antagonists & inhibitors
5.
Life Sci ; 64(2): 113-23, 1999.
Article in English | MEDLINE | ID: mdl-10069489

ABSTRACT

Parasympathetic hyperactivity is found in some infants presenting faint episodes and could be responsible of certain Sudden Infant Death Syndrome cases. Therefore it was interesting to look for a noninvasive biochemical indicator of parasympathetic activity. A parasympaticomimetic syndrome associated with muscarinic receptor stimulation, which has been followed during 48 h, was obtained in the awake rat by reserpine injection (6.25 mg/kg at T0 and T24h), and a model of prolonged parasympatholytic syndrome, by administration of diphemanil-methylsulfate (DPMS), a muscarinic receptor inhibitor, in drinking water (mean daily dosis: 150 mg/kg). Significant bradycardia and tachycardia were respectively observed. In the reserpine-treated rats we found significantly increased cyclic guanosylmonophosphate (cGMP) urinary excretion between T24h and T48h, when compared with vehicle-treated controls (+87% in one experiment, +135% in the other, when expressed in pmol/microg creatinine); norepinephrine urinary excretion between T24h and T48h was decreased (-44%); the increase in cGMP urinary excretion was not significantly modified by the NO-synthase inhibitor, L-nitroarginine-methyl-ester. In the DPMS-treated rats, we observed a significantly decreased cGMP (-20%) and increased norepinephrine urinary excretion (+61%). Thus cGMP excretion varied in opposite directions in the reserpine- and DPMS-treated rats. The link between these modifications in cGMP excretion and muscarinic receptor stimulation or blockade has still to be fully demonstrated. Urinary cGMP excretion could be tested as screening parameter in infants at risk of faint episodes associated with bradycardia.


Subject(s)
Autonomic Nervous System Diseases/urine , Cyclic GMP/urine , Parasympathetic Nervous System/physiopathology , Parasympatholytics/pharmacology , Piperidines/pharmacology , Reserpine/pharmacology , Animals , Autonomic Nervous System Diseases/chemically induced , Autonomic Nervous System Diseases/physiopathology , Dose-Response Relationship, Drug , Heart Rate/drug effects , Hemodynamics/drug effects , Male , Muscarinic Agonists/administration & dosage , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/administration & dosage , Muscarinic Antagonists/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/physiology , Nitric Oxide Synthase/antagonists & inhibitors , Norepinephrine/urine , Parasympathetic Nervous System/drug effects , Parasympatholytics/administration & dosage , Piperidines/administration & dosage , Rats , Rats, Sprague-Dawley , Reserpine/administration & dosage , Syndrome
6.
Diabetologia ; 39(6): 641-8, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8781759

ABSTRACT

Diabetic microangiopathy is characterized by a thickening of capillary basement membranes associated with type IV collagen accumulation. An increase in type IV collagen content of the aortic wall is also observed in macroangiopathy. In order to analyse the importance of the polyol pathway in the development of the collagen metabolism alterations seen in diabetic angiopathy and their prevention by aldose reductase inhibitors, we have studied the effects of sorbinil on the high glucose-induced stimulation of type IV collagen biosynthesis in human umbilical vein endothelial cells. Primary cultures were exposed to high glucose (16.7 mmol/l), with and without 0.11 mmol/l sorbinil, for 3 or 6 days after beginning of confluence. We measured the soluble type IV collagen secreted into the culture medium and the insoluble type IV collagen accumulated in the extracellular matrix and cells, by ELISA. We also studied [14C]proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. High glucose decreased the number of cells and increased the amount of type IV collagen in the culture supernatant and in the extracellular matrix and cell fraction. It also increased proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. Sorbinil corrected all these high glucose-induced alterations. The corrective effects of sorbinil on the proliferation and on type IV collagen metabolism of endothelial cells cultured in high glucose may be attributed to prevention of polyol pathway dysregulation.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Collagen/biosynthesis , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Glucose/pharmacology , Imidazoles/pharmacology , Imidazolidines , Animals , Carbon Radioisotopes , Cell Survival/drug effects , Cells, Cultured , Collagen/drug effects , Collagen/immunology , Culture Media, Serum-Free , Diabetic Angiopathies/etiology , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix/chemistry , Humans , Osmolar Concentration , Proline/analysis , Proline/metabolism , Proteins/metabolism , Umbilical Veins/cytology
7.
Biochem Cell Biol ; 74(5): 659-67, 1996.
Article in English | MEDLINE | ID: mdl-9018373

ABSTRACT

Since diabetic microangiopathy and macroangiopathy are characterized by type IV collagen accumulation in vascular basement membranes, it was of interest to study type IV collagen production and type IV collagenase secretion by endothelial cells (EC) cultured in high glucose and to evaluate the role of protein kinase C (PKC) activation in the alterations induced by high glucose. Primary cultures of human umbilical vein EC were exposed to high glucose concentration for 3 days at the beginning of confluence. The number of EC decreased with glucose concentration from 5 to 50 mM. At 16.7 mM glucose concentration, the amount of type IV collagen, determined by a two-step ELISA, increased in the culture supernatant and in the insoluble fraction associated with the extracellular matrix and cells; proline incorporation was more markedly elevated in the collagenous than in the total proteins of the culture supernatant and of the extracellular matrix and cell extracts. Gelatin zymography of the culture supernatant showed that EC mainly produce a 72-kDa gelatinase known to degrade type IV collagen. At 16.7 mM glucose concentration, total gelatinase activity per millilitre of culture supernatant was reduced and the 72-kDa gelatinase activity measured on the zymogram scan was lowered. When EC were exposed to 16.7 mM glucose, the specific PKC inhibitor GF 109203X corrected the increases in type IV collagen concentration and in proline incorporation into the collagenous or total proteins present in he culture supernatant or in the extract of the insoluble fraction, including the extracellular matrix and cells. Our results show that soluble and insoluble type IV collagen accumulation by EC cultured at high glucose concentration is not only associated with increased synthesis of the collagenous and total proteins but also with decreased total 72-kDa gelatinase activity in the extracellular fluid. The observed effects of GF 109203X are in favor of the involvement of PKC activation in the type IV collagen accumulation.


Subject(s)
Collagen/biosynthesis , Endothelium, Vascular/metabolism , Gelatinases/biosynthesis , Glucose/toxicity , Indoles/pharmacology , Maleimides/pharmacology , Protein Kinase C/antagonists & inhibitors , Cell-Free System/enzymology , Cells, Cultured , Collagen/blood , Collagen/drug effects , Culture Media , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Gelatinases/drug effects , Humans , Indoles/toxicity , Maleimides/toxicity , Mannitol/pharmacology , Molecular Weight , Umbilical Veins
8.
C R Seances Soc Biol Fil ; 189(6): 967-85, 1995.
Article in French | MEDLINE | ID: mdl-8763028

ABSTRACT

Three stages can be distinguished during the glycation process: initiation with the formation of Amadori product; spreading with glyco-oxidation reactions; terminal formation of advanced glycation end products (AGEs). Some AGEs have been isolated and characterized: pyrraline linked to one aminoacid, pentosidine linked to two aminoacids and forming a cross-link between peptidic chains. The AGE-induced cross-links alter the biophysical properties of the proteins with increased stiffness of the fibrous proteins and resistance to proteases. Glycation of the glomerular basement membrane (GBM) macromolecules modifies the architecture of the glomerular filtration barrier. Type IV collagen is the major constituent of the GBM and the mesangial matrix and is a substrate for prolonged glycation, due to its long half-life. In the GBM, AGE level (particularly pentosidine level per mg collagen) increases with age; it is higher in diabetic or uremic patients than in age-matched controls. In insulin-dependent diabetes mellitus, a correlation has been shown between the pentosidine level of skin collagen and the severity of vascular complications. Glycation inhibits the homotypic polymerization interactions between two type IV collagen molecules through their NC1 ends. Glycation also affects the heterotypic interactions between different GBM macromolecules: the affinity of glycated fibronectin for type IV collagen is diminished. Besides, glycation modifies the interactions between type IV collagen and adjacent cells: mesangial and endothelial cells are less adherent on a glycated type IV collagen matrix and their morphology modified. GBM treated with dimethylmalonimidate, which induces cross-links between amines as does advanced glycation, are more permeable to proteins.


Subject(s)
Diabetes Mellitus/metabolism , Kidney Glomerulus/metabolism , Aging , Basement Membrane , Glycosylation , Humans , Kidney Glomerulus/physiology , Macromolecular Substances
9.
C R Seances Soc Biol Fil ; 187(2): 247-57, 1993.
Article in French | MEDLINE | ID: mdl-8019906

ABSTRACT

In Diabetes Mellitus, type IV collagen biosynthesis is increased: the alpha 1(IV) procollagen specific mRNA concentration is elevated, particularly in the kidney, and the type IV collagen protein is accumulating is the thickened basement membranes. Aldose reductase inhibitors like sorbinil do prevent basement membrane thickening and type IV collagen overproduction. The latter seems related to intracellular sorbitol accumulation and also to protein kinase C activation. Autocrine or paracrine TGF beta may be involved in the type IV collagen oversecretion. The secreted type IV collagen is subject to posttranslational alterations, especially glycation which leads to advanced glycation end-products and covalent crosslinks. This decreases collagen extractability and susceptibility to collagenases and favours basement membrane thickening. Disaccharide unit-specific alpha-glucosidase activity is inhibited by glucose (Kp = 7.5 mM). Type IV collagenase activity secreted by endothelial cells cultured at high glucose concentrations appears to be diminished. Therefore type IV collagen catabolism may be decreased in Diabetes Mellitus.


Subject(s)
Collagen/metabolism , Diabetes Mellitus/metabolism , Animals , Basement Membrane/metabolism , Collagen/biosynthesis , Collagen/chemistry , Disaccharides/metabolism , Glycosylation , Polysaccharides/metabolism , Rats
10.
Biochem Biophys Res Commun ; 175(2): 617-24, 1991 Mar 15.
Article in English | MEDLINE | ID: mdl-2018506

ABSTRACT

RI-HB is an extracellular heparin binding protein regulated by retinoic acid and essentially expressed during embryogenesis. This study reports the cloning and sequencing of the cDNA that encodes RI-HB. The sequence of RI-HB contains 121 amino acid residues and is very rich in basic amino acids and cysteines. This sequence was compared to those of HBGAM and MK protein, two other heparin binding proteins exhibiting growth and/or neurotrophic activities. Northern blot analysis indicates that RI-HB mRNA is strongly expressed during early chicken embryogenesis and that it is induced by retinoic acid treatment of chicken fibroblasts and myotubes in culture.


Subject(s)
Avian Proteins , Carrier Proteins/genetics , Animals , Base Sequence , Blotting, Northern , Chick Embryo , Cloning, Molecular , DNA/genetics , Gene Expression/drug effects , Heparin/metabolism , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , RNA, Messenger/genetics , Restriction Mapping , Tretinoin/pharmacology
11.
C R Seances Soc Biol Fil ; 185(6): 520-6, 1991.
Article in French | MEDLINE | ID: mdl-1822401

ABSTRACT

An heparin binding protein (RIHB) was purified from chick embryos. Essentially expressed during early embryogenesis it is mainly localized within basement membranes. Its synthesis and that of the RIHB mRNA are induced by retinoic acid in chicken myoblasts cell culture. This protein belongs to the same family that HBGAM or Pleiotropin and MK protein two other heparin binding proteins exhibiting growth and/or neurotrophic activities.


Subject(s)
Carrier Proteins/classification , Heparin/metabolism , Tretinoin/pharmacology , Animals , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Chick Embryo , Gene Expression Regulation
12.
Anal Biochem ; 185(2): 308-12, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2187375

ABSTRACT

This paper describes an original methodology for determining macromolecular antigen levels by polarization of fluorescence. it involves the use of fluorescent derivatives of Fab fragments of a monoclonal antibody (Mr 50,000), whose fluorescence polarization rises significantly when it combines with a macromolecular antigen. An experimental system (Fab anti-aldosterone and aldosterone--bovine serum albumin (BSA)) is studied to test this methodology, which was then used to develop an immunoassay for human immunoglobulin M (IgM), using anti-mu chain Fabs. In the two assays, the binding stoichiometry of Fab/antigen was 10/1 and 8/1 for aldosterone--BSA and IgM, respectively. The lower limit of detection of the IgM assay was 0.8 microgram/ml and thus it was applicable to clinical detection of IgM concentrations.


Subject(s)
Immunoglobulin M/immunology , Macromolecular Substances , Aldosterone/immunology , Animals , Antibodies, Monoclonal/immunology , Cattle , Electrophoresis, Polyacrylamide Gel/methods , Fluorescence Polarization/methods , Fluorescent Antibody Technique , Humans , Immunoassay/methods , Immunoglobulin Fragments/immunology , Immunoglobulin G/immunology , Isoquinolines , Mice , Papain/immunology , Reference Standards , Serum Albumin, Bovine/immunology
13.
Clin Chim Acta ; 164(1): 71-82, 1987 Apr 15.
Article in English | MEDLINE | ID: mdl-3581482

ABSTRACT

The development of non-competitive microtitre plate enzyme-linked immunosorbent assays for steroids was investigated using immobilised steroid as immunosorbent and enzyme-labelled second antibodies. An assay for testosterone with polyclonal anti-testosterone immunoglobulins was optimised with respect to a number of parameters but remained unsatisfactory for clinical assays. The results were applied to developing an aldosterone assay using monoclonal anti-aldosterone immunoglobulins. The latter method was used for the determination of urinary aldosterone and the results are compared with those obtained by a classical radioimmunoassay. Problems concerned with the use of sulphur-containing proteins and with the presence of low affinity antibodies in polyclonal preparations are discussed.


Subject(s)
Aldosterone/urine , Testosterone/analysis , Animals , Antibodies, Monoclonal , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulins , Immunosorbents , Radioimmunoassay
14.
Anal Biochem ; 159(1): 179-86, 1986 Nov 15.
Article in English | MEDLINE | ID: mdl-3812998

ABSTRACT

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.


Subject(s)
Fluorescence Polarization/methods , Immunoassay/methods , Isoquinolines , Steroids/analysis , Estriol/analogs & derivatives , Estriol/analysis , Estriol/chemical synthesis , Fluorescent Dyes , Testosterone/analogs & derivatives , Testosterone/analysis , Testosterone/chemical synthesis
15.
J Steroid Biochem ; 19(6): 1811-6, 1983 Dec.
Article in English | MEDLINE | ID: mdl-6200695

ABSTRACT

A series of derivatives of estrone with fluorescent dyes (dansyl or coumarin) coupled at different positions on the steroid molecule, have been synthesized. These derivatives were tested for their quantum yields and their binding properties were determined with respect to rat alpha 1 fetoprotein. Derivatives at C-3 (estrone-3-hemisuccinate-dansyl-cadaverine and estrone-3-dansyl) compete with estrone for binding to the fetal protein; however derivates of estrone at C-6 (estrone-6-carboxymethyloxime-dansyl-cadaverine) and at C-17 (estrone-17-carboxymethyloxime-coumarine, estrone-17-carboxymethyloxime-dansyl-cadaverine and estrone-17-dansyl-hydrazine) compete poorly or not at all. The association constant of the radioactive derivative estrone-3-dansyl [3H] with rat alpha 1 fetoprotein was measured directly: the same number of high affinity binding sites (0.6) as that for estrone was found with an apparent association constant of 3.7 X 10(6) M-1. In addition to the high affinity binding sites, a low affinity class of binding sites was found which corresponds to the binding of the dansyl fraction of the fluorescent steroid derivative.


Subject(s)
Estrone/analogs & derivatives , Fluorescent Dyes , alpha-Fetoproteins/metabolism , Animals , Binding Sites , Binding, Competitive , Coumarins/metabolism , Dansyl Compounds/metabolism , Estrone/metabolism , Fetal Blood/analysis , Rats , Spectrometry, Fluorescence , Structure-Activity Relationship
18.
Biochim Biophys Acta ; 621(1): 63-71, 1980 Jan 24.
Article in English | MEDLINE | ID: mdl-6153275

ABSTRACT

The phase partition system dextran/polyethylene glycol (2 : 1, w/w) was chosen to separate rat alpha 1-foetoprotein from rat serum albumin, which is its main contaminant due to their having close physicochemical properties. The optimization of this method necessitated a systematic study of the behaviour of rat serum albumin in the system under consideration. This article describes the optimum conditions, in terms of pH, ionic strength and the concentration of polymer solutions, for the purification and recovery of alpha 1-foetoprotein. After a prepurification of rat foetal serum by CM-cellulose chromatography, a single partition step permitted the recovery of 15% of the total alpha 1-foetoprotein present in the rat serum. The purity of this alpha 1-foetoprotein was demonstrated by its binding parameters and by analytical gel electrophoresis.


Subject(s)
Serum Albumin/analysis , alpha-Fetoproteins/isolation & purification , Animals , Dextrans , Hydrogen-Ion Concentration , Methods , Osmolar Concentration , Polyethylene Glycols , Protein Binding , Rats
20.
Steroids ; 30(1): 129-37, 1977 Jul.
Article in English | MEDLINE | ID: mdl-919013

ABSTRACT

An enzyme-linked immunoassay of testosterone is described. The conjugation of testosterone with high specific activity horseradish per-oxidase and a highly sensitive assay for this enzyme having previously been studied, here we describe the immobilization of the anti-testosterone antibody and the development of assay conditions permitting the determination of 50 pg to 1.5 ng testosterone in one working day. In this work attention has been paid to keeping the assay method as simple as possible. The method is discussed in terms of other enzyme-linked immunoassays for steroids.


Subject(s)
Testosterone/analysis , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Estradiol/immunology , Gonadal Steroid Hormones/immunology , Radioimmunoassay , Testosterone/immunology
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