Free Radical Biology & Medicine, Vol. 31, No. 11, pp. 1352–1359, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
Original Contribution POLYPHOSPHATE ACCUMULATION AND OXIDATIVE DNA DAMAGE IN SUPEROXIDE DISMUTASE-DEFICIENT ESCHERICHIA COLI MAY A. AL-MAGHREBI
LUDMIL T. BENOV
The Department of Biochemistry, Faculty of Medicine, Kuwait University, Safat, Kuwait (Received 29 May 2001; Accepted 9 August 2001)
Abstract—Inorganic polyphosphate is a ubiquitous, linear polymer of phosphate residues linked by high-energy phosphoanhydride bonds. In response to starvation, polyP levels are increased up to 100-fold. It has been proposed that chelation of transition metals by polyP might reduce their toxicity, and that polyP accumulation is vital for survival in stationary phase. SOD-deficient E. coli is unable to survive in stationary phase. We found that deletion of the cytoplasmic SODs does not impair the cell’s capability of synthesizing polyP. However, transient accumulation of polyphosphate correlated with increased resistance to H2O2 and protection of DNA against oxidative damage. The reason for this protective effect of polyP is the induction of HPII catalase and DNA repair enzymes as members of the rpoS regulon. PolyP did not directly protect DNA against oxidative damage in vitro and acted as a pro-oxidant by stimulating the production of hydroxyl radical in the Fenton reaction. It is thus suggested that accumulation of poly P and rpoS induction cannot compensate for the lack of cytosolic SODs for survival in stationary phase. © 2001 Elsevier Science Inc. Keywords—Polyphosphate, Superoxide dismutase-deficient, Oxidative DNA damage, Hydrogen peroxide, Oxidative stress, Free radicals
. Based on these similarities between the ppk and the SOD mutants, it might be assumed that the SOD-deficient cells, like the ppk mutants, are unable to accumulate polyP. In the experiments reported herein we demonstrate that the SOD mutants accumulate as much polyP as the parental, SOD-proficient strain. Accumulation of polyP protected DNA against oxidative damage and made the cells more resistant to H2O2. This effect of polyP, however, was entirely dependent on the induction of the rpoS regulon, and was not due to polyP acting as a free radical scavenger or metal chelator.
Inorganic polyphosphate is a chain of phosphate residues linked by high-energy phosphoanhydride bonds. It has been found in every living organism, from bacteria to mammals . Among the known functions of polyP are substitution for ATP, chelation of metals, reservoir for phosphate, and regulatory role in the physiological adjustments to growth, development, stress, and lack of nutrients . The importance of polyP has been clearly demonstrated by an E. coli mutant (ppk) lacking the enzyme polyphosphate kinase and thus unable to accumulate polyP . The ppk mutant was deficient in functions expressed in stationary phase and failed to survive . It also showed low resistance to H2O2 and the redox-cycling agent menadione . Mutants lacking cytoplasmic superoxide dismutases show similar inability to survive in stationary phase, and high sensitivity toward redox-cycling agents and H2O2
MATERIALS AND METHODS
Reagents and plasmid Sodium phosphate glass was obtained from Sigma (Germany). To get rid of metal contaminations and low molecular weight phosphate, sodium phosphate glass was dialyzed 24 h against 1 mM EDTA followed by 48 h against deionized water using dialysis tubing with exclusion limit of 3500. Polyphosphoric acid (Sigma) was first
Address correspondence to: Ludmil Benov, Department of Biochemistry, Faculty of Medicine, Kuwait University, P. O. Box 24923 Safat, 13110 Kuwait; Tel: (965) 531-9489; Fax: (965) 533-8908; E-Mail: [email protected]
Polyphosphate and oxidative DNA damage
neutralized with NaOH, and chilled on ice, which caused precipitation of the high molecular weight sodium polyphosphate. The high molecular weight polyphosphate crystals were separated by filtration at 4°C, dissolved in deionized water, and treated as above. No difference between the two types of polyP was observed with respect to HO• production. The pcDNA3 circular plasmid (5,446 bp) was purified using nucleobond midi prep kit (Clontech). Media LB medium contained 10 g Bacto-Tryptone, 5 g yeast extract, and 10 g NaCl per liter and was adjusted to pH 7.0 with ⬃ 1.5 g of K2HPO4. When used for plating, the LB medium was solidified with 1.5% Bacto-Agar. M9CA medium consisted of minimal A salts , 0.2% casamino acids, 0.2% glucose, 3 mg pantothenate, and 5 mg of thiamine per liter. MOPS medium contained 50 mM MOPS, pH 7.0, 0.1 mM K2PO4, and 4 mg/ml glucose . Strains The strains of E. coli used were as follows: GC4468 ⫽ parent and QC1799 ⫽ GC4468 ⌬ sodA3, ⌬ sodB-kan, QC1817 ⫽ GC4468 ⌬ sodA3, ⌬ sod B-kan, ⌬ sox::cat , (D. Touati, Institute Jacques Monod, CNRS, Universite Paris, France); UM1, a catalase-deficient mutant and GSH7, parental , were obtained from I. Fridovich (Duke University Medical Center, Durham, NC, USA). AS430 ⫽ GC4468 ⌬oxyR::spec, was provided by J. Imlay (University of Illinois at Urbana-Champaign, Urbana, IL, USA); BJ4 ⫽ parental, and BJ4 rpoS (rpoS⫺)  were a gift from K. Krogfelt (Statens Serum Institut, Copenhagen, Denmark). Stationary phase viability Overnight LB cultures were diluted 200-fold in LB medium and were kept for the entire length of the experiment at 37°C, with aeration ensured by shaking at 200 rpm. Aliquots were removed at intervals for enumeration of surviving cells by diluting, plating, and counting. Plates were incubated aerobically at 37°C for 24 – 48 h before counting. Growth conditions for accumulation of polyP For accumulation of polyP the cells were incubated as described by Rao et al. . Briefly, the strains were grown aerobically overnight at 37°C in LB medium and were then diluted 200-fold into M9CA and grown to mid
log phase. Growth was followed by measuring A600 nm. For incubation in MOPS medium the cells were washed and re-suspended in this medium to the original volume. Cells were disrupted by a French Press and debris were removed by centrifugation. PolyP assay Nucleic acids were removed by precipitation with 1% streptomycin sulfate. Proteins were eliminated either with phenol/chloroform  or by adding 2% perchloric acid. Similar results were obtained from either treatment. In the second case, after 30 min, the samples were centrifuged and perchloric acid was neutralized with KOH. The samples were chilled on ice and the precipitate was removed by centrifugation. After removal of the proteins, the samples were filtered through a 3000 cut-off Centricon filter. The higher molecular weight residue was washed two times with distilled water, and was resuspended in distilled water to the original volume. Aliquots were mixed with an equal volume of 2N HCl and heated for 60 min at 100°C in glass-stoppered tubes. Phosphate content was assayed as described by Cowling and Birnboim . The assay was tested by adding specific amounts of high molecular weight polyP to cell free extracts and demonstrated satisfactory recovery and reproducibility. In parallel polyP was assayed as described by Clark et al. , and similar results were obtained. Throughout the text, the concentration of polyP is expressed in terms of orthophosphate. Hydroxylation assay PolyP effect on hydroxyl radical production was assayed as described by Goscin and Fridovich . The system contained either FeSO4 plus ascorbate or FeSO4 plus xanthine oxidase (15 nM) and xanthine (200 M). FeSO4 was added to final concentrations of 1, 10, 20, 50, 100, and 500 M. Resistance to H2O2 H2O2 killing was performed essentially as described by Carlioz and Touati . Mid log phase M9CA cultures, or cultures incubated in MOPS medium for 3 h, were diluted to A600 ⫽ 0.1 with M9 salts and were incubated with 2.5, 5.0, 10.0, 20.0, or 30.0 mM H2O2 for exactly 30 min. H2O2 concentration of the stock solution was adjusted using extinction coefficient at 240 nm ⫽ 43.6 M⫺1 cm⫺1 . Measurement of H2O2 consumption H2O2 degradation by cells was monitored at 240 nm. To 460 l of 100 mM phosphate buffer, pH 6.5 were
M. A. AL-MAGHREBI and L. T. BENOV
added 500 l of cell suspension, diluted with the same buffer to exactly A600 ⫽ 0.1, and 40 l of 250 mM H2O2. The decrease of the H2O2 concentration was followed on a recording spectrophotometer. To avoid cell sedimentation and fluctuations in H2O2 concentration, the samples were stirred every 60 s. H2O2 consumption was expressed in moles of H2O2 consumed per 10 min per 103 cells.
accumulate polyP. For H2O2 killing, cells grown in M9CA and MOPS were divided into two halves. Only one half was treated with 150 M KCN for 15 min at room temperature before exposure to H2O2 killing and plating for cell viability determination as described above. Plasmid DNA was isolated from both CN-treated and untreated cells using the alkaline lysis method . Isolated plasmid DNA was precipitated with ethanol and resuspended in TE.
Catalase/peroxidase activity determination The crude extracts were assayed for HPI and HPII catalases as described by Visick and Clarke . In brief, after measuring the total catalase activity, the crude extracts were heated in a 55°C water bath for 15 min and the assay was repeated again. This gives the activity of the heat stable HPII. HPI activity was estimated by subtracting HPII form the total activity. The o-dianisidine peroxidase was assayed as described by Claiborne and Fridovich .
Agarose gel electrophoresis DNA samples were resolved on 1% agarose gel electrophoresis containing 0.3 g/ml ethidium bromide. The gel was run in TAE buffer (40 mM Tris-Acetate, 1 mM EDTA, pH 8.0) at 60 – 80 volts for 1 h. DNA bands were visualized in UV transiluminator and photographed. Photographs were scanned and DNA damage was calculated as percent of remaining supercoiled DNA to total DNA using Scion Image analysis software. All experiments were repeated at least three times with 3–5 replicates. Bars on figures represent SEM
DNA damage by iron/ascorbate Two g of purified plasmid DNA dissolved in TE (10 mM Tris, 1 mm EDTA, pH 8.0) was incubated at room temperature for 60 min with 10 mM ascorbate and 100 M Fe (II) in 100 mM K2PO4 (pH 6.0). Purified polyP was added to a final concentration of 1 mM in a total reaction volume of 200 l. Fifty l aliquots were removed at time intervals of 0, 30, and 60 min and kept at ⫺20°C before analysis on agarose gel electrophoresis. Control reactions were carried out simultaneously, but without Fe.
SOD⫺ mutant survival and polyP accumulation in stationary phase SodAsodB E. coli rapidly loses viability in stationary phase (Fig. 1A). The number of viable cells dropped from ⬎ 109 to ⬍ 104 cells/ml for the time of observation. Obviously, this is not related to deficiency of polyP synthesis because at entering stationary phase the SOD mutant contained as much polyP as the parental strain (Fig. 1B).
DNA damage by xanthine/xanthine oxidase The reaction was carried out as described by Brawn and Fridovich . Two g plasmid DNA was incubated with 50 –200 M xanthine, 5–24 nM xanthine oxidase, 100 M EDTA, and 1 and 10 mM purified polyP in the presence of 50 mM K2PO4 (pH 7.8). FeSO4 was added to a final concentration of 5 or 10 M after removing a 50 l sample at time 0 from a 300 l total reaction volume. Reactions were performed at 37°C with shaking under aerobic conditions. Reaction samples were removed at 20 min intervals and stored at ⫺80°C until the other samples were ready for electrophoresis. DNA damage in vivo SOD-mutant cells were transformed with pcDNA3 plasmid at 37°C. The transformed cells were then grown in M9CA overnight and transferred to MOPS for 3 h to
PolyP enhances the generation of HO• Addition of polyP (10 mM) to a system containing Fe(II) and ascorbate caused about 4-fold increase in the hydroxylation of 4-nitrophenol (Fig. 2A). Hydroxylation was inhibited by mannitol, an HO• scavenger, thus confirming the production of HO•. Similar results were found when O2•⫺was produced by xanthine oxidase/ xanthine (Fig. 2B). PolyP stimulated hydroxylation of 4-nitrophenol at all the Fe(II) concentrations tested between 1 and 500 M. No effect of polyP was observed in the absence of iron. Accumulation of PolyP increases cell resistance to H2O2 Raising the content of polyP made the SOD mutants significantly more resistant to H2O2 (Fig. 3). At the same
Polyphosphate and oxidative DNA damage
Fig. 1. Stationary phase survival and accumulation of polyP. Overnight LB cultures of GC4468 (SOD-proficient) and QC1799 (SOD-deficient) were diluted 200-fold in LB medium and were grown aerobically at 37°C and 200 rpm. Growth was monitored by measuring A600 nm. For cell survival, the cultures were kept at the same conditions for 96 h. At the indicated time intervals aliquots were diluted and plated for counting colonies. Panel (A): Cell survival. Line 1, GC4468; line 2, QC1799. Panel (B): PolyP content. Bar 1, GC4468 at mid log phase (A600 nm ⫽ 1.193); bar 2, GC4468 in stationary phase; bar 3, QC1799 at mid log phase (A600 nm ⫽ 1.026); bar 4, QC1799 in stationary phase.
time, the high-polyP cells showed a higher rate of H2O2 consumption (⬃ 2.5-fold) (Fig. 4). H2O2 might be decomposed either enzymatically or by polyP-transition metal complex(es). PolyP chelates metals and some of the polyP-metal complexes might have such capability . In an attempt to distinguish between these two possibilities, we used CN⫺ to inhibit the enzymatic decomposition of H2O2. Preincubation with 150 M CN⫺ for 15 min eliminated the H2O2 decomposition capability of both the high and low polyP cells (Fig. 4), without affecting the plating efficacy. Such a concentration of CN⫺ inhibited respiration up to about 65%. Induction of rpoS-dependent hydroperoxidases The inhibitory effect of CN⫺ suggests that the increased capability of the high polyP cells to decompose
Fig. 2. Hydroxylation of 4-nitrophenol. The reaction mixture contained 8.3 mM 4-nitrophenol in 83.0 mM potassium phosphate buffer, pH 6.0 and the following additions: Panel (A): Line 1, 500 M FeSO4 ⫹ 30 mM ascorbate ⫹ 10 mM PolyP; line 2, FeSO4 ⫹ ascorbate only; line 3, 500 M FeSO4 ⫹ 30 mM ascorbate ⫹ 10 mM PolyP ⫹ 100 mM mannitol; line 4, FeSO4 ⫹ ascorbate ⫹ 100 mM mannitol; line 5, no additions. Panel (B): Line 1, 20 M FeSO4 ⫹ 200 M XTH ⫹ 15 nM XO ⫹ 10 mM polyP; line 2, 1 M FeSO4 ⫹ 3 mM ascorbate ⫹ 10 mM polyP; line 3, 20 M FeSO4 ⫹ 200 M XTH ⫹ 15 nM XO; line 4, 1 M FeSO4 ⫹ 3 mM ascorbate; line 5, no additions.
H2O2 is a result of enzyme(s) induction. Indeed, the total catalase activity dramatically increased in the high polyP cells. This high catalase activity was mainly due to induction of HPII. The increase in HPI activity was relatively small and did not contribute significantly to the total induction of catalase activity (Fig. 5). The o-dianisidine assay also confirmed that HPI changed little during polyP accumulation (data not shown). Effect of CN⫺ on H2O2 killing If the resistance to H2O2 is solely dependant on catalase induction, then the difference in survival between the high polyP and the low polyP cells would disappear if catalase activity is blocked with CN⫺. Indeed, protec-
M. A. AL-MAGHREBI and L. T. BENOV
Fig. 3. Lethality of H2O2. QC1799 was grown aerobically in M9CA medium to a density of A600 nm ⬃ 0.4. Half of the suspension was centrifuged, washed, and resuspended in MOPS medium to raise the polyP content. After 3 h the aliquots were diluted with M9 salts to A600 nm ⫽ 0.1 and were incubated 30 min with H2O2 before final dilution and plating. The rest of the suspension was used for assaying polyP. Line 1, high polyP; line 2, low polyp.
tion against high H2O2 concentrations was completely abolished by inhibition of the catalases. At lower H2O2 concentrations (⬍ 5.0 mM) high-polyP cells treated with CN⫺ survived better than the low-polyP ones (Fig. 6). This protection was not related to recovery of catalase activity during the incubation. To be sure that blocking respiration  is not the cause for the CN⫺ effect, the experiment was repeated with a catalase-deficient strain UM 1, and the same results were obtained (not shown). For reasons that are not understood, we did not observe the H2O2-concentration dependence reported by other authors .
Fig. 4. Decomposition of H2O2 by intact cells. High and low polyP QC1799 suspensions were incubated for 15 min with or without 150 M KCN. H2O2 decomposition was monitored spectrophotometrically as described in the Materials and Methods section. Bar 1, QC1799, low polyP; bar 2, QC1799, high polyP; bar 3, as 1, ⫹ CN⫺, bar 4, as 2, ⫹ CN⫺, bar 5, UM1 (catalase-deficient).
Fig. 5. HPI and HPII catalases in high and low PolyP cells. HPI and HPII activities in high and low polyP cells were assayed as described in the Materials and Methods section. Bars 1 and 2, low polyP; bars 3 and 4, high polyP.
Oxidative DNA damage in vivo To check if polyP protects DNA against oxidative damage, SOD⫺ cells were transformed with pcDNA3 plasmid and were exposed to the same concentrations of H2O2. Exposure of low polyP cells to H2O2 induced damage of the plasmid DNA. The percentage of the intact (supercoiled) DNA dropped from 96 to 16%, while that of the nicked, linear, and fragmented DNA increased to 84%. Blocking catalase activity by preincubating the cells with 150 M KCN and subsequent exposure to H2O2 caused total DNA damage (100%) at ⬎ 10 mM H2O2. Accumulation of polyP appeared to prevent DNA damage. Even after incuba-
Fig. 6. Effect of CN⫺ on the killing of high polyP and low polyP cells by H2O2. QC1799 transformed with pcDNA3 plasmid were grown to A600 nm ⫽ 0.5. Half of the suspension was additionally incubated for 3 h in MOPS medium. Aliquots were preincubated with 150 M KCN for 15 min, and were then incubated with H2O2 for 30 min. The insert shows the same cells, but without KCN treatment. Line 1, high polyP; line 2, low polyP.
Polyphosphate and oxidative DNA damage
Table 1. DNA Damage In Vitro Without polyP
N ⫹ L (%)
N ⫹ L (%)
0 30 60
78 31 11
12 69 89
83 37 9
17 63 91
Two g of purified plasmid DNA was incubated at room temperature with 10 mM ascorbate and 100 M Fe (II) in 100 mM K2PO4 (pH 6.0). Purified polyP was added to a final concentration of 1 mM expressed as orthophosphate. Fifty l aliquots were removed at time intervals of 0, 30, and 60 min and kept at ⫺20°C prior to analysis on agarose gel electrophoresis. DNA damage was assessed by the conversion of the supercoiled plasmid DNA into nicked and linear forms and is expressed as a percentage of the total DNA. SC ⫽ supercoiled; N ⫽ nicked; L ⫽ linear.
tion with 20 mM H2O2, the remaining percentage of the intact DNA was 93%. However, blocking the catalases enhanced DNA damage, which was much less pronounced if the polyP content was high (30% vs. 16% of the total DNA remained intact after the exposure, see above). PolyP does not affect oxidative DNA damage directly The fact that high polyP cells show less DNA damage does not necessarily mean that polyP directly protects DNA. Accumulation of polyP coincides with induction of various genes, some of which are involved in the cellular response to oxidative stress and oxidative DNA damage. To determine whether polyP can directly affect DNA, 4-nitrophenol in the HO• assay system was replaced by plasmid DNA, pcDNA3. A system containing Fe(II) ⫹ ascorbate caused DNA damage, as judged by the conversion of the supercoiled plasmid DNA into nicked and linear forms (Table 1). Addition of PolyP, however, did not affect DNA damage. The same result was obtained when O2•⫺ was generated by xanthine oxidase ⫹ xanthine. PolyP protective effect is rpoS-dependent SoxRS, oxyR, and rpoS regulons control the expression of a large number of genes involved in cellular response to a diverse number of stresses, including oxidative stress and starvation [19 –21]. To check if the effect of polyP depended on any one of these regulons soxRS⫺, oxyR⫺, and rpoS⫺ mutants were used. Inactivation of soxRS, oxyR, or rpoS did not affect the accumulation of polyP. PolyP protective effect against H2O2 killing was observed in both, the soxRS⫺ and the oxyR⫺ strains even when catalase activity was blocked (Fig. 7). Inactivation of rpoS, however, completely abolished the
Fig. 7. H2O2 killing of soxRS⫺, oxyR⫺, and rpoS⫺ strains. All conditions were as on Fig. 6. The cells were diluted to A600 nm ⫽ 0.1 and were challenged with 2.5 mM H2O2 for 30 min. oxyR⫺ cells were plated anaerobically to ensure growth.
protective effect of polyP. The respective parental strains were also checked and demonstrated increased H2O2 tolerance with the increase of the polyP content (not shown). DISCUSSION
Recent studies demonstrate that polyP has important physiological functions. In bacteria it is necessary for survival in stationary phase and resistance to oxidative stress . PolyP chelates metals like Fe and Cu, and it has been hypothesized that chelation of such metals by polyP might reduce their toxicity . If so, metal chelation by polyP would play a crucial role in protection against superoxide-induced cell damage. The increased level of superoxide liberates Fe(II) from O2•⫺-sensitive [4Fe-4s] clusters  followed by continuous deposition of iron from the Fe-storage proteins into the cluster by the cluster reconstitutory process [23,24]. Cycling of O2•⫺ attack and [4Fe-4S] reconstitution can allow a single enzyme to release large amounts of free Fe(II) during aerobic growth . It has been demonstrated that sodAsodB E. coli cells contain ⬃ 8 times more free iron than the respective SOD-proficient cells . These excess amounts of Fe(II) in or near DNA and concomitant increase in HO• formation generated by Fenton chemistry are responsible for the observed enhancement in DNA damage and the increased susceptibility to H2O2mediated killing seen in the mutants lacking cytoplasmic SODs. We expected that by chelating the “free” iron, polyP will divert HO• away from the biologically important targets and thus will reduce cell damage. In vitro, however, polyP enhanced the generation of HO• by the Fenton reaction if small molecules like 4-nitrophenol
M. A. AL-MAGHREBI and L. T. BENOV
were used as HO• probes, but had no effect on DNA damage. A plausible explanation is that the polyP-Fe complex is accessible to relatively small molecules like 4-nitrophenol or Coumarin-3-carboxylic acid , but not to the bulky, negatively charged DNA molecule. In vivo, polyP accumulation led to increased H2O2 tolerance. Because polyP did not exert direct protection, this low susceptibility to H2O2 killing could only mean that protective and repair enzymes are induced. Indeed, HPII catalase activity was much higher in the high-polyP cells. Both HPI and HPII are members of the rpoS regulon. HPI is expressed under aerobic and anaerobic conditions and its synthesis is induced when cells are exposed to sublethal levels of H2O2. HPII is produced at low levels when cells are in exponential phase and is the principal hydroperoxidase in aerobically grown, stationary phase cultures . Induction of HPII in the highpolyP cells protected against high and low concentrations of H2O2, but it appeared not to be the only mechanism of protection. Even after the elimination of catalases, the high-polyP cells showed better survival when challenged with low H2O2 concentrations. Killing by low H2O2 concentrations has been attributed to DNA damage . It might therefore appear that polyP effect is mediated through an inducible, DNA-protective or DNA-repair factor. All three stress-response regulons, soxRS, oxyR, and rpoS induce such factors, but the protective effect of polyP was not eliminated by inactivation of oxyR or soxRS. In contrast, polyP protection was completely absent in the rpoS⫺ strains. In E. coli, rpoS encodes a transcription factor s, which controls the expression of more than 50 genes involved in cellular response to diverse number of stresses, including starvation, heat shock, acid shock, osmotic stress, and oxidative DNA damage . Obviously, starvation for amino acids induced both the accumulation of polyP and the synthesis of proteins under rpoS control, and the second, rather than the first was the cause for increased resistance to H2O2 and protection against oxidative DNA damage. This is in agreement with the findings of Shiba et al.  that transient polyP accumulation is capable of inducing the expression of the rpoS regulon at the transcriptional level. Acknowledgements — This work was supported by a grant MB031 from Kuwait University. We are grateful to I. Fridovich (Duke University Medical Center, Durham, NC, USA), J. Imlay (University of Illinois at Urbana-Champaign, Urbana, IL, USA), D. Touati (Institute J. Monod, CNRS, Universite Paris, France), and K. Krogfelt (Statens Serum Institut, Copenhagen, Denmark), who generously provided the strains used in this study. We are also grateful to I. Fridovich for the help in the preparation of the manuscript.
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