Electrostatic interactions between model mitochondrial membranes

Colloids and Surfaces B: Biointerfaces 41 (2005) 121–127

Electrostatic interactions between model mitochondrial membranes Stephanie Nichols-Smith, Tonya Kuhl∗ Department of Chemical Engineering and Materials Science, University of California, One Shields Avenue, Davis, CA 95616-5294, USA Received 15 October 2003; received in revised form 1 November 2004; accepted 1 November 2004 Available online 7 January 2005

Abstract Lipids are very diverse in both their respective structures and functions; and cells exquisitely control membrane composition. One intriguing issue is the specific role of lipids in modulating the physical properties of membranes. Cardiolipin (CL) is a unique four-tailed, doubly negatively charged lipid found predominately within the inner mitochondrial membrane, and is thought to be influential in determining the inner mitochondrial membrane potential and permeability. To determine the role of cardiolipin in modulating the charge properties of membranes, this study investigated the electrostatic interactions between mixed cardiolipin and phosphatidylcholine bilayers as a function of cardiolipin concentration. For physiologically relevant concentrations of cardiolipin, the surface charge density of the membrane was found to increase linearly with increasing concentration of cardiolipin. However, only a fraction of the cardiolipin molecules predicted to carry a charge from pK-values were ionized. Clearly environmental factors, beyond that of pH, play a role in determining the charge of bilayers containing cardiolipin. © 2004 Elsevier B.V. All rights reserved. Keywords: Surface force; Bilayer; Cardiolipin; Ionization; Electrostatics

1. Introduction Cardiolipin (CL) is a unique phospholipid found only in membranes of bacteria and mitochondria, i.e., those whose function is to generate an electrochemical potential for substrate transport and ATP synthesis [1,2]. Cardiolipin is a dimeric phospholipid with two phosphatidyl moieties linked by a central glycerol group. It has four acyl chains of varying lengths and degrees of saturation and a small headgroup, which contains two acidic sites [2]. Ionization levels of the headgroup have been found to be pH dependent but there are contradictory reports in the literature, particularly at neutral pH. From titrations, Haines and Dencher (2002) determined pK1 = 4 while pK2 = 8, indicating a single negative charge carried at neutral pH [1]. Some studies have assumed two negative charges at neutral pH based on pK-values [3], while others consider both acidic sites to have the same pKa and to be fully ionized at pH 8 [4]. A consensus on the charge of car∗

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0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.11.003

diolipin is yet to be reached. Moreover, the charge properties of membranes containing cardiolipin have not been directly studied. Cardiolipin is the most unsaturated lipid in the human body [5]. Its concentration in mitochondria increases from 3 mol% at birth to adult levels of approximately 9% by the end of the first year of life [6]. Decreased mitochondrial function due to a decline in cardiolipin content is associated with aging [7]. For example Hagen et al. found older rats had a significant reduction in mitochondrial function and ambulatory activity, along with a decline in cardiolipin levels compared to young rats [8]. This decline could be reversed by giving the older rats acetyl-l-carnitine, which increased the cardiolipin levels to near that of young rats, significantly higher than that of untreated older rats. These studies clearly established a link between mitochondrial function and cardiolipin levels. Biosynthesis of cardiolipin has also been shown to decrease in a model of lipid-induced apoptosis [9]. Previous experiments utilizing the Langmuir trough probed the thermodynamic behavior of mixed cardiolipin– egg phosphatidylcholine systems [10]. Comparison of the

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area per molecule in a mixed system with that of the pure species indicated that under low ionic conditions, attractive interactions were present between the lipid molecules for all concentrations above 9% CL. In contrast, repulsive interactions and a relative maximum are uniquely observed at 5% CL. At physiological salt conditions, all molar ratios exhibited attraction with a relative minimum at 5% CL indicating an increased stability over that of 9% CL. Complimentary micropipette aspiration measurements of PC vesicles demonstrated that the vesicle membrane mechanical properties were unaffected upon the inclusion of 5% CL. Increasing the amount of CL to 9.2% decreased both the lysis tension and the area compressibility. Decreases in the area compressibility indicated less energy was required to stretch the membrane and correlates with the creation of inner mitochondrial membrane folds. Decreased lysis tensions indicated membranes containing CL were less stable and ruptured at lower tension values. These thermodynamic and mechanical properties of lipid monolayers and bilayers correlate with the lower proton leakage and increased fluidity of membranes containing cardiolipin [11,12]. Changes in cardiolipin levels should also modulate the degree of surface charge carried by the membrane. Previously, the surface forces apparatus (SFA) technique has been used to determine the surface charge density and surface potential of membranes containing various charged lipid components. In a study of phosphatidylglycerol bilayers, the surface charge density in pure DMPG and DSPG systems as a function of aqueous electrolyte conditions was determined [13]. With monovalent elecrolyte, NaCl, between 0.3 and 9.2 mM, the surface charge density remained constant, consistent with each gel-phase DSPG molecule being fully ionized. In contrast, in fluid DMPG membranes, only 37.6% of the molecules were ionized at 1.5 mM NaCl. These findings suggest fluid bilayers have a significantly diminished chargecarrying capacity. In similar studies, the surface charge density of mixed DLPC and DLPG (3:1, PC:PG) was measured [14]. At pH 5.6 with 0.3 mM NaCl solution, the dissociation of PG was only 2.2%. Increasing the electrolyte concentration to 20 mM increased the dissociation to 17%. Again, the dissociation of lipids in fluid-state membranes was greatly diminished, but could be increased by screening the charges at higher salt concentrations. Very low ionization levels (∼1%) have also been observed for single component bilayers of dihexadecyl phosphate (DHP), even at salt concentrations of 10 mM [15]. On the other hand, examinations of phosphatidic acid established a strong dependence between ionic strength and the surface potential or degree of dissociation. In this study, the surface forces apparatus (SFA) technique was used to directly measure the interaction forces between model egg-phosphatidylcholine membranes as a function of CL concentration. In the absence of proteins, the membrane potential and surface charge density determined from these studies can be unambiguously attributed to the ionization of the cardiolipin molecules.

2. Materials Egg phosphatidylcholine (EPC) dissolved in chloroform and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) in powder form were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. Bovine heart cardiolipin (CL) dissolved in methanol was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used without further purification. Lipid structures are shown in Fig. 1. Chloroform, HPLCgrade, purchased from Acros (NJ, USA), was used to dilute and store the lipid solutions. Sodium nitrate 99.995% was purchased from Aldrich (St. Louis, MO, USA). Ntris[Hydroxymethyl]methyl-2-aminoethane-sulfonic acid (TES) was purchased from Sigma at a purity of 99%. Water was purified by a Barnstead nanopure filtration system (Dubuque, IA, USA) to a resistivity of 18.2 M
cm. Lipid solutions were prepared in chloroform at a concentration of 1 mg/ml and varying molar ratios between CL and EPC. For reference, the major lipid components of the human mitochondrial membrane are as follows: phosphatidylcholine (PC), 34%; phosphatidylethanolamine (PE), 43%; cardiolipin, 18%; and phosphatidylinositol (PI), 5% (weight percentages of the total weight of lipids) [16]. 2.1. Surface forces apparatus A MarkII surface forces apparatus was utilized in these experiments [17]. The substrates were molecularly smooth, back-silvered, mica glued onto two cylindrically curved silica disks. The upper surface was mounted on a fixed support while the lower surface was mounted on a double cantilever spring, displaceable vertically by a micrometer screw. Motors were used to finely control the movement of the lower surface. The silver layer on each disk not only partially transmits light directed normally through the surfaces, but also constructively interferes, such that distances between the surfaces could be measured by observation of the displacement of fringes of equal chromatic order (FECO) within a spectrometer [18]. Forces were measured through displacement of the spring with known spring constant. The movement of the lower surface was related to the movement of the FECO fringes at a distance sufficiently beyond the range of forces. Comparison of the applied motion, known from this calibration with the actual motion observed from FECO images, enables the displacement of the spring to be measured directly, thereby determining the force between the surfaces. The measured radius of curvature for each contact position was used to normalize the force profile. The Derjaguin approximation relates the interaction energy per unit area for two flat plates with the force relationship between two crossed cylinders. E(D) =

F (D) 2πR

(1)

where E is energy per unit area, F the force between the cylinders, and R the radius of the contact position [19]. The

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Fig. 1. Structure of lipids: cardiolipin, EPC, and DPPE. Note that EPC and cardiolipin are natural lipids with a distribution of acyl chain lengths and saturations. Both DPPE and EPC are zwitterionic, whereas cardiolipin has two negative charges in its headgroup associated with sodium counterions.

Derjaguin approximation is valid at small distances where D  R. The radius of curvature was measured for two crosssections at 90◦ and the geometrical mean was used in calculations. For these experiments, the radius of curvature was 1.5 ± 0.5 cm. The SFA was filled with an aqueous solution containing 0.5 mM NaNO3 at pH 5.3 ± 0.2. The measured electrolyte concentration was found to vary slightly, 0.5 ± 0.2 mM. These small variations in the effective electrolyte may have resulted from salt contamination in the SFA and/or the transfer of the membrane surfaces after LB deposition. Effects from sodium dissociation from CL were negligible. In the vesicle adsorption experiments, the aqueous solution contained 0.5 mM NaNO3 , and 10 mM TES buffer solution at pH 7.

external particles from falling onto the surface and to avoid air currents from disrupting the monolayer or the pressure sensor. Fig. 2 shows the surface pressure–area (Π–A) curves of EPC and mixtures of 5, 9.2, 15 and 100 mol% CL. Lipids were compressed at a constant rate of 25 cm2 /min, corresponding to a linear barrier speed of 1.25 cm/min, until collapse. To check reproducibility of the isotherm analysis, experiments were performed on independent mixtures made from separate stock solutions of CL and EPC. Average isotherms were then obtained by arithmetically averaging the independent

2.2. Langmuir trough A Teflon® Langmuir–Blodgett trough with dimensions 20 cm × 30 cm, (Type 611, Nima, UK) was used to measure surface pressure–area per molecule isotherms. The surface pressure was measured using a filter paper Wilhelmy plate. Surface pressure and trough area were recorded simultaneously using software provided by Nima. Based upon the volume deposited, average molecular weight, and solution concentration, the average area per molecule was calculated. All trough work was carried out at a room temperature of 22 ± 1 ◦ C. The trough was enclosed in a chamber to prevent

Fig. 2. Pressure vs. area (Π–A) isotherms for mixed CL/EPC monolayers at molar ratios of pure EPC, 5% CL:95% EPC, 9.2% CL:90.8% EPC, 15% CL:85% EPC, 100% CL, on a subphase of nanopure water at a room temperature of 22 ± 1 ◦ C. Selected error bars shown are ±1 standard deviation.

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isotherms from separate solutions. The isotherms shown are the average isotherms with selected error bars corresponding to ±1 standard deviation. As expected, the isotherms indicate that CL:EPC mixtures are in the fluid state with no evidence of phase separation.

2.5. Electrostatic forces

2.3. Langmuir–Blodgett deposition

d2 Y = sinh Y dx2

Langmuir–Blodgett (LB) deposition was used to construct supported bilayers on mica. The supported bilayers were composed of an inner leaflet of DPPE and an outer leaflet of EPC containing CL. The DPPE monolayer was ˚2 deposited at a constant pressure of 39 mN/m (about 43 A per molecule). To deposit the inner layer, mica-coated disks were submersed beneath the surface of the water prior to the addition of the lipids at the air–water interface. The deposition took place as the disks were raised up through the interface. The DPPE leaflet binds strongly to mica and provides a smooth hydrophobic surface for deposition of the outer CL:EPC monolayer [20]. After depositing the DPPE monolayer, the surfaces were allowed to dry overnight at room temperature before deposition of the outer CL:EPC monolayer. The outer bilayer leaflet consisted of EPC and 5, 9.2, or 15% CL by mole. For all mole fractions, the monolayer was deposited at 25 mN/m. This outer layer was deposited by lowering the DPPE-coated disks from air down through the interface into the water. Transfer ratios of 1.00 ± 0.05 were obtained using large (30–40 cm2 ) DPPE-coated mica test sheets. A transfer ratio of 1 indicates that the deposited lipids maintain their packing area during deposition. Once the complete bilayer was formed, the surfaces were kept submerged and mounted in the SFA. 2.4. Vesicle adsorption Small unilamellar vesicles were prepared from 9.2% CL:90.8% EPC. Lipid solutions in chloroform were evaporated using a rotavaporator, then hydrated with degassed water and frozen. The solution was cycled through freeze–thaw 12–15 times and extruded through 100 nm-pore filters. In experiments where CL:EPC monolayers were formed by vesicle adsorption, DPPE monolayer contact was first determined in air. Afterwards, the SFA was filled with buffer solution. The surfaces were separated by 0.5–1 mm and 60 ␮l of vesicle solution of 10 mg/ml was injected between the DPPE-coated mica surfaces leading to an overall concentration of 0.002 mg/ml in the SFA. Bilayers were formed when CL:EPC monolayers spontaneously self-assembled on the hydrophobic DPPE monolayers. An incubation period of 2 h followed injection of vesicles before force runs commenced. In some cases, incubation occurred for longer time periods or at a higher concentration of vesicles to prevent hemifusion between incomplete membranes [21].

The electrostatic double-layer force was calculated using a numerical iterative solution to the nonlinear Poisson Boltzmann equation (NLPB) (this program was graciously supplied by Alexis Grabbe) [22]. (2)

where x and Y are defined as Y=

zeψ kT

(3)

x=

D κ−1

(4)

where κ−1 is the Debye length, z the valency of the ion (monovalent in this case), e the charge on an electron, ψ the electrical double-layer potential, k the Boltzmann constant, and ˚ T the absolute temperature. At large distances (D > 100 A), all forces were attributed to the electrostatic double-layer. An exponential curve was fit to the data to determine the Debye length and effective salt concentration of the system. The surface charge density and potential of the CL:EPC bilayers were then calculated by solving the NLPB equation [22]. The Derjaguin approximation (Eq. (1)) was used to convert the NLPB solution between flat plates to the force between a sphere and a flat, normalized by the radius of curvature. 2.6. Determination of membrane thickness and D = 0 To maintain equilibrium between free monomers in solution and the LB-deposited bilayers during experiments, the apparatus solution was presaturated with lipid of the outer leaflet. In addition, experiments were completed within 72 h of assembling bilayer surfaces in the apparatus. At the end of each experiment, the surfaces were separated and the apparatus solution was drained, thereby removing the outer leaflet of the bilayer. In air, the hydrophobic inner DPPE layers were brought into contact to determine the thickness change attributable to the outer leaflets and their hydration. Contact between the two bilayers was defined as D = 0. The negative charges on cardiolipin originate in the headgroup, thus D = 0 was also chosen as the outer Helmholtz plane from which the diffuse double layer originates. The experimental thickness of two CL:EPC monolayers can be compared to the theoretical thickness for anhydrous bilayers using the volume of the hydrocarbon chains and the headgroups along with the known deposition area per molecule. Experimental details are given in Table 1. The total bilayer thickness is given as T = 2[2Vhydrocarbon + Vheadgroup ]/Area. The volume of a single gel-state hydrocarbon chain is, Vhydrocarbon = (27.4 + 26.9n) for an n-carbon chain [23]. Assuming the density of a gelstate hydrocarbon chain is 0.87 g/cm3 while a fluid-state hydrocarbon chain is 0.70 g/cm3 [24], the total volume of

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Table 1 SFA experimental details

5% CL–95% EPC 9.2% CL–90.8% EPC 15% CL–85% EPC

Area per ˚ 2) molecule (A

Drainage ˚ thickness (A)

73 ± 2 73 75

69 ± 3 68 70

SFA-measured area per molecule at deposition and drainage thickness of outer leaflets.

the hydrocarbon chains in the case of 5% CL:95% EPC is ˚ 3 . Since only 5% CL was present, the volume of a PC 1293 A headgroup was used to approximate the headgroup volume, ˚ 3 [24]. At a deposition area per molecule Vheadgroup = 324.5 A ˚ 2 , the theoretical anhydrous bilayer thickness is 44 A. ˚ of 73 A The majority of lipids present in the bilayer system are phosphatidylcholine, known to be well hydrated by approximately ˚ of bound-water [25]. Combining the theoretical bi+10–12 A layer thickness with the predicted hydration for PC, the ex˚ which is comparable pected bilayer thickness is ∼64–68 A, to the experimentally measured drainage thicknesses shown in Table 1. 3. Results and discussion Forces were measured between supported lipid bilayers of cardiolipin and egg phosphatidylcholine. As the percentage of cardiolipin in the outer leaflet of the bilayer increases, the force due to electrostatic double-layer repulsion is expected to increase due to the additional negative charges of cardiolipin. Representative force curves are shown in Fig. 3 for each of the three different bilayer concentrations. Measurements for the different bilayers have been separated vertically by a factor of 103 and 106 for comparison. In the semi-logarithmic plot, a straight line on the graph corresponds to an exponential decay in the force, as expected for interacting double-layers of charged bilayers. Variations in the decay slope are due to small differences in the effective electrolyte concentration (0.5 ± 0.2 mM). At small distances, the measured force increases considerably due to the electrostatic double-layer repulsion, headgroup hydration and thermal fluctuations of the lipid molecules in and out of the bilayer plane. The long-range electrostatic repulsion was fit using a numerical solution to the nonlinear Poisson Boltzmann equation [22]. Results are given in Table 2. As the mole fraction of cardiolipin increases, the surface charge density of the bilayer increases. The measured surface charge density can be directly converted from mC/m2 to an area per electronic charge.

Fig. 3. Measured force vs. distance on a semi-log plot for LB-deposited bilayers as a function of CL concentration; open symbols indicate approach, solid symbols indicate separation. Solid lines are exact numerical calculations of the theoretical double-layer repulsion with constant surface charge boundary conditions (Eqs. (2)–(4)). At contact, the membranes are adhesive due to van der Waals interactions. Weak hydrophobic interactions may also be present. The inset shows a linear relationship between the measured surface charge density with the CL concentration.

Comparison between the total areas between CL molecules, considering two ionization sites per CL, with the area per electronic charge yields the ionization. Simple prediction of each CL carrying two negative charges significantly overestimates the amount of charge to be carried by each surface. For example the shift from 5 to 9.2% CL would suggest a near doubling of the surface charge whereas only a 27% increase was shown. Similarly increasing from 5 to 15% CL would suggest a tripling of the surface charge density while an increase of 62% was observed. As the concentration of CL increases, their lateral packing density in the bilayer increases. The overall charge density of the membrane goes up, but as a result of lateral proximity, the fraction of CL ionized becomes more and more suppressed. The inset of Fig. 3 shows that there is a linear relationship between the mole fraction of cardiolipin and the membrane surface charge density. The slope (0.32 mC/m2 per % CL, R = 0.999) indicates a given increase in cardiolipin content corresponding to a 32% increase in the surface charge density. Focusing on the most physiologically relevant concentration, 9.2%, a series of force curves is shown in Fig. 4. In

Table 2 SFA-measured experimental results

5% CL–95% EPC 9.2% CL–90.8% EPC 15% CL–85% EPC

σ (mC/m2 )

ψ (mV)

˚ 2 per CL A

˚ 2 per e− A

Ionization (%)

5.1 ± 3.0 6.5 ± 3.3 8.3 ± 2.5

66.2 ± 18.8 81.0 ± 28.3 93.0 ± 15.8

1460 790 500

3140 2460 1930

23 16 13

Surface charge density, surface potential, area between cardiolipin molecules, area between electronic charges, and ionization based on a possible charge of 2e− per cardiolipin.

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Fig. 4. Measured force vs. distance on a semi-log plot for LB-deposited bilayers of 9.2% CL:90.8% EPC in 0.5 mM NaNO3 . Solid and dashed lines are the theoretical double-layer repulsion with constant surface charge and constant surface potential boundary conditions, respectively.

addition to the forces predicted based on a constant surface charge density of 7 mC/m2 , the solution for a constant surface potential of 87.7 mV is also shown. Data points obtained from the approach of the surfaces are distinguished from those obtained from the separation. Upon separation, the force curve does not follow the approach force curve due to attractive van der Waals’ and hydrophobic interactions between the bilayers at short distances. Following adhesive contact, the lower surface ‘jumps out’ when the gradient of the force exceeds the mechanical spring constant and then traces the same electrostatic double-layer repulsion path as the approach force curve. The van der Waals force is related to the distances between the surfaces and the Hamaker constant through Eq. (5) Fad −A = 2 R 6Deq

(5)

where A is the Hamaker constant, Fad /R the adhesive force at the minimum, and Deq the distance at the adhesive energy well [26]. The average adhesion measured for the three CL concentrations was Fad /R = 4.6 ± 1.6 mN/m. Using an approximate Hamaker constant of 7 × 10−21 J for PC bilayers ˚ [20]. This is would require a pull-off distance of Deq = 4 ± 1 A significantly reduced from previous work on saturated PC bi˚ was determined and layers, where a pull-off distance of 14 A the adhesive force was 0.6 mN/m [20]. Although force profiles for all CL concentrations were reproducible with multiple contacts, in some cases the adhesive minimum was found to increase slightly with increasing contact time. Taken together, these findings suggest that hydrophobic interactions between opposing lipid bilayers give rise to an increased adhesion. The most likely reason for this increased hydrophobic interaction is inadequate screening of the hydrocarbon tails by the PC or CL headgroups. Saturated, fluid state PC ˚ 2 at headgroups have a typical cross-sectional area of 65 A our deposited surface pressure of 25 mN/m [27]. In comparison, our Langmuir-deposited lipids had cross-sectional areas ˚ 2 for 5, 9.2, and 15% CL, respectively. of 73, 73, and 75 A

Fig. 5. Measured force vs. distance on a semi-log plot for vesicle-adsorbed bilayers at 9.2% CL:90.8% EPC in 2.9 mM electrolyte. Solid line corresponds to a constant surface charge of 8 mC/m2 .

The increased area per headgroup with increasing CL concentration is due to less efficient packing arrangements. As a result, there is less shielding of the hydrophobic core and a coincident increase in the adhesion. The exposure of more hydrophobic groups would also act to suppress ionization of CL in the membrane due to the lower dielectric of hydrocarbon compared to water (ε∼4 versus 78) [28]. Such effects are observed in charged surfactant systems where the degree of headgroup dissociation is approximately 25% when the surfactant molecules are associated in micelles, i.e., when surrounded by the low dielectric constant environment of the hydrocarbon chains [29]. Similar experiments carried out using vesicle adsorption to form the model mitochondrial membrane yielded comparable results. In the vesicle adsorption experiments for 9.2% CL, the effective salt concentration was 2.9 mM and the surface charge density was fit to 8 mC/m2 . Force–distance curves for vesicle adsorption are shown in Fig. 5. The thickness change upon removal of the CL:EPC outer monolayer was ˚ commensurate to the observed thickness of the bilay65 A, ers deposited using Langmuir–Blodgett deposition. This confirms our findings are intrinsic to the properties of the bilayer and not an artifact of the method of formation.

4. Conclusion The surface charge density observed between model inner mitochondrial membranes comprised of cardiolipin and egg phosphatidylcholine cannot be predicted a priori based on the surface charge thought to be carried by each cardiolipin molecule at a given pH. At 5% cardiolipin, the surface charge density is 5.1 ± 3.0 mC/m2 , and the fraction of cardiolipin lipids carrying two negative charges is 23%. While at 15% cardiolipin, the surface charge density is 8.3 mC/m2 and the fraction fully ionized is 13%, only a small portion of the cardiolipin molecules predicted to carry a charge from pKvalues are ionized. Moreover, the surface charge density was determined to increase linearly with the concentration of cardiolipin. Clearly environmental factors, beyond that of pH,

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play a role in determining the charge of bilayers containing cardiolipin.

Acknowledgement This work was generously supported by the Searle Scholars Program/the Chicago Community Trust (01-L-108).

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