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1.
Molecular simulations were performed for phospholipid bilayers as model cell membranes to understand their dynamic and structural properties, in particular how those are affected in the presence of the cell-preservation agents trehalose and DMSO. Simulations covered a large range of temperatures and concentrations of trehalose and DMSO. A number of properties were examined, including the area per head group, lipid-tail order parameter, density profile, conformation, and binding of trehalose. Our results indicate that trehalose specifically interacts with the phospholipid bilayer by binding to the polar head groups (H-bonds). This binding serves as a bridge between the lipids and prevents their aggregation. The hydrophilic nature of trehalose also makes it a water substitute when binding to the phospholipids. At high trehalose concentrations, trehalose forms a glassy matrix and preserves the bilayer structure by 'freezing' it in its natured state, as the core of the bilayer is found to remain fluid. Simulations with DMSO revealed that DMSO can penetrate and freely diffuse through the bilayer, which is attributed to its unique characteristic of interacting favorably with both hydrophilic and hydrophobic domains.  相似文献   

2.
SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells. Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids. In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined. The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids. These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer. It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein. This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis. These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide. This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol. The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.  相似文献   

3.
The interaction of aqueous dimyristoyl-phosphatidylcholine liposomes with the polypeptides gramicidin A, poly-L-lysine, valinomycin, and gramicidin S was investigated by means of laser-Raman spectroscopy. Auxiliary data were obtained with differential scanning calorimetry. Studies were carried out over the temperature range of 0--50 degrees C, encompassing the gel phase, the transition region, and the liquid crystalline phase of the liposomes. Conformational changes in the phospholipid molecules were investigated by measuring the intensity of the 1062-cm-1 Raman band which is assigned to C-C stretching vibrations of trans segments. Three different types of phospholipid-polypeptide interactions were indicated by the observed Raman data. They are interpreted as (a) orderly penetration of the phospholipid bilayer by a hydrophobic polypeptide; (b) polar interactions involving primarily the head groups of the phospholipid; and (c) disorderly hydrophobic binding between a polypeptide and the hydrocarbon domain of the phospholipid.  相似文献   

4.
The interaction of the basic polypeptide poly-L-lysine with the negatively charged phospholipid dipalmitoylphosphatidylglycerol was studied using Raman spectroscopy. The nature of the interaction appeared to depend on the molar ratio of the constituents. At up to one lysine group per lipid molecule, the bilayer was stabilized by the polypeptide that underwent a conformational transition toward an ordered alpha-helical structure, in which the electrostatic interactions were probably maximal. The stabilization of the bilayer was detected by an increase in both the temperature of the thermotropic transition of the lipid and the interchain vibrational coupling of the methylene C-H vibrations. At higher poly-L-lysine concentration, hydrophobic interactions must have been involved to explain the binding of excess polypeptide. There seemed to be a penetration of poly-L-lysine in the bilayer that increased with the polypeptide concentration. Under these conditions, the chain-packing lattice gradually changed from hexagonal to either orthorhombic or monoclinic symmetry. We believe that this change of structure is associated with the interdigitation of the acyl chains.  相似文献   

5.
We have examined the hypothesis that hydrophilic portions of membrane-bound proteins which lie on either side of the phospholipid bilayer may be degraded at a different rate than are the hydrophobic portions of membrane proteins which are within the bilayer. Plasma membrane fractions from cells of the Maden-Darby canine kidney cell line and rat liver were digested with papain and pronase to cleave a mixture of peptides which is enriched in hydrophilic amino acids. It is proposed that these peptides are derived from regions of membrane-bound proteins which lie outside the bilayer. The residual particulate protein is enriched in hydropholic amino acids and presumably contains the portion of membrane-bound proteins which are in direct contact with the bilayer. A double-isotope method was used to assess the relative degradation rates of these two protein fractions. There was no measurable difference in protein degradation rates between the two fractions and the initial plasma membranes. These results suggest that the intramolecular heterogeneity which results from insertion of membrane-bound proteins into a bilayer is not a factor in their degradation.  相似文献   

6.
The deuterated phospholipid, 1,2-dipalmitoyl-d62-phosphatidylcholine is shown by Raman spectroscopic measurements to be useful for obtaining information concerning phospholipid conformation in complex phospholipid and lipidprotein mixtures. The Raman bands of the deuterated phospholipid are assigned, and the sensitivity of these vibrational modes to conformational changes in the bilayer is demonstrated. Deuteration of the alkyl chains reveals the CH vibrations of the head group. A change in these bands is observed at the melting temperature and is assigned to alteration of the glycerol backbone conformation upon melting.  相似文献   

7.
Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.  相似文献   

8.
We have investigated effects of dimethylsulfoxide (DMSO) on the phase stability of multilamellar vesicles of the ether-linked 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine (DHPC-MLV), which is known to be in the interdigitated gel (LbetaI) phase in excess water at 20 degrees C. The results of X-ray diffraction experiments indicate that the DHPC membrane was in the Lbeta, phase at X> or =0.12 (X=mole fraction of DMSO in DMSO/water mixture). The result of differential scanning calorimetry indicate that the gel to liquid-crystalline phase transition temperature increased, but the LbetaI to Pbeta, phase transition temperature decreased with an increase in DMSO concentration. These results show that DMSO stabilizes the bilayer gel phase rather than the LbetaI phase at its low concentration. The solubility of phosphorylcholine, which is the same structure as the headgroup of DHPC, decreased with an increase in DMSO concentration, indicating that the interaction free energy of the hydrophilic segments of the membrane with solvents increases with an increase in DMSO concentration. On the basis of the thermodynamic analysis, the mechanism of the stabilization of the bilayer gel phase of DHPC-MLV by DMSO is discussed. The decrease in the repulsive interaction between the headgroups of the phospholipid induced by the low concentrations of DMSO in water plays an important role in this stabilization.  相似文献   

9.
Two major types of helical structures have been identified in lipid-associating proteins, being either amphipathic or transmembrane domains. A conformational analysis was carried out to characterize some of the properties of these helices. These calculations were performed both on isolated helices and in a lipid environment. According to the results of this analysis, the orientation of the line joining the hydrophobic and hydrophilic centers of the helix seems to determine the orientation of the helix at the lipid/water interface. The calculation of this parameter should be useful to discriminate between an amphipathic helix, parallel to the interface and a transmembrane helix orientated perpendicularly. The membrane-spanning helices are completely immersed in the phospholipid bilayer and their length corresponds to about the thickness of the hydrophobic core of the DPPC bilayer. The energy of interaction, expressed per phospholipid is significantly higher for the transmembrane compared to the amphipathic helices. For the membrane-spanning helices the mean energy of interaction is higher than the interaction energy between two phospholipids, while it is lower for most amphipathic helices. This might account for the stability of these protein-anchoring domains. This computer modeling approach should usefully complement the statistical analysis carried out on these helices, based on their hydrophobicity and hydrophobic moment. It represents a more refined analysis of the domains identified by the prediction techniques and stress the functional character of lipid-associating domains in membrane proteins as well as in soluble plasma lipoproteins.  相似文献   

10.
The effects of phosphocreatine (PCr) and its analogues (creatine, phosphocreatinine, phosphoarginine and inorganic phosphate) on liposomal and erythrocyte membranes and on the sarcolemmal membrane of cardiomyocytes were studied. The ESR spectrum of the spin-labeled probe, 5-doxyl-stearate, incorporated into the membrane were recorded for analysis of the structural order of the phospholipid bilayer of these membranes. PCr and its analogues had no effect on the structure of the phospholipid bilayer in liposomes; this effect was temperature-independent. However, in erythrocyte and sarcolemmal membranes the rigidity of the membranes was increased by these compounds (except for creatine) at temperatures above 38-40 degrees C. Analysis of these and literary data revealed that cardiac cell membranes may be the site of protective action of PCr on the ischemic myocardium. The lack of effect on liposomes may suggest that the membrane-stabilizing effect of PCr depends on the presence of membrane proteins. The compounds under study may influence the lipid-protein interactions by increasing the rigidity of membrane phospholipids. These membranotropic effects may be due to the interaction of charged molecules of the compounds with polar heads of phospholipids and/or polar groups of proteins in the membrane interphase which, in turn, may influence the packing of hydrophobic fatty acid chains.  相似文献   

11.
Kinetics of the interaction of hemin liposomes with heme binding proteins   总被引:1,自引:0,他引:1  
As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.  相似文献   

12.
In this paper analyses are made of the thermodynamic and geometric properties of the predicted association between amphipathic helixes and phospholipid vesicles. From thermodynamic considerations it is proposed that a major driving force for such an association is the negative free energy gained by the transfer of a number of hydrophobic residues (contained within the non-polar faces of amphipathic helixes), from water to the interior of a phospholipid bilayer. The mechanism proposed is that in the aqueous state a potentially amphipathic sequence forms a non-helical hydrophobic patch on the surface of the apolipoprotein. Formation of an amphipathic helix and simultaneous burial of the hydrophobic residues in the surface of a phospholipid bilayer provides the driving force for lipid association. From this model an estimate of the upperlimit for the hydrophobically driven free energy of lipid association (?40?65 kcal/mol) is calculated for the 4 apolipoproteins with known sequences.On the basis of geometrical considerations a model for an intermediate state of high density lipoprotein (HDL) synthesis is proposed. This model consists of a cholesterol-containing phospholipid bilayer disc whose ‘naked’ hydrophobic edges are shielded from the aqueous phase by amphipathic helixes of the apolipoproteins. Exposure of these ‘bicycle tire’ micelles to the enzyme lecithin: cholesterol acyl transferase (LCAT) is postulated to result in the formation of mature spherical HDL particles with cholesteryl ester forming a neutral lipid core.  相似文献   

13.
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
We have employed four lipids in the present study, of which two are cationic and two bear phosphatidylcholine (PC) headgroups. Unlike dipalmitoylphosphatidylcholine, the other lipids employed herein do not have any ester linkage between the hydrocarbon chains and the respective lipid backbones. Small unilamellar vesicles formed from each of the PC and cationic lipids with or without varying amounts of cholesterol have been examined using the steady-state fluorescence anisotropy method as a function of temperature. The anisotropy data clearly indicate that the order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect is similar irrespective of the electrostatic character of the lipid employed. The influence of cholesterol inclusion on multi-lamellar lipid dispersions has also been examined by 1H-nuclear magnetic resonance spectroscopy above the phase transition temperatures. With all the lipids, the line widths of (CH2)n protons of hydrocarbon chains in the NMR spectra respond to the addition of cholesterol to membranes. The influence on the bilayer widths of various lipids upon inclusion of cholesterol was determined from X-ray diffraction studies of the cast films of the lipid-cholesterol coaggregates in water. The effect of cholesterol on the efflux rates of entrapped carboxyfluorescein (CF) from the phospholipid vesicles was determined. Upon incremental incorporation of cholesterol into the phospholipid vesicles, the CF leakage rates were progressively reduced. Independent experiments measuring transmembrane OH- ion permeation rates from cholesterol-doped cationic lipid vesicles using entrapped dye riboflavin also demonstrated that the addition of cholesterol into the cationic lipid vesicles reduced the leakage rates irrespective of lipid molecular structure. It was found that the cholesterol induced changes on the membrane properties such as lipid order, linewidth broadening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3beta-OH of cholesterol. These findings emphasize the importance of hydrophobic interaction between lipid and cholesterol and demonstrate that it is not necessary to explain the observed cholesterol induced effects on the basis of the presence of hydrogen bonding between the 3beta-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region.  相似文献   

15.
There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of beta-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Abeta(1-42), i.e., Abeta(25-35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Abeta(25-35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Abeta(25-35).  相似文献   

16.
This work examines the extent to which alterations in the composition of the phospholipid bilayer of the erythrocyte membrane influences the stability of the association of the ‘cytoskeletal network’ to the rest of the membrane. Rates of spectrin-actin dissociation at low ionic strength were used as a measure of the stability, and composition of the phospholipid bilayer was altered by the action of the enzyme phospholipase A2. Hydrolysis of all the phosphatidylcholine of the outer leaflet of the bilayer had no effect on dissociation rates, whether or not the hydrolysis products were extracted with albumin. Hydrolysis of inner leaflet phospholipids increased the rates by up to 2-fold if the hydrolysis products were not extracted; for ?50% hydrolysis, the rates were unaffected if the hydrolysis products were extracted. The moderate magnitudes of the increases in dissociation rates indicate that interactions between the ‘cytoskeletal network’ and the phospholipid bilayer are not a decisive factor in maintaining the stability of the membrane, at least under low ionic strength conditions.  相似文献   

17.
Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.  相似文献   

18.
C C Lo  J H Hsu  Y C Sheu  C M Chiang  W g Wu  W Fann    P H Tsao 《Biophysical journal》1998,75(5):2382-2388
Cobra cardiotoxins (CTXs) are able to adopt a three-fingered beta-strand structure with continuous hydrophobic patch that is capable of interacting with zwitterionic phospholipid bilayer. In addition to the four disulfide bonds that form the rigid core of CTXs, Asp57 near the C-terminus interacts electrostatically with Lys2 near the N-terminus (Chiang et al. 1996. Biochemistry. 35:9177-9186). We indicate herein, using circular dichroism and the time-resolved polarized tryptophan fluorescence measurement, that Asp57 to Asn57 (D57N) mutation perturbs the structure of CTX molecules at neutral pH. The structural stability of the D57N mutant was found to be lower, as evidenced by the reduced effective concentration of the 2,2,2-trifluoethanol (TFE)-induced beta-sheet to alpha-helix transition. Interestingly, the single mutation also allows a greater degree of molecular unfolding, because the rotational correlation time of the TFE-induced unfolding intermediate is larger for the D57N mutant. It is suggested that the electrostatic interaction between N- and C-termini also contributes to the formation of the functionally important continuous hydrophobic stretch on the distant end of CTX molecules, because both the binding to anilinonaphthalene fluorescent probe and the interaction with phospholipid bilayer were also reduced for D57N mutant. The result emphasizes the importance of the hydrophobic amino acid residues near the tip of loop 3 as a continuous part of the three-fingered beta-strand CTX molecule and indicates how a distant electrostatic interaction might be involved. It is also implicated that electrostatic interaction plays a role in expanding the radius of gyration of the folding/unfolding intermediate of proteins.  相似文献   

19.
Copper‐Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo‐SOD1 aggregates may be due to their ability to cause defects within cell membranes by co‐aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane‐bound copper chaperones. But how apo‐SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc‐binding loops in apo‐SOD1 dimers play a critical role in the binding of 1‐octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo‐SOD1 monomer also associates with phospholipid bilayer via its zinc‐binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane‐bound copper chaperone. The orientation also suggests how membrane‐bound monomers could act as seeds for membrane‐associated SOD1 aggregation. Proteins 2014; 82:3194–3209. © 2014 Wiley Periodicals, Inc.  相似文献   

20.
The first direct experimental evidence that gramicidin A (gA), a transmembrane peptide, facilitates the translocation of unlabeled lipids in a phospholipid bilayer was obtained with sum-frequency vibrational spectroscopy (SFVS). SFVS was used to investigate the effect of gA on lipid flip-flop in a planar 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipid bilayer. The kinetics of lipid translocation were determined by an analysis of the SFVS intensity versus time at different temperatures in the presence of 2 mol % gA. The rate constants of DSPC flip-flop increase from 2 to 10 times relative to the pure DSPC system. The results indicate that facial lipid exchange can be induced by a hydrophobic transmembrane helix. The increase in lipid flip-flop rates is correlated to an increase in the gauche content of the lipid tails. The results suggest that membrane defects induced by the presence of integral membrane proteins may play a large role in modulating the rate of lipid flip-flop.  相似文献   

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