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First published online 6 June 2006
doi: 10.1242/jcs.03007
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Research Article |
Departments of Physiology and Biophysics, Biochemistry and Molecular Biology, and Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, Faculty of Medicine, Calgary, AB, T2N 4N1, Canada
* Author for correspondence (e-mail: jcoorsse{at}ucalgary.ca)
Accepted 4 April 2006
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Rafts, Microdomains, Ceramide, Lysenin, Cholesterol, Secretory vesicles
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Introduction |
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) and these also appear to be the sites of Ca2+-triggered membrane fusion (Lang et al., 2001; Ohara-Imaizumi et al., 2004; Gil et al., 2005; Churchward et al., 2005). Fusion of vesicles with areas of the plasma membrane rich in cholesterol (CHOL) and sphingolipids, which can self associate to form lipid rafts (Simons and Ikonen, 1997; Brown and London, 2000; Simons and Ehehalt, 2002; Ramstedt and Slotte, 2002; Scherfeld, 2003; Wang and Silvius, 2003), might be more energetically favorable than fusion with non-raft domains (Coorssen and Rand, 1990; Chamberlain et al., 2001; Churchward et al., 2005). An attractive hypothesis is that rafts form platforms to concentrate certain proteins that are important for exocytosis and membrane fusion itself, thereby governing their interaction or segregation (Chamberlain et al., 2001; Lang et al., 2001; Ohara-Imaizumi et al., 2004; Churchward et al., 2005; Salaun et al., 2005). CHOL, a key component of membrane microdomains, plays a crucial role in the process of Ca2+-triggered membrane fusion, acting as both a membrane organizer contributing to the efficiency of fusion and, by virtue of its intrinsic negative curvature (Coorssen and Rand, 1990; Chen and Rand, 1997), as a specific molecule acting directly to facilitate Ca2+-triggered membrane merger (Churchward et al., 2005). The data indicate a more dynamic role for CHOL than previously envisaged, and demonstrate separation of the fundamental ability to fuse from the efficiency (Ca2+ sensitivity and rate) of the process, suggesting that the latter is largely dependent on microdomain organization (Churchward et al., 2005). Microdomains are postulated to maintain the localization of `efficiency' factors, including Ca2+ sensors and other protein and lipid components, so that these directly facilitate the fundamental fusion machine that is of low intrinsic Ca2+ sensitivity, thus bringing the Ca2+ activity of the mechanism into the recognized physiological range (Coorssen et al., 2003; Churchward et al., 2005).
Several studies have demonstrated that lipids have a strong self-organizing capacity; indeed, lipid immiscibility can drive phase separation and thereby domain formation within model lipid assemblies (Joost et al., 2001). Together with CHOL, the sphingolipids are recognized to form liquid-ordered microdomains that segregate from the more fluid regions of membranes (Brown and London, 2000; Samsonov et al., 2001; Wang and Silvius, 2003). In particular, sphingomyelin (SM) has been shown to form tight hydrophobic interactions with CHOL and to have a key role in the formation of lipid rafts (Simons and Ikonen, 1997; Brown and London, 2000; Simons and Ehehalt, 2002; Ramstedt and Slotte, 2002; Scherfeld, 2003; Wang and Silvius, 2003). CHOL also appears to stabilize the coexistence of segregated phases when mixed with SM (Samsonov et al., 2001).
Sphingomyelinase (SMase) is an enzyme that hydrolyzes SM to ceramide (Cer) and thereby alters membrane lipid composition (Zxa et al., 1998). Thus, in addition to its structural role, SM is also a source of Cer that has been implicated in cell signaling events (Zhang and Kolesnik, 1994; Cremesti et al., 2002). Cer is also known to induce negative monolayer curvature, and its tendency to form domains appears to be important in membrane restructuring processes (Montes et al., 2002; Saez-Cirion et al., 2002). Moreover, Cer can also induce trans-bilayer lipid motion (Contreras et al., 2005). This ability of Cer to promote lipid flip-flop is dependent on the presence of CHOL and phosphatidylethanolamine, which are also known to facilitate inverted phase formation (Coorssen and Rand, 1990). In addition, Cer production results in extensive aggregation and leakage of vesicles containing SM and CHOL as a result of the transient formation of nonlamellar inverted phases (Ruiz-Argüello et al., 1996; Grassme et al., 2001).
Reduction of cellular SM results in disintegration of membrane microdomains. The marked differences in the physical properties of SM and Cer affect the local structural organization of the membrane during SMase-driven hydrolysis (Fanani, 2002). Lysenin, a uniquely selective reagent that has been well characterized in recent years, was used to elucidate the influence of SM on membrane structural organization (Ishitsuka and Kobayashi, 2004; Ishitsuka et al., 2005). Isolated from the earthworm Eisenia foetida, lysenin is a 41 kDa protein that binds SM with high selectivity in that binding is dependent on local SM density (Yamaji et al., 1998). Thus, lysenin binding was found to be not only selective for SM, but also dependent on local SM organization (Ishitsuka and Kobayashi, 2004; Ishitsuka et al., 2005), defining this as a powerful probe with which to study the membrane-active roles of SM.
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; Coorssen et al., 2003; Szule and Coorssen, 2003; Hibbert et al., 2005; Churchward et al., 2005). Here, we test the hypothesis that CHOL- and SM-enriched membrane microdomains underlie the efficiency of the fusion mechanism but not the fundamental ability to fuse (Churchward et al., 2005). Rather than addressing this by manipulating only CHOL (Churchward et al., 2005), we reasoned that this model could also be tested by disrupting rafts using SMase. Our working model predicts that such selective disruption of endogenous rafts, without affecting CV membrane CHOL density, should specifically alter the efficiency of fusion without inhibiting the fundamental ability to fuse (Churchward et al., 2005). The results are consistent with this interpretation, indicating a crucial role for raft integrity in ensuring the efficiency of fusion, whereas the intrinsic ability of the native membranes to fuse is unaffected. |
Results |
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; Churchward et al., 2005) with respect to that of the untreated controls (Fig. 1a, Fig. 2a, Fig. 3a). Molecular analyses confirmed a dose-dependent decrease in the total CV membrane SM content (Fig. 1c). Although this could not be measured directly after treatments with 20 U/ml SMase (see Materials and Methods), linear extrapolation suggests that endogenous SM concentrations in the CV membrane are reduced by 
80% under this condition. Notably, although fusion kinetics were dose-dependently inhibited at t>1 second, the fast initial rate of fusion (48.8±1.9% per second; n=5) was not affected by any of the SMase treatments (Fig. 1b).
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Previous studies indicated that the competence of CV fusion was dependent on membrane microdomains rich in CHOL, and that microdomain disruption selectively affects the rate and Ca2+ sensitivity of fusion (Churchward et al., 2005). In an effort to rescue CV fusion competence, we supplied exogenous CHOL using the carrier 2-hydroxypropyl-ß-cyclodextrin (hpßcd) (Churchward et al., 2005). CVs initially subjected to SMase treatments were subsequently incubated with CHOL-loaded hpßcd (2 mM) for 30 minutes. Supplementation of CHOL into SM-depleted CVs resulted in full recovery of Ca2+ sensitivity (not shown) and kinetics of fusion (Fig. 2). These results are thus consistent with recent findings that the Ca2+ sensitivity and rate of fusion are dependent upon microdomain integrity (Churchward et al., 2005).
The specificity of the previously established correlation between CHOL depletion and inhibition of fusion was confirmed using methyl-ß-cyclodextrin (mßcd) as a tool for selective CHOL removal and disruption of lipid rafts (Churchward et al., 2005). To test the relationship between fusion and lipid rafts further, CHOL was depleted with mßcd either before or after SMase-mediated SM hydrolysis. CVs treated only with 2 mM mßcd showed a significant rightward shift in Ca2+ sensitivity (EC50=139.9±5.7 µM [Ca2+]free; n=8), a 40.9±4.2% inhibition in the extent of fusion, and a complete inhibition of fast initial fusion kinetics (Fig. 3a,b), consistent with previous studies (Churchward et al., 2005). Subsequent treatment with SMase had no further effect on the extent or Ca2+ sensitivity of fusion (Fig. 3a). By contrast, treating CVs with mßcd following SM hydrolysis did not further decrease the Ca2+ sensitivity, and only a slight inhibition of fusion was observed (Fig. 3a); the extent of fusion was 90.1±3.6% (n=5, P<0.01). Although there was no detectable CHOL in supernatants of SMase-treated and control CVs (not shown), after treatments with 2 mM mßcd, with or without a subsequent exposure to SMase, 25.6±0.8 and 27.1±0.6 µM of CHOL, respectively, were found in the buffer. Thus, raft disruption with SMase alone is selective, and does not result in a loss of CV CHOL.
By contrast, CVs treated first with SMase and then with mßcd had a much higher Cer concentration (363.0±3.0 zmol/CV) than did CVs treated first with mßcd and then with SMase (155.0±26.5 zmol/CV); untreated CVs and CVs treated with only SMase had 131.0±20.5 zmol/CV and 347.0±10.0 zmol/CV of Cer, respectively (Fig. 3c). Thus, an initial treatment with mßcd obviated the subsequent treatment with SMase, resulting in no more Cer than in controls.
Following incubation with the protein lysenin (0.6 nM), which binds and sequesters SM with high selectivity (Fanani et al., 2002; Ishitsuka and Kobayashi, 2004; Ishitsuka et al., 2005), the extent of fusion was inhibited by 24.9±1.4% (n=8; Fig. 4a). Lysenin had no effect on the Ca2+ sensitivity or kinetics of fusion (Fig. 4a,b). However, following treatment of CVs with SMase, adding more than twice the initial dose of lysenin (1.5 nM) did not affect the extent, Ca2+ sensitivity (Fig. 4a) or rate of fusion (not shown) relative to SMase-treated CVs. Thus, after SMase treatment, lysenin could no longer effectively bind to CVs.
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Discussion |
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; Coorssen et al., 2003; Hibbert et al., 2005; Churchward et al., 2005), we find that raft integrity underlies the efficiency of fast, Ca2+-triggered native membrane fusion, but not the fundamental ability to fuse. Using selective methods, including enzymatic digestion and sequestration within the membrane (Churchward et al., 2005), we show that the integrity of microdomains rich in SM-CHOL correlates directly with Ca2+ sensitivity and late fusion kinetics. In terms of the Ca2+ sensitivity of triggered fusion, SM does not appear to contribute directly, but rather through its role as a microdomain organizer. Thus, unlike its neighboring CHOL molecules, SM is not an essential component of the minimal native fusion machine. As a microdomain organizer, SM is associated with Ca2+ sensing and/or the interaction of additional proteinaceous or lipidic components that support the physiological Ca2+ sensitivity of triggered membrane merger.
Previous studies indicated that a decrease in membrane CHOL content perturbed CHOL-rich microdomains, which in turn modulated the efficiency of the fusion mechanism (Churchward et al., 2005). If SM hydrolysis, like CHOL depletion, has a direct impact on microdomain structural integrity (without affecting membrane CHOL density), it was postulated that SMase would have a selective effect on the efficiency of fusion (Ca2+ sensitivity and rate). Rather than the mixed effects seen upon CHOL depletion (Churchward et al., 2005), here we show that microdomain disruption selectively affects the efficiency of fusion but not the fundamental ability to fuse (Figs 1, 2, 3). Treatment with SMase caused a dose-dependent reduction in CV membrane SM, and a parallel inhibition of the Ca2+ sensitivity and late kinetics of fusion (Fig. 1), without affecting CHOL concentrations. As the resulting Ca2+activity curves are all translationally invariant, with no loss in the total extent of fusion, and there was no effect on the initial fast rate of fusion (Fig. 1b), there is consequently no evidence of a fundamental alteration in the minimal native fusion mechanism. Rather, the efficiency of the underlying mechanism has been perturbed. The progressive rightward shift in the Ca2+ sensitivity of fusion with SM hydrolysis indicated that the fundamental mechanism remained potent in terms of the ability to effect bilayer merger, but that Ca2+ sensors were either lost (dispersed) or reduced in sensitivity. Previous studies indicated that some sensors are more directly associated with the minimal fusion machinery, are not lost during microdomain disruption (Churchward et al., 2005), but are sensitive to proteolysis (Coorssen et al., 2003).
Disruption of membrane microdomains was confirmed by two independent methods: SMase activity after treatments with mßcd (Fig. 3) and the binding of lysenin (Fig. 4). The SM-binding protein lysenin (Yamaji et al., 1998; Fanani, 2002; Ishitsuka and Kobayashi, 2004; Ishitsuka et al., 2005) was used as a tool to sequester SM in the membrane, without affecting the integrity of microdomains. Polyene antibiotics have previously been used in a comparable manner to assess the roles of CHOL (Churchward et al., 2005). As it is not expected to disrupt rafts, even a saturating dose of lysenin was found to have no effect on either the Ca2+ sensitivity or kinetics of fusion. By contrast, lysenin selectively inhibited the extent of fusion, most probably by sterically hindering the interaction of CV membranes. As the binding of lysenin is dependent on the local SM density (1 lysenin to 5 SM), this large protein selectively recognizes SM in clusters or domains. Following treatment of CVs with SMase, even more than double the initial dose of lysenin failed to inhibit fusion (Fig. 4a); domain disruption thus results in little or no binding of lysenin.
The structural integrity of microdomains is often disrupted to assess their functional role in biological processes. Changes in CV fusion efficiency upon lipid raft disruption were confirmed by depletion of CHOL using mßcd (Fig. 3) (Churchward et al., 2005). Effective blockade of SMase activity by pretreating CVs with mßcd confirmed the disruption of microdomains enriched in SM-CHOL; intact rafts with densely packed SM result in highly efficient SMase activity, whereas dispersal of microdomain constituents following mßcd treatment results in no significant SM hydrolysis (Fig. 3c). Thus, treating with SMase after rafts had already been disrupted had no further effect on fusion. By contrast, CVs treated with SMase prior to mßcd treatments showed only 10% inhibition in the ability to fuse (e.g. extent), whereas the inhibition caused by mßcd alone was 40% (Fig. 3a); however, these curves did not differ in their diminished Ca2+ sensitivity, consistent with the correlation of domain disruption and reduced fusion efficiency. However, it is also clear that the generation of endogenous Cer, by treating the CVs with SMase prior to mßcd, `protected' against the loss of the ability to fuse that is associated with reduced membrane CHOL densities (Churchward et al., 2005). Consistent with previous work (Pörn and Slotte, 1995), the data here show that reduction of membrane SM density has no effect on the level of membrane CHOL; thus, CHOL still facilitates formation of energetically favorable fusion intermediates (Coorssen and Rand, 1990; Montes et al., 2002; Churchward et al., 2005). Quantitative high-performance thin-layer chromatography (HPTLC) analyses indicated a greater than twofold increase in the level of CV Cer following effective SM hydrolysis (Fig. 3c); this amounts to 21.4x104 molecules of Cer per CV relative to a baseline level of 7.9x104 molecules per CV. With its small hydroxyl headgroup, Cer has specific structural and negative curvature effects on membranes, contributing to the formation of transient, high-curvature membrane intermediates associated with bilayer merger (Ruiz-Argüello et al., 1996; Cremesti et al., 2002; Montes et al., 2002; Saez-Cirion et al., 2002; Van Blitterswijk et al., 2003; Contreras et al., 2005). SMase-mediated Cer accumulation in the membrane can drive structural reorganization within the lipid matrix and the formation of Cer-enriched clusters (Cremesti et al., 2002; Montes et al., 2002; Saez-Cirion et al., 2002). As Cer tends to cluster with itself and with CHOL (Van Blitterswijk et al., 2003), it is effectively localized to contribute negative curvature to the fusion mechanism and thus to protect against the effects of CHOL depletion, comparable with the fusion-facilitating effects of other membrane components having inherent negative curvature (Churchward et al., 2005). It is thus also possible that different types of secretory vesicles or granules utilize alternate negative curvature components such as phosphatidylethanolamine, or its plasmalogen equivalent (Glaser and Gross, 1994), or combinations of these and CHOL to facilitate membrane merger.
The selective effect of SM perturbation on late fusion kinetics is also consistent with the dispersal of domain-enriched efficiency components. Fast initial fusion kinetics are unaffected as Cer can to some extent locally substitute for CHOL (Ruiz-Argüello et al., 1996; Cremesti et al., 2002; Montes et al., 2002; Saez-Cirion et al., 2002; Van Blitterswijk et al., 2003; Contreras et al., 2005), and total CHOL levels are not affected by SMase. However, with the progressive dispersal of microdomain constituents, which are dispersed still further on the large initial fusion products, termed fusosomes (Zimmerberg et al., 2000), there is a progressively lower efficiency for the remaining CVs to fuse with the large fusosomes and hence a slowing of fusion kinetics. Comparable with previous results [fig. 7B in Churchward et al. (Churchward et al., 2005)], increasing native CHOL levels enhanced late fusion kinetics (Fig. 2), indicating some recovery of native component interactions and/or sensitivities. This is particularly notable in that perturbation of native CHOL levels causes far more substantial right-shifts in Ca2+ sensitivity (Fig. 3a) (see also Churchward et al., 2005), comparable with those encountered after potent protein hydrolysis (Coorssen et al., 2003), than does the most extensive loss of SM (Fig. 1). This strongly suggests the specific interaction of CHOL with membrane components that promote physiological Ca2+ sensitivity. CHOL might thus have a third effect on triggered native membrane fusion, essentially acting as a cofactor of specific efficiency components, perhaps to maintain functional conformations (Lee, 2003; Scanlon et al., 2001; Kallen et al., 2002; Graziani et al., 2006).
We establish that membrane microdomain integrity underlies the physiological efficiency (Ca2+ sensitivity and, in part, kinetics) of fusion, but not the fundamental ability of the minimal native mechanism to effect membrane merger. The data are consistent with a fundamental fusion machine of low intrinsic Ca2+ sensitivity (Coorssen et al., 2003) and with a role for microdomains in maintaining the localization and perhaps activity of Ca2+-sensing components that facilitate the membrane fusion process under physiological conditions (Churchward et al., 2005). This suggests that specific membrane rafts and components (Chamberlain et al., 2001; Lang et al., 2001; Ohara-Imaizumi et al., 2004; Chen et al., 2005; Churchward et al., 2005; Gil et al., 2005; Predescu et al., 2005; Salaun et al., 2005) have evolved to promote a more fundamental (and conserved) fusion mechanism (Coorssen et al., 2003; Szule and Coorssen, 2003; Churchward et al., 2005). This basic interplay between local membrane composition and triggered vesicular release might thus underlie the devastating consequences of microdomain disruption that appear to occur in several disease states (Simons and Ehehalt, 2002; Puglielli et al., 2003; Sawamura et al., 2004; Puglielli et al., 2005).
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Materials and Methods |
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CV preparation and fusion assays
Sea urchin CVs were isolated from the unfertilized eggs of Strongylocentrotus purpuratus as previously described (Coorssen et al., 1998). All experiments were carried out in baseline intracellular media (BIM) with the addition of 2.5 mM ATP and protease inhibitors, and CVs in suspension were counted using a hemocytometer (Coorssen et al., 2003; Churchward et al., 2005). Standard end-point and kinetic fusion assays were carried out as previously described (Coorssen et al., 2003; Churchward et al., 2005). The resulting Ca2+ activity curves for fusion were fit using the sigmoidal cumulative log-normal model to establish both the extent and Ca2+ sensitivity of fusion (Coorssen et al., 1998; Coorssen et al., 2003; Churchward et al., 2005). For each analysis, n indicates the number of separate experiments; four replicates per experiment were used for each condition tested.
CV treatments
SMase was dissolved in phosphate-buffered saline (PBS) to a concentration of 1 U/µl, and delivered from this stock solution to final concentrations of 1.0, 5.0, 10.0 and 20.0 U/ml in CV suspensions (in BIM, pH 7.4) having an optical density (OD405) of 1.00±0.05. CVs were treated with SMase for 30 minutes at 30°C, followed by centrifugation to isolate CVs. In some experiments, 100 nM D609 was also used to inhibit trace contaminating phospholipase C activity in the SMase. CHOL was removed from membranes using our established mßcd protocol (Churchward et al., 2005). Addition of CHOL to CHOL-depleted CVs was achieved using CHOL-loaded hpßcd, as previously described (Churchward et al., 2005). Stock solutions of mßcd and CHOL-loaded hpßcd (100 mM) were prepared by dissolving in BIM (pH 6.7) and were added to CV suspensions (OD 1.00) at a final concentration of 2 mM; treatment was for 30 minutes at 25°C, followed by centrifugation. The carrier hpßcd was specifically chosen to rescue membrane CHOL levels because it has no significant effect on CV fusion activity at the concentration used (Churchward et al., 2005). For all fusion assays, isolated CVs were suspended in BIM (pH 6.7) to OD 0.4. Aliquots of CV suspensions were stored at –80°C for molecular analyses. Lysenin was dissolved in ddH2O and added directly to CV suspensions. Lysenin treatments of CV suspensions (OD 0.4) were carried out for 30 minutes at 25°C prior to fusion assays.
CHOL and SM assays
Following CV treatments with mßcd, resulting supernatants were cleared of membrane fragments by ultracentrifugation at 125,000 g for 3 hours at 4°C. Total CHOL in supernatants was determined using the Amplex Red Cholesterol Assay kit according to manufacturer's instructions (Molecular Probes). The amount of SM in controls and SMase-treated (1, 5 and 10 U/ml) CV membrane samples was assayed using the Amplex Red SMase assay kit; at the highest dose of SMase used (20 U/ml), it was difficult to wash the exogenous enzyme fully from the CV membranes, obviating the assay. CV membranes were isolated as previously described (Hibbert et al., 2005), suspended in 2% Triton X-100, and total SM was assayed enzymatically at a fixed SMase concentration. All measurements were carried out using the Wallac Victor2V microplate reader (Perkin Elmer).
Lipid analysis
CV membrane lipids were extracted with methanol and chloroform according to Bligh and Dyer (Bligh and Dyer, 1959), with modifications (Churchward et al., 2005), and separated by automated HPTLC on silica gel 60 HPTLC plates (Merck). Plates were washed with CH3OH:ethyl acetate (6:4) and then activated at 110°C for 30 minutes prior to use. Dried, isolated CV lipids were dissolved in chloroform:methanol (9:1) and applied to the HPTLC plates using the CAMAG LINOMAT IV. Lipid species were then resolved in one 90 mm step with dichloromethane:methanol:acetic acid (100:2:5, v/v/v) (Herget et al., 2000) using the CAMAG AMD 2 multi-development TLC unit (Churchward et al., 2005). Subsequent quantitative charring of the HPTLC plates was then carried out as described (Herget et al., 2000). Briefly, HPTLC plates were dried at 180°C for 10 minutes, cooled to room temperature, and exposed to 10% CuSO4 in 8% H3PO4 aqueous solution for 15 seconds. Charring was performed at 180°C for 10 minutes. Ceramides were identified according to their position on the HPTLC plate, and quantified by comparing integrated fluorescent signals to that of standards resolved in parallel. Total separated lipids were imaged (Ex 540 nm/Em 620 nm) with the PROXPRESS multi-wavelength fluorescent imager (Perkin Elmer) as previously described (Churchward et al., 2005).
Statistical analysis
All data are presented as mean ± s.e.m. Two-sample two-tailed Student's t tests were used to test for differences between the experimental conditions; P<0.05 was used to define significance.
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Acknowledgments |
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