Proc. Natl. Acad. Sci. USAVol. 92, pp. 7342-7346, August 1995Neurobiology

Neurosecretory vesicles can be hybrids of synaptic vesicles andsecretory granules

(small dense core vesicles/sympathetic neurons)

RUDOLF BAUERFEIND, RuTH JELINEK, ANDREA HELLWIG, AND WIELAND B. HUrTNER*Institute for Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany

Communicated by Philip Siekevitz, The Rockefeller University, New York, NY, March 27, 1995

ABSTRACT We have investigated the relationship of theso-called small dense core vesicle (SDCV), the major cate-cholamine-containing neurosecretory vesicle of sympatheticneurons, to synaptic vesicles containing classic neurotrans-mitters and secretory granules containing neuropeptides.SDCVs contain membrane proteins characteristic of synapticvesicles such as synaptophysin and synaptoporin. However,SDCVs also contain membrane proteins characteristic ofcertain secretory granules like the vesicular monoaminetransporter and the membrane-bound form of dopaminef3-hydroxylase. In neurites of sympathetic neurons, synapto-physin and dopamine 8-hydroxylase are found in distinctvesicles, consistent with their transport from the trans-Golginetwork to the site of SDCV formation in constitutive secre-tory vesicles and secretory granules, respectively. Hence,SDCVs constitute a distinct type ofneurosecretory vesicle thatis a hybrid of the synaptic vesicle and the secretory granulemembranes and that originates from the contribution of boththe constitutive and the regulated pathway of protein secre-tion.

Two distinct types of neurosecretory vesicles are thought tomediate the regulated release of neurotransmitters and neu-ropeptides (1). One type is the synaptic vesicles that store andrelease classic neurotransmitters such as glutamate, acetylcho-line, glycine, and y-aminobutyric acid but do not containsecretory proteins. We shall refer to these as classic synapticvesicles because of the data described below and the conceptderived therefrom. Classic synaptic vesicles are thought tooriginate from early endosomes after delivery of their mem-brane proteins via the constitutive pathway of protein secre-tion (for review, see ref. 2). The other type is the secretorygranules (in neurons also called large dense core vesicles) thatstore and release neuropeptides, originate from the trans-Golgi network, and constitute the regulated pathway of proteinsecretion (3). In certain neurons and endocrine cells, secretorygranules also store and release biogenic amines such ascatecholamines (4).An enigma has been the nature of the so-called small dense

core vesicles (SDCVs) that are found in certain nerve cells,such as catecholaminergic sympathetic neurons, and store andrelease biogenic amines (e.g., catecholamines) (for review, seeref. 4). SDCVs do not contain secretory proteins (5) and showan electron dense core only after certain chemical fixations(for example, permanganate or dichromate) that prevent theloss of catecholamines (6, 7).The relation of SDCVs to classic synaptic vesicles and

secretory granules has been controversial (1, 4, 8, 9). Oneconcept has been that SDCVs, because of their ability to storeand take up catecholamines like certain secretory granulessuch as chromaffin granules, are simply formed from themembrane of secretory granules after their exocytosis (4, 9).

This implies that only the regulated, but not the constitutive,pathway of protein secretion contributes to the biogenesis ofSDCVs. This concept has been extended to the biogenesis ofsynaptic vesicles in general (4, 9, 10) but has not provided asatisfactory explanation for the very low abundance, in secre-tory granules, of certain membrane proteins that are majorcomponents of classic synaptic vesicles. The other concept,which originated from the demonstration that the classicsynaptic vesicle membrane is distinct from that of the secretorygranule, has viewed the SDCVs, which lack secretory proteinsand morphologically resemble classic synaptic vesicles, assynaptic vesicles that simply contain vesicular amine transport-ers instead of uptake systems for classic neurotransmitters (1,8). Since only the constitutive, but not the regulated, pathwayof protein secretion is thought to contribute to the biogenesisof classic synaptic vesicles from early endosomes (2), thisconcept has not provided a satisfactory explanation for thepresence, in SDCVs, of certain secretory granule membraneproteins such as cytochrome b561 (11). Herein we show thatSDCVs are hybrids of the classic synaptic vesicle and thesecretory granule membranes.

MATERIALS AND METHODSElectron Microscopy. Rat vas deferens was fixed with (i) 1%

glutaraldehyde in 100 mM sodium cacodylate (pH 7.2) (caco-dylate buffer) overnight (Fig. 1A); (ii) 2% (wt/vol) paraform-aldehyde in 200 mM Hepes-NaOH (pH 7.4) for 5 hr, followedby a postfixation with 8% paraformaldehyde in 200 mMHepes-NaOH (pH 7.4) for 16 hr (Fig. 1B); (iii) dichromatefixative [3% (wt/vol) potassium dichromate/4.8% paraform-aldehyde/0.1 M sodium acetate, pH 5.8] for 1 hr (Fig. 1 C andD); or (iv) 3% (wt/vol) potassium permanganate in 150 mMsodium phosphate (pH 7.4) for 1 hr (Fig. 1E). For Eponembedding, fixed samples were washed in cacodylate bufferand postfixed in 1% OS04 plus 1.5% (wt/vol) magnesiumferricyanide for 1 hr. After washes in cacodylate buffer andwater, samples were incubated for 30 min in 1.5% (wt/vol)magnesium uranyl acetate in water. Samples were then dehy-drated in ethanol and embedded in Epon. Ultrathin sectionswere contrasted with lead citrate and uranyl acetate. Ultrathincryosections were prepared from samples fixed with paraform-aldehyde or dichromate and immunogold-labeled as described(12, 13), by using the mouse monoclonal antibody againstsynaptophysin (14) (Boehringer Mannheim) (1 jig/ml), asecondary rabbit anti-mouse IgG antibody (Cappel) (50 ,ug/ml), and protein A-colloidal gold (9 nm), prepared as de-scribed by Slot and Geuze (15), at an OD520 of 0.098.

Subcellular Fractionation. A post-10,000 x g high-speedmembrane pellet from vas deferens, prepared from 12-16 ratsas described (5, 16), was resuspended in 1 ml of in vitro buffer(250 mM sucrose/10 mM KCl/10 mM MgCl2/10 mMHepes-KOH, pH 7.2) and centrifuged for 5 min at 14,000 x g,V

Abbreviation: SDCV, small dense core vesicle.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Presence of synaptophysin on SDCVs byimmunoelectron microscopy. Rat vas deferens wasfixed with aldehyde (A and B), dichromate (C andD), or permanganate (E), followed by the prepara-tion of Epon sections (A, C, and E) or cryosectionswith (B) or without (D) immunogold labeling forsynaptophysin. The sections show axonal varicositiesinnervating smooth muscle. Note the presence ofdense cores in SDCVs (arrowheads) after fixationwith dichromate and permanganate, but not alde-hyde, whereas the dense cores in secretory granules(arrows) are observed irrespective of the type offixation. In cryosections prepared after aldehydefixation, gold particles indicative of synaptophysinimmunoreactivity are associated with SDCVs but notsecretory granules (B). (Inset) Immunogold-labeledvesicular profiles (50-60 nm in diameter) at highermagnifications. No synaptophysin immunoreactivitycould be detected in cryosections prepared afterdichromate fixation (data not shown). (Bars: A-E,200 nm.)

The pellet was resuspended in 1 ml of in vitro buffer andcentrifuged for 5 min at 14,000 X gay, and the supernatantsfrom the two 14,000 x g centrifugations were pooled. Thepooled supernatant (1.0-1.6 ml) was supplemented with (finalconcentrations) 5 mM creatine phosphate (from a 0.8 Mstock), creatine phosphokinase at 40 units/ml (from a 3200units/ml stock), and 5 mM ATP (from a 100 mM stock). Insome experiments, the supplemented supernatant was splitinto two aliquots and 3 ,uM reserpine (from a 200 ,uM stock inethanol) or the corresponding amount of ethanol only wasadded. The supplemented supernatant was then preincubatedfor 5 min at 32°C, followed by the addition of 1-[7,8-3H]noradrenaline at 7.2 ,uCi/ml (180 nM) (40 Ci/mmol; 1 Ci= 37 GBq; Amersham TRK.584) and a 30-min incubation. Atthe end of the incubation, as shown in Fig. 2, samples wereeither subjected to immunoadsorption or directly applied to alinear 0.6-1.6 M sucrose gradient (Beckman SW40 rotor) andcentrifuged at 25,000 rpm for 5 hr, with the brake off.Immunoadsorption was performed as described (17) by usingimmunobeads (Bio-Rad) precoated without or with the mousemonoclonal antibody against synaptophysin at 10 ,ug/ml (14).At the end of the immunoadsorption, the unbound materialwas loaded onto the linear sucrose gradient. After equilibriumsucrose gradient centrifugation, fractions (0.5 ml) were col-

lected from the bottom of the gradient. Aliquots of eachfraction were (i) analyzed for [3H]noradrenaline by liquidscintillation counting or (ii) subjected to SDS/PAGE andimmunoblot analysis using the following antibodies: the rabbitanti-hSgIIi_2o peptide antiserum against secretogranin II (18)(1:200 dilution); a rabbit antiserum (gift of T. Flatmark,Bergen, Norway) against a synthetic 21-mer peptide, corre-sponding to the cytoplasmically located portion [Gln2-Ala22(19)] of the uncleaved signal peptide (20) of rat dopamine3-hydroxylase, specific for the membrane-bound, but not the

soluble, form of dopamine f3-hydroxylase (1:500 dilution); themouse monoclonal antibody against synaptophysin (14) (0.2,g/ml); a rabbit antiserum against synaptoporin (21) (1:500dilution); and mouse monoclonal antibody 41.1 against syn-aptotagmin (22) (1 ,ug/ml). Rabbit primary antibodies weredetected by incubation with 125I-labeled protein A (0.12 ,uCilml; NEN). Monoclonal primary antibodies were detected byincubation with rabbit anti-mouse IgG (Cappel) at 1 ,ug/mland 1251-labeled protein A (0.12 ACi/ml; NEN). Immunore-activity was quantitated by using a Fuji BioImaging analyzer.

Immunofluorescence. Primary culture of sympathetic neu-rons from the superior cervical ganglion of newborn rats wasperformed as described (23). After 11 days in culture, cellswere fixed and processed for double immunofluorescence as

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FIG. 2. Comparison of SDCVs and secretory granules after theirseparation by equilibrium sucrose gradient centrifugation. A mem-

brane fraction containing secretory granules (solid arrow) and SDCVs(open arrow) was prepared from rat vas deferens and subjected tocatecholamine uptake followed by immunoadsorption of vesicles withcontrol immunobeads (solid circles) or immunobeads coated withsynaptophysin antibody (open circles). The nonimmunoadsorbedmembranes were then analyzed by equilibrium sucrose gradient cen-trifugation (fraction 1 = bottom of gradient), and fractions were

subjected to catacholamine determination (A) and immunoblot anal-ysis for secretogranin II (B), the membrane-bound form of dopamine,B-hydroxylase (C), synaptophysin (D), synaptoporin (E), and synap-totagmin (F). A also shows catecholamine uptake in the presence ofreserpine (crosses) into vesicles subjected directly to equilibriumsucrose gradient centrifugation; in this experiment, catecholaminerecovery in the absence of reserpine (data not shown) was 20-25%higher than in the nonimmunodepleted sample (solid circles) shown inA. a.u., Arbitrary units.

described (24), by using the mouse monoclonal antibodyagainst synaptophysin (14) (0.5 ,.g/ml) followed by a fluores-cein isothiocyanate-conjugated secondary antibody and therabbit antisera against secretogranin II (18) (1:50 dilution) orthe membrane-bound form of dopamine j3-hydroxylase (1:100dilution) followed by tetramethylrhodamine P isothiocyanate-conjugated secondary antibodies. The confocal laser scanningmicroscope used was a Leica TCS4D. Scanning of opticalsections in the x and y axes was performed with a pinholesetting of 80, and a single pixel corresponded to 110 nm.

RESULTSSDCVs Contain Synaptophysin, an Integral Membrane

Protein Characteristic of Classic Synaptic Vesicles. We firstexamined whether SDCVs contain the integral membraneprotein synaptophysin, a major component of classic synapticvesicles but not secretory granules (8, 25). SDCVs wereidentified in sympathetic neurons of the rat vas deferens byusing permanganate (Fig. 1E) or dichromate (Fig. 1 C and D)fixation (6, 7). After such fixation, both the numerous SDCVs(arrowheads) and the few secretory granules (arrows) ob-served in axonal varicosities are characterized by electron-dense cores that result from the fixation of catecholamines andcondensed secretory proteins. In contrast, after aldehydefixation (Fig. 1 A and B), secretory granules (arrows) but notSDCVs (arrowheads) appear with electron-dense cores be-cause of the differential effect of this fixation on the condensedsecretory proteins and the catecholamines. Immunogold la-beling with an anti-synaptophysin antibody of cryosectionsprepared after aldehyde fixation (Fig. 1B) revealed immuno-reactivity over membrane profiles corresponding to SDCVs(arrowheads, compare with Fig. 1D) but not over thosecorresponding to secretory granules (arrows). Hence, SDCVscontain the classic synaptic vesicle marker protein synapto-physin.

Coexistence ofClassic Synaptic Vesicle and Secretory Gran-ule Membrane Proteins in SDCVs. Rat vas deferens SDCVsand secretory granules were separated from each other byequilibrium sucrose gradient centrifugation and the gradientfractions were analyzed for the presence of various markerproteins (Fig. 2, solid circles). Two populations of vesiclescapable of [3H]noradrenaline uptake in vitro were observed(Fig. 2A). The sensitivity of this uptake to reserpine (Fig. 24,crosses), a specific inhibitor of vesicular but not plasmamembrane catecholamine uptake (26), indicates that both ofthese catecholamine-containing vesicles possess the vesicularamine transporter. The vesicles in the denser fractions of thegradient were identified as secretory granules because of thepresence of secretogranin II (Fig. 2B), a secretory proteinpresent in the matrix of secretory granules in a wide variety ofendocrine cells and neurons (27). The catecholamine-containing vesicles in the lighter fractions of the gradient wereidentified as SDCVs because of the presence of synaptophysin(Fig. 2D), which is specifically associated with SDCVs as shownin Fig. 1B.Both secretory granules and SDCVs contained synaptotag-

min (Fig. 2F), a protein implicated in calcium-dependentexocytosis (28). Synaptoporin, which like synaptophysin is anintegral membrane protein characteristic of classic synapticvesicles but not secretory granules (21), was associated with theSDCVs of the rat vas deferens but not the secretory granules(Fig. 2E). In contrast, the membrane-bound form of dopaminef-hydroxylase, a protein characteristic of certain secretorygranules but not classic synaptic vesicles (29), was found notonly in secretory granules of the rat vas deferens but also inSDCVs (Fig. 2C).To determine whether the various proteins detected in the

SDCV-containing fractions of the gradient were indeed local-ized in the same membrane vesicle as the classic synaptic

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vesicle marker synaptophysin, we subjected a membrane frac-tion from vas deferens to immunoadsorption by using immu-nobeads coated with an anti-synaptophysin antibody, followedby equilibrium sucrose gradient centrifugation of the nonad-sorbed membrane vesicles (Fig. 2, open circles). This immu-noadsorption resulted in the removal of only a small portion ofthe proteins studied from the sucrose gradient fractions con-taining secretory granules. [This is consistent with previousobservations that the abundance of synaptophysin on secretorygranules is very low (8, 25) but may be sufficient for theirpartial immunoadsorption (10).] In contrast, the immunoad-sorption led to the almost complete depletion of each of theproteins analyzed from the sucrose gradient fractions contain-ing SDCVs, indicating that each of these proteins resided insynaptophysin-containing vesicles. We conclude that SDCVscontain membrane proteins of both classic synaptic vesicles(synaptophysin and synaptoporin) and secretory granules (ve-sicular monoamine transporter and membrane-bound form ofdopamine P-hydroxylase) and, thus, are hybrids of these twotypes of neurosecretory vesicles (for synaptotagmin, see Dis-cussion).

Delivery of Classic Synaptic Vesicle and Secretory GranuleMembrane Proteins to SDCVs via Distinct Vesicles. How arethe membrane proteins characteristic of either classic synapticvesicles or secretory granules delivered to SDCVs? To obtaina first clue toward answering this question, we studied thelocalization of synaptophysin and dopamine f3-hydroxylase inthe neurites of primary sympathetic neurons in culture, byusing double immunofluorescence and confocal laser scanningmicroscopy (Fig. 3). Consistent with the results of subcellularfractionation and immunoadsorption, synaptophysin and do-pamine 3-hydroxylase were colocalized in varicosities andterminals (data not shown), where SDCVs are known toaccumulate. However, in the thin segments of the neurites,analysis of the fine immunoreactive puncta, which are likely tocorrespond to vesicles en route to varicosities and terminals,revealed that synaptophysin and dopamine ,3-hydroxylase werelocalized in distinct vesicles (Fig. 3A). As SDCVs of cat-echolaminergic neurons characteristically contain both synap-tophysin and dopamine /3-hydroxylase (see Fig. 2), neither ofthese vesicles was SDCVs. The vesicles immunoreactive fordopamine ,B-hydroxylase contained secretogranin II (data notshown) and, thus, were likely to be secretory granules. Incontrast, the vesicles immunoreactive for synaptophysin lackedsecretogranin II (Fig. 3B) and, thus, were not secretorygranules. Given the previous observation that in the neuroen-docrine cell line PC12, newly synthesized synaptophysin isdelivered from the trans-Golgi network to the cell periphery inconstitutive secretory vesicles (2), at least some of thesesynaptophysin-positive but secretogranin II- and dopaminef3-hydroxylase-negative vesicles may well be constitutive se--El.

FIG. 3. Localization of synaptophysin, the membrane-bound formof dopamine f3-hydroxylase, and secretogranin II in the neurites of ratsympathetic neurons in primary culture studied by double immuno-fluorescence and confocal laser scanning microscopy. (A) Synapto-physin (green) and dopamine 3-hydroxylase (red). (B) Synaptophysin(green) and secretogranin II (red). The two micrographs are singleoptical sections. Note that in the thin segments of the neurites, the finepuncta of immunoreactivity for synaptophysin (A and B) (arrows) aredistinct from those for the membrane-bound form of dopamine,3-hydroxylase (A) (arrowheads) and secretogranin II (B) (arrow-heads). (Bar in B = 5 ,um.)

cretory vesicles (although other types of vesicles cannot beexcluded). Our results are consistent with the hypothesis thatboth the constitutive (synaptophysin) and the regulated (do-pamine 83-hydroxylase) pathway of protein secretion contrib-ute to the biogenesis of SDCVs (for details, see Fig. 4).

DISCUSSIONOur study resolves a long-standing controversy about thebiogenesis of synaptic vesicles and calls for a modification ofthe classification of neurosecretory vesicles. Because SDCVshave been regarded as model synaptic vesicles by some inves-tigators (4, 9), the fact that they contain secretory granulemembrane components (5, 11) has led to the general hypoth-esis that synaptic vesicles are formed from the membrane ofsecretory granules after their exocytosis. On the other hand,the characterization of highly purified brain synaptic vesicles,which for the vast majority are classic synaptic vesicles as theydo not contain biogenic amines but classic neurotransmitters,has established that their membrane protein composition isclearly distinct from that of secretory granules (1, 25). This has

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FIG. 4. Proposed biogenesis of the SDCV, a hybrid between theclassic synaptic vesicle and the secretory granule membranes. Char-acteristics of classic synaptic vesicles and secretory granules areindicated by the use of open and solid type, respectively. In theperikaryon, the membrane proteins characteristic of either classicsynaptic vesicles (e.g., syn4ptophysin) or secretory granules [e.g.,dopamine f3-hydroxylase (DBH) or vesicular monoamine transporter(VMAT)] are segregated, in the trans-Golgi network, into distinctsecretory vesicles-i.e., the constitutive secretory vesicle and thesecretory granule, respectively. After their exocytosis at the cellperiphery, both the membrane proteins characteristic of classic syn-aptic vesicles and secretory granules are intemalized into early endo-somes. In neurons that form secretory granules and classic synapticvesicles (e.g., glutamatergic neurons), the membrane proteins char-acteristic of secretory granules and classic synaptic vesicles are segre-gated, in early endosomes, into vesicles recycling back to the trans-Golgi network and into classic synaptic vesicles, respectively (notillustrated). In contrast, in neurons that form secretory granules andSDCVs (e.g., catecholaminergic sympathetic neurons), the membraneproteins characteristic of secretory granules and classic synapticvesicles are assembled, in early endosomes, to generate SDCVs.

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led to the realization that the biogenesis of classic synapticvesicles should be independent from that of secretory granules(1, 25), a concept supported by experimental evidence (2) andalso consistent with the fact that in many neurons the abun-dance of secretory granules simply seems too low for thisorganelle to provide the membrane for classic synaptic vesicles.Our observations that the SDCV is neither derived only fromthe secretory granule membrane nor a classic synaptic vesiclelacking secretory granule membrane components but, rather,is a vesicle composed of membrane constituents of both classicsynaptic vesicles (synaptophysin and synaptoporin) and secre-tory granules (vesicular amine transporter and membrane-bound form of dopamine f-hydroxylase) demonstrate thateach of the two previous concepts as to the nature of SDCVswas partially correct, but incomplete. The present data andprevious observations (5, 11, 29, 30) require that the conceptof the existence of two types of neurosecretory vesicles, eachoriginating independently from the other via either the con-stitutive (classic synaptic vesicle) or the regulated (secretorygranule) pathway of protein secretion, needs to be modified(see Fig. 4) to include the SDCV as a hybrid type of neuro-secretory vesicle.

Synaptophysin and the membrane-bound form of dopaminef3-hydroxylase, though colocalized in SDCVs, were found indistinct vesicles in the neurites of sympathetic neurons. Weinterpret this as an indication that both constitutive secretoryvesicles (containing synaptophysin but lacking dopamine (-hy-droxylase and secretogranin II) and secretory granules (con-taining dopamine f-hydroxylase and secretogranin II butlacking synaptophysin) deliver membrane proteins to the siteof SDCV biogenesis. Our concept, though speculative atpresent, is illustrated in Fig. 4. Based on our observations, wepropose that the membrane proteins of SDCVs (i) are segre-gated from each other into constitutive secretory vesicles andsecretory granules upon exit from the trans-Golgi networkand, after exocytosis of these vesicles, (ii) meet again in earlyendosomes [into which both classic synaptic vesicle (31) andsecretory granule (32) membrane proteins are internalizedafter exocytosis] to assemble into the SDCV membrane. Thisconcept not only is consistent with our data but also providesa satisfactory explanation for the differential distribution ofsynaptophysin and various secretory granule membrane pro-teins in sympathetic axons observed by other investigators (11,30, 33).

It remains to be determined whether synaptotagmin, whichis found in secretory granules, classic synaptic vesicles, andSDCVs, exits from the trans-Golgi network only in secretorygranules or also in constitutive secretory vesicles (Fig. 4). It isworth noting that the relative proportion of synaptotagminimmunoreactivity in SDCVs vs. secretory granules (Fig. 2F)was the same as that of dopamine ,B-hydroxylase immunore-activity (Fig. 2C). This would be consistent with the possibilitythat in SDCV-containing neurons, synaptotagmin is deliveredto the site of SDCV formation predominantly via secretorygranules. However, in many neurons the abundance of secre-tory granules in comparison to classic synaptic vesicles may beso low as to necessitate delivery of synaptotagmin to the siteof classic synaptic vesicle formation via the same traffic routeas synaptophysin (2).

We thank Drs. H. Betz, T. Flatmark, H.-H. Gerdes, and R. Jahn forantibodies; Dr. Hermann Rohrer for advice on the culturing ofsympathetic neurons; Dr. Matthew Hannah for help with confocal

microscopy and comments on the manuscript; and Alan Summerfieldfor excellent photography and artwork. W.B.H. was supported by agrant from the Deutsche Forschungsgemeinschaft (SFB 317).

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