Cell Tissue Res (1999) 295:409-417

© Springer-Verlag 1999

REGULAR ARTICLE

H. Tollemer · C. A. Teitsma · J. Leprince · T. Bailhache F. Vandesande · O. Kah · M. C. Tonon · H. Vaudry

Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus Received: 21 April 1998 / Accepted: 5 October 1998

Abstract Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQNH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalaThis work was supported by grants from INSERM (U413 to H.V.), CNRS (UPRES-A 6026 to O.K.), the LARC-Neuroscience network (to O.K. and H.V.), an INSERM-MVG exchange program (to F.V. and H.V.) and the Conseil Régional de Haute-Normandie (to M.C.T. and H.V.). H.T. was a recipient of a fellowship from the EU Human Capital and Mobility Program. C.A.T. was a recipient of a fellowship from the European Union (FAIR GT95–2549). J.L. was a recipient of a fellowship from ORIL laboratories and the Conseil Régional de HauteNormandie. H. Tollemer · J. Leprince · M. C. Tonon · H. Vaudry (✉) European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, F-76821 Mont-Saint-Aignan, France Tel.: +33 235 14 6624; Fax: +33 235 14 6946; e-mail: [email protected] C. A. Teitsma · T. Bailhache · O. Kah Laboratory of Reproductive Molecular Endocrinology, CNRS UPRES-A 6026, INRA, Institut Rennais d’Ecologie et Biologie des Poissons, Campus de Beaulieu, F-35042 Rennes, France F. Vandesande Laboratory of Neuroendocrinology, Zoological Institute, University of Leuven, B-3000 Leuven, Belgium

mus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities. Key words Proopiomelanocortin · Post-translational processing · Novel neuropeptides · Immunohistochemistry · HPLC analysis · Oncorhynchus mykiss (Teleostei)

Introduction Proopiomelanocortin (POMC) is the precursor of several biologically active neuropeptides including α-melanocytestimulating hormone (α-MSH), adrenocorticotropin (ACTH) and β-endorphin (Eipper and Mains 1980). POMC is predominantly synthesized in corticotrope cells of the pars distalis and in melanotrope cells of the pars intermedia (Civelli et al. 1982). In addition, the POMC gene is expressed in discrete populations of neurons which, in mammals, are located in the hypothalamic arcuate nucleus (Jacobowitz and O’Donohue 1978; Bugnon et al. 1979; Pelletier and Leclerc 1979; Vaudry et al. 1980) and the commissural nucleus of the solitary tract of the brainstem (Bronstein et al. 1992). The occurrence of neurons containing POMC-derived peptides has also been demonstrated in the brain of fish (Follénius and Dubois 1977; Dubois et al. 1979; Vallarino 1985; Vallarino et al. 1989a–c, 1992), amphibians (Doerr-Schott et al. 1981; Yui 1983; Benyamina et

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Fig. 1 Schematic representation of the structure of trout pre-POMCA. Vertical bars indicate pairs of basic amino acids. The black zone represents the signal peptide sequence. Hatched zones represent regions which are well conserved across species. Open zones indicate

sequences with low conservation. The dotted zone represents the Cterminal extension of POMC-A. The primary structure of this 25-amino acid peptide is shown, and the three pairs of basic amino acids are indicated in bold letters and underlined

Fig. 2 Schematic frontal sections (rostrocaudal: A–E) through the nucleus lateralis tuberis, pars anterior, of the brain of Oncorhynchus mykiss depicting the distribution of immunoreactive EQWGREEGEEand YHFQ-NH2-containing perikarya (open circles) and fibers (dots) (Ir infundibular recess, NAH nucleus anterior hypothalami, NDL nu-

cleus dorsolateralis thalami, NDM nucleus dorsomedialis thalami, NLL nucleus lobi lateralis, NLTa nucleus lateralis tuberis, pars anterior, NLTl nucleus lateralis tuberis, pars lateralis, NPGl nucleus preglomerulosus, pars lateralis, NPv nucleus periventricularis, NRL nucleus recessus lateralis, P pituitary, Ppn pituitary pars nervosa)

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al. 1986; Delbende 1986; Vallarino 1987), reptiles (Dores et al. 1984; Vallarino 1984, 1986) and birds (Blähser and Heinrichs 1982). The sequence of the POMC cDNA has been determined in representative species of agnathans (Heinig et al. 1995; Takahashi et al. 1995), gnathostomes (Kitahara et al. 1988; Salbert et al. 1992; Okuta et al. 1996; Amemiya et al. 1997; Dores et al. 1997), amphibians (Martens et al. 1982; Pan and Chang 1989; Hilario et al. 1990) and mammals (Nakanashi et al. 1979; Drouin and Goodman 1980; Whitfeld et al. 1982; Boileau et al. 1983; Uhler and Herbert 1983; Patel et al. 1988; Keightley et al. 1991; Mol et al. 1991). Two POMC cDNAs have been characterized in some tetraploid species, i.e., the African clawed toad Xenopus laevis (Martens et al. 1982), the rainbow trout Oncorhynchus mykiss (Salbert et al. 1992) and the sockeye salmon Oncorhynchus nerka (Okuta et al. 1996). Although the structure of the biologically active domains of α-MSH/ACTH and β-endorphin have been highly conserved during evolution, several differences are noted between the organizations of fish and tetrapod POMC molecules. In particular, the γ-MSH sequence contained in the N-terminal domain of mammalian and amphibian POMCs (Nakanashi et al. 1979; Martens et al. 1982; Pan and Chang 1989; Hilario et al. 1990) is lacking in salmonid POMCs (Kitahara et al. 1988; Salbert et al. 1992; Okuta et al. 1996). Moreover, one of the trout POMC variants (POMC-A) exhibits a unique C-terminal extension which has not been found in any other POMC molecules characterized so far (Fig. 1). It has been recently shown that this 25-residue polypeptide, which possesses three potential dibasic cleavage sites, is actually processed in the trout pituitary to generate two novel decapeptides, EQWGREEGEE and ALGERKYHFQ-NH2 (Tollemer et al. 1997a,b). Although the POMC-A gene is actively expressed in the trout hypothalamus (Salbert et al. 1992), the possible existence of peptides derived from the C-terminal extension of POMC-A has not yet been examined in the brain. The aim of the present study was to determine the distribution of EQWGREEGEE- and ALGERKYHFQ-NH2-immunoreactive neurons in the central nervous system of the trout and to characterize these two novel POMC-A-derived decapeptides in brain extracts.

Materials and methods Animals Adult rainbow trout (Oncorhynchus mykiss, 118 specimens) of both sexes were obtained from a fish farm (Montville, France). For immunohistochemical studies, the animals were kept in the laboratory in a recirculating water system at 12–15°C under an artificial light regimen mimicking the natural photoperiod (46° N), and the animals were fed daily a trout diet (Aqualife, Nersac, France). For biochemical studies, the animals were decapitated immediately after arrival in the laboratory and the brains were rapidly dissected. Animal manipulations were performed according to the recommendations of the French Ethics Committee and under the supervision of authorized investigators.

Materials The following five peptides were synthesized by the solid-phase methodology as previously described (Tollemer et al. 1997b): EQWGREEGEE and its N-terminally tyrosylated analogue YEQWGREEGEE, ALGERKYHFQ-NH2, YHFQ-NH2 and YHFQG. All peptides were purified by reversed-phase high-performance liquid chromatography (HPLC) and their identity was verified by fast atom bombardment-mass spectrometry. Antisera against EQWGREEGEE, YHFQNH2, YHFQG and α-MSH used for immunohistochemistry and radioimmunoassay have been raised in rabbits (Vaudry et al. 1978; Tollemer et al. 1997a). Bovine serum albumin (BSA; fraction V) was obtained from Boehringer (Paris, France). Na125I was purchased from Amersham (Les Ulis, France). Acetonitrile was from Carlo Erba (Milan, Italy). Trifluoroacetic acid (TFA) and 4-chloro-1-naphthol were from Sigma Chemical (St. Louis, MO). Perhydrol was obtained from Merck (Darmstadt, Germany). Immunohistochemical procedure The fish were anesthetized by immersion in 0.04% (v/v) phenoxyethanol and perfused through the aortic bulb with 100 ml 0.65% NaCl. The perfusion was continued with 300 ml 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB; pH 7.4). The brain with the attached pituitary was dissected and postfixed for 3 h at 4°C in the same fixative solution. The tissues were incubated overnight at Fig. 3 Low magnification of adjacent frontal sections through the nucleus lateralis tuberis immunostained with antibodies against EQWGREEGEE (A) or YHFQ-NH2 (B) (P pituitary, asterisk third ventricle). Scale bars 100 µm

412 4°C in PB containing 15% sucrose. They were then placed in an embedding medium (OCT Tissue Tek, Nussloch, Germany) and frozen at –80°C. The tissues were cut at 12 µm in the coronal plane with a cryomicrotome (Microm, Francheville, France) and collected on gelatincoated glass slides. Tissue sections were preincubated for 45 min with 0.5% milk powder in 0.1 M veronal buffer containing 0.2% Triton X100 (Merck, Darmstadt, Germany) (VB; pH 7.6). Thereafter, the sections were incubated overnight in a moist chamber at room temperature with antibodies against EQWGREEGEE, YHFQG, YHFQ-NH 2 diluted 1:2000 or α-MSH diluted 1:1000 in PB. Sections were rinsed 3 times in VB and incubated for 90 min at room temperature with a peroxidase-conjugated goat anti-rabbit antibody (1:200; Biosys, Compiègne, France). After two rinses in VB and one rinse in 0.05 M TRIS buffer (pH 7.6), the sections were processed for peroxidase visualization using 4-chloro-1-naphthol dissolved in 0.05 M TRIS buffer (48 mg/100 ml) containing 0.02% (v/v) perhydrol (30% H2O2). After formation of a dark blue precipitate, the reaction was stopped by adding distilled water. The slides were mounted in PB/glycerol (1:1) and examined with an Olympus Provis microscope. To demonstrate the specificity of the immunostaining, alternate sections were treated with the antibodies against EQWGREEGEE preincubated with synthetic EQWGREEGEE or with the antibodies against YHFQ-NH2 preincubated with ALGERKYHFQ-NH2, YHFQ-NH2 or YHFQG (10–5 M each).

Preparation of tissue extracts Trout brains were dissected into five regions: telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The tissue samples were each immersed in 1 ml boiling 2 N acetic acid and maintained in a boiling water bath for 10 min to ensure inactivation of proteolytic enzymes. The tissues were homogenized at 4°C using a glass Potter homogenizer. The tissue homogenates were sonicated for 1 min and centrifuged at 13 000×g for 30 min at 4°C. The peptide material contained in the supernatants was concentrated on three SepPak C18 cartridges (Waters, St. Quentin en Yvelines, France) connected in series. Bound material was recovered by elution with acetonitrile-water-TFA (60:39.96:0.04; vol/vol) and kept at –20°C until chromatographic analysis. Radioimmunoassay procedure The peptide concentrations were measured in duplicate by using a double antibody RIA technique as previous described (Vaudry et al. Fig. 4 Pairs of adjacent sections through the nucleus lateralis tuberis incubated with antibodies against EQWGREEGEE (A) or YHFQNH2 (B) and with the same antibodies preincubated with 10–5 M EQWGREEGEE (C) or with 10–5 M ALGERKYHFQ-NH2 (D), respectively. The arrows indicate perikarya stained with the two antisera (asterisk third ventricle). Scale bars 50 µm

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Fig. 5 Pairs of adjacent sections through the nucleus lateralis tuberis immunostained with antibodies against EQWGREEGEE (A), YHFQNH2 (B) or α-MSH (C,D). The arrows indicate perikarya stained with antisera against EQWGREEGEE (A) and α-MSH (C), or YHFQ-NH2 (B) and α-MSH (D) (asterisk third ventricle). Scale bars 50 µm 1978). Briefly, synthetic YEQWGREEGEE, ALGERKYHFQ-NH2 and YHFQG (1 µg each) were iodinated by the chloramine-T method and separated from free iodine on Sep-Pak C18 cartridges with a step gradient of acetonitrile (6–44%) in 0.1% TFA. Radioiodinated YEQWGREEGEE, ALGERKYHFQ-NH2 and YHFQG were eluted at 14%, 16% and 12% acetonitrile, respectively, and kept at –20°C in glycerol (1:1, vol/vol). The RIAs were performed as previously described (Tollemer et al. 1997a) using synthetic EQWGREEGEE, ALGERKYHFQ-NH2 and YHFQG as reference standards and 6000 cpm tracer per tube. The RIA for ALGERKYHFQ-NH2 was performed using an antiserum against YHFQ-NH2, which shows substantial cross-reactivity with the decapeptide (Tollemer et al. 1997a). The final dilutions of the antisera against EQWGREEGEE (code number 729-1601), YHFQ-NH2 (code number 644-1506) and YHFQG (code number 643-1506) were 1:60 000, 1:16 000 and 1:1000, respectively. The detection limits of the EQWGREEGEE, ALGERKYHFQ-NH2, YHFQ-NH2 and YHFQG RIAs were 5.6, 100, 72 and 3.5 pg/tube, respectively. The intra-assay coefficients of variation were 2.7%, 3.2%, 1.9% and 6.8%, respectively, and the inter-assay coefficients of variation were 4.3%, 5.2%, 6.4% and 10.8%, respectively. Reversed-phase HPLC analysis Sep-Pak-prepurified tissue extracts from 50 trout brains were partially evaporated and injected onto a 0.4×25-cm C18 HPLC column (Merck, Darmstadt, Germany) equilibrated with a solution of acetonitrile-water-TFA (12:87.9:0.1, v/v/v). The concentration of acetonitrile

was held at 12% for 10 min, raised to 28% over 20 min, and finally raised to 80% over 20 min. The synthetic peptides YHFQ-NH2, EQWGREEGEE, YHFQG and ALGERKYHFQ-NH2, used as standards, were chromatographed in the same conditions. Fractions (1 ml) were collected, evaporated in a Speed-Vac Concentrator (Savant Instruments, Hicksville, NY) and radioimmunoassayed in duplicate.

Results Immunocytochemistry localization The overall distribution of EQWGREEGEE- and YHFQNH2-immunoreactive cell bodies and fibers is schematically presented in Fig. 2. A population of neurons located in the nucleus lateralis tuberis, pars anterior was consistently immunostained with the antibodies against EQWGREEGEE (Fig. 3A) and YHFQ-NH2 (Fig. 3B). The most rostral immunoreactive cell bodies were found just anterior to the opening of the third ventricle and extended caudally up to the anterior part of the pituitary stalk. On a single 12-µmthick section, the number of cells varied from 2 to 20. Comparison of adjacent sections alternatively incubated with each antiserum showed that at least part of these neurons expressed both peptides (Fig. 4A,B) although the antibodies against YHQF-NH2 produced a more intense staining than those against EQWGREEGEE. The antiserum against YHFQG only produced a faint staining of a few neurons in the nucleus lateralis tuberis (data not shown). In the anteri-

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or part of the nucleus, the cell bodies immunoreactive to EQWGREEGEE and YHFQ-NH2 had large dendrites and axons which projected dorsally through the ventral hypothalamus, the nucleus anterior tuberis, the thalamus and the torus semicircularis of the midbrain tegmentum. In the caudal part of the nucleus, the axons were orientated more laterally, surrounding the lateral recess before reaching the thalamic region. The nucleus ventromedialis thalami contained a high density of fibers immunostained with the antisera against EQWGREEGEE and YHQF-NH2. Immunopositive fibers were also visualized in the lateral preoptic area, the dorsal telencephalon, the optic tectum and the medulla oblongata. Preincubation of the antibodies against EQWGREEGEE and YHFQ-NH2, respectively, with EQWGREEGEE and or YHFQ-NH2 suppressed ALGERKYHFQ-NH2 immuno- staining (Fig. 4C,D). In contrast, the immunoreaction was not abolished when the antibodies against YHFQ-NH2 were preincubated with YHFQG (data not shown). Labeling of consecutive sections with antibodies against EQWGREEGEE (Fig. 5A) YHFQ-NH2 (Fig. 5B) or αMSH (Fig. 5C,D) showed that all three peptides were contained in the same population of neurons. HPLC analysis

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Measurement of EQWGREEGEE- and ALGERKYHFQNH2-LI in crude tissue extracts revealed that the two peptides were present in the diencephalon, telencephalon and mesencephalon while the cerebellum and medulla oblongata were virtually devoid of immunoreactivity. Characterization of these immunoreactive peptides was performed by combining reversed-phase HPLC analysis with RIA detection. The gradient of acetonitrile used (Fig. 6) made it possible to resolve the synthetic peptides EQWGREEGEE, YHFQ-NH2, YHFQG and ALGERKYHFQ-NH2 (retention times 18.24, 16.85, 21.63 and 26.51, respectively). In extracts of diencephalon, telencephalon and mesencephalon the EQWGREEGEE RIA detected a major peak that coeluted with synthetic EQWGREEGEE (Fig. 6A–C). Several minor immunoreactive peaks exhibiting longer retention times were also observed. The ALGERKYHFQ-NH2 RIA detected a predominant form which had the same retention time as ALGERKYHFQ-NH2 in diencephalon, telencephalon and mesencephalon extracts (Fig. 6D–F). A few minor Fig. 6 Reversed-phase HPLC analysis of EQWGREEGEE- (A–C) or YHFQ-NH2-like immunoreactivity (D–F) in trout brain extracts. Tissue extracts consisting of 50 diencephalons (A,D), 50 telencephalons (B,E) or 50 mesencephalons (C,F) were prepurified on Sep-Pak cartridges and chromatographed on a Lichrosorb C18 column. The concentrations of EQWGREEGEE- and ALGERKYHFQ-NH2-immunoreactive material in the HPLC fractions were measured by specific radioimmunoassays. The arrows indicate the retention time of synthetic YHFQ-NH2 (16.85), EQWGREEGEE (18.24 min), YHFQG (21.63 min) and ALGERKYHFQ-NH2 (26.51 min). The recoveries of EQWGREEGEE, ALGERKYHFQ-NH2 and YHFQ-NH2 were 74.6%, 93.4% and 39%, respectively. The dashed lines show the concentration of acetonitrile in the eluting solvent

immunoreactive species were also resolved but none of them coeluted with YHFQ-NH2. One of these minor peaks exhibited the same retention time as YHFQG (Fig. 6D,E), but no immunoreactive species could be detected with the YHFQG RIA (data not shown).

Discussion Trout POMC-A possesses a C-terminal extension of 25 residues which has never been found in any other POMCs including trout POMC-B (Salbert et al. 1992). This unusual extension comprises three pairs of basic amino acids suggesting that trout POMC-A may generate several novel peptides (Fig. 1). Indeed, we have recently isolated and sequenced, from a trout pituitary extract, two decapeptides derived from the C-terminal region of POMC-A, indicating that, in the hypophysis, this C-terminal tail is actually processed (Tollemer et al. 1997b). However, the occurrence of these novel POMC-A-derived peptides in the trout brain has not yet been investigated. The present study has shown the presence of neurons immunostained with antibodies raised against the decapeptide EQWGREEGEE and the tetrapeptide YHFQ-NH2 in the rostral part of the nucleus lateralis tuberis. In contrast, these neurons were not labeled with antibodies against the pentapeptide YHFQG, suggesting that the Cterminal Gln residue is α-amidated. Consistent with this notion, immunoabsorption of the YHFQ-NH2 antibodies with ALGERKYHFQ-NH2 or YHFQ-NH2 resulted in complete loss of the immunoreaction, whereas preincubation of the antibodies with YHFQG did not suppress immunostaining. Labeling of consecutive sections with the different antisera revealed that most, if not all, α-MSH-immunoreactive cells also contained EQWGREEGEE- and YHFQ-NH2-like immunoreactivity. Since POMC-A and POMC-B both have the potential to generate α-MSH, this observation suggests that, in the trout diencephalon, the two POMC genes are probably expressed by the same population of neurons. Combination of HPLC analysis with RIA detection has revealed that two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQNH2 were found in the trout diencephalon, telencephalon and mesencephalon, indicating that, in the brain, the two Lys-Arg dibasic sites flanking the decapeptide EQWGREEGEE at its N- and C-terminal ends are actually processed. In contrast, we did not detect any peptide coeluting with YHFQ-NH2, although the cross-reactivity of the antibodies against YHFQ-NH2 was 10 times higher with YHFQ-NH2 than with ALGERKYHFQ-NH2 (Tollemer et al. 1997a). These data demonstrate that the Arg-Lys pair of basic residues within the ALGERKYHFQ-NH2 sequence is not cleaved, as previously reported in the trout pituitary (Tollemer et al. 1997a). In agreement with this finding, it has been observed that prohormone convertases are far less efficient for processing at the Arg-Lys pair than at the LysArg pair (Steiner et al. 1992). In crude brain tissue extracts,

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we did not detect any YHFQG-like immunoreactivity, confirming that the C-terminal Gln residue of trout POMC-A is α-amidated. In all brain regions studied, the amount of ALGERKYHFQ-NH2 was 4.5–5.5 times higher than the amount of EQWGREEGEE. This observation suggests that the LysArg site upstream from the decapeptide EQWGREEGEE may not be fully processed. If that proves true, trout POMC-A may generate C-terminally extended forms of βendorphin. In support of this hypothesis, HPLC analysis resolved several peaks exhibiting a longer retention time than synthetic EQWGREEGEE, some of which might correspond to extended β-endorphin forms. Alternatively, a substantial amount of EQWGREEGEE may be processed at the single Arg5 residue by a Kex2-like prohormone convertase. Indeed, the sequence of trout POMC-A in this region fulfills the rules governing mono-arginyl cleavage, i.e., (1) presence of a basic residue at position –6 and (2) absence of a hydrophobic aliphatic residue at position +1 (Nakayama et al. 1992). In conclusion, the present data indicate that, in the trout brain, the C-terminal extension of POMC-A is actually processed to generate two decapeptides, EQWGREEGEE and ALGERKYHFQ-NH2. These novel peptides, which are contained in fibers widely distributed in the trout brain, may either exert biological activities by their own or modulate the effect of other POMC-derived neuropeptides. Acknowledgements The authors wish to thank D. Tranchand-Bunel for raising the antibodies.

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