Differential Expression of Membrane-Associated Proteoglycans in the Skeletal Muscle of Turkeys with Different Growth Rates1

C. Liu,* D. C. McFarland,† K. E. Nestor,* and S. G. Velleman*2

*Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster 44691; and †Department of Animal and Range Sciences, South Dakota State University, Brookings 57007

ABSTRACT Heparan sulfate proteoglycans (HSPG) are 14 to ED 22 and declined sharply from ED 22 to ED 24 key components of the cell membrane and extracellular to a very low level. In contrast, the SYN 3 and GPC 1 matrix of skeletal muscle cells. Two major groups of mem- expression showed a continuous decline from ED 14 to brane-associated HSPG found in skeletal muscle are syn- ED 24. The F line had higher SYN 2 (ED 14, 18, 20, 22), decans (SYN) and glypicans (GPC), both of which can SYN 3 (ED 22), and SYN 4 (ED 22) expression than the regulate growth factor activities and, thus, modulate cell RBC2 line. In myogenic satellite cells, initiating differenti- proliferation and differentiation. In the current study, the ation resulted in a decrease in SYN 2 expression and an mRNA expression of a group of membrane-associated increase in GPC 1 expression. Both SYN 3 and SYN 4 HSPG (SYN 2 through 4 and GPC 1) was investigated in expression stayed almost constant through both the pro- embryonic pectoralis major muscle [embryonic days (ED) liferation and differentiation stages. The proliferating sat- 14, 16, 18, 20, 22, 24] and myogenic satellite cells isolated ellite cells from the F line displayed higher SYN 4 expres- from males of a turkey genetic line selected for increased sion than those from the RBC2 line. Collectively, the re- 16-wk BW (F line) and an unselected randombred control sults from the current study suggest that membrane- (RBC2 line) from which the F line was developed. The associated HSPG are differentially expressed in both em- mRNA expression was measured by a real-time quantita- bryonic p. major muscle tissue and satellite cells isolated tive PCR approach. The SYN 2 and SYN 4 expression from F-line and RBC2-line male turkeys, implying their exhibited a similar pattern during embryonic p. major distinct roles in myogenesis and differing influence on muscle development, which remained constant from ED muscle growth properties. Key words: heparan sulfate, syndecan, glypican, muscle, satellite cell, turkey 2006 Poultry Science 85:422–428

INTRODUCTION fiber number is determined, around the perinatal period (Smith, 1963). However, postnatal muscle growth contin- Skeletal muscle formation is a complex process that ues by fusion of another group of myogenic stem cells, involves the proliferation, alignment, and fusion of myo- satellite cells, with the existing fibers. This process of genic precursor cells to form multinucleated myotubes postnatal muscle growth is called hypertrophy. Satellite that can differentiate into muscle fibers. Embryonic myo- cells are undifferentiated mononuclear myogenic cells genic precursor cells are actively dividing and differenti- that reside between the basal lamina and the plas- ating into mature muscle fibers, which leads to muscle malemma of muscle fibers (Mauro, 1961). In adult tissue, growth through hyperplasia. In almost all animals and satellite cells, normally quiescent, can be activated in re- birds, muscle fiber formation is complete, and the muscle sponse to stress signals (e.g., injury) to proliferate and fuse to form new fibers or repair damaged ones (Schultz et al., 1985). 2006 Poultry Science Association, Inc. The proliferation and differentiation of muscle cells Received June 10, 2005. Accepted October 25, 2005. are, in part, regulated by the (ECM) 1Salary and research support to S. G. Velleman was provided by components. The ECM that surrounds muscle cells con- state and federal funds appropriated to Ohio Agricultural Research and sists of fibrous (e.g., collagens) embedded in a Development Center, The Ohio State University. Research support to S. G. Velleman was from the Cooperative State Research, Education, mixture of nonfibrous macromolecules, proteoglycans. and Extension Service, U.S. Department of Agriculture under Proteoglycans are a group of macromolecules that consist Agreement No. 2003-35206-13696, and research support to S. G. Velle- of a core attached covalently by one or more man and D. C. McFarland was provided by the Midwest Poultry Con- sortium. carbohydrate chains called glycosaminoglycans (GAG). 2Corresponding author: [email protected] Glycosaminoglycans can be classified into heparan sulfate

422 HEPARAN SULFATE PROTEOGLYCAN EXPRESSION 423 (HS), chondroitin sulfate, dermatan sulfate, and keratan MATERIALS AND METHODS sulfate based on their structure and sequence. Heparan sulfate proteoglycans (HSPG) are present on Animals the cell surface or in the ECM and can interact with many compounds, including ECM constituents, adhesion mole- The F- and RBC2-line turkeys used in the present study cules, and growth factors (Brandan and Larraı´n, 1998). were maintained at the Ohio Agricultural Research and They exist in both membrane-associated forms [e.g., syn- Development Center. The F line was selected for in- decans (SYN) and glypicans (GPC)] and secreted forms creased 16-wk posthatch BW, and the RBC2 line was an (e.g., perlecan and agrin). Membrane-associated HSPG unselected randombred control line from which the F line are attached to the cell membrane by either a transmem- was developed (Nestor, 1977a,b). To avoid sex effects, brane core protein or a glycosylphosphatidylinositol an- only male turkeys were used in the current study. chor. Two major groups of membrane-associated HSPG, SYN and GPC, are found in skeletal muscle. Syndecans Sample Collection and Sex Identification are a family of at least 4 transmembrane HSPG whose structure consists of a signal peptide, an extracellular Pectoralis major muscle tissue was isolated from 5 male domain with several consensus sequences for GAG at- individuals of each line at embryonic days (ED) 14, 16, tachment, a hydrophobic transmembrane domain, and a 18, 20, 22, and 24. After isolation, the muscle was quick- short cytoplasmic tail. Syndecans 1 through 4 are all found frozen in liquid nitrogen and stored at −70°C until use. in skeletal muscle (Larraı´n et al., 1997; Brandan and Lar- Sex identification of the turkey embryos was performed raı´n, 1998; Fuentealba et al., 1999). Six members of the using a PCR method adapted from D’Costa and Petitte GPC family have been identified in mammals, and only (1998). Only embryonic p. major muscle tissue from males GPC 1 has been reported in skeletal muscle (Campos et was used in the present study. al., 1993). The GPC 1 core protein is characterized by a cysteine-rich globular domain, a more extended GAG Cell Culture domain, and a glycosylphosphatidylinositol anchor that tethers the molecule to the cell membrane. Satellite cells were isolated from the pectoralis major The presence of HS is required for stable binding of muscle tissue of F- and RBC2-line male turkeys (Velleman the fibroblast growth factor (FGF) ligand to the receptor et al., 2000) and were plated on 35-mm, gelatin-coated 6- tyrosine kinase (Rapraeger et al., 1991). The participation well plates (Corning Inc., Corning, NY) at a density of of HS in the ternary FGF-receptor signaling complex is 35,000 cells per well. The cells were cultured as described facilitated through HS-binding motifs on both the FGF by Liu et al. (2002). Two sample plates were taken out ligand and the receptor tyrosine kinase (Schlessinger et from the incubator at each time point (72 h of proliferation al., 2000). Syndecans 1 and 3 have been reported to modu- and 0, 24, 48, 72, and 96 h of differentiation). The medium late FGF2 activity during mouse C2C12 muscle cell differ- was removed from the plates, and the cells were rinsed entiation (Larraı´n et al., 1998; Fuentealba et al., 1999). with sterile PBS (170 mM NaCl, 3 mM KCl, 10 mM Fibroblast growth factor 2 is a known stimulator of muscle Na2HPO4,and2mM KH2PO4; pH 7.08). All sample plates cell proliferation and an inhibitor of differentiation (Dol- were kept at −70°C until use. lenmeier et al., 1981). Thus, the expression of HSPG may affect muscle growth characteristics through regulating Total RNA Extraction and cDNA Synthesis FGF signaling. To explore the relationship between HSPG expression Total RNA was extracted from the pectoralis major and muscle growth characteristics, a genetic line of turkey muscle tissue using the method developed by Chomczyn- (F line) selected for increased 16-wk BW and a ran- ski and Sacchi (1987) and from the cultured satellite cells dombred control line (RBC2 line) from which the F line using TRIzol reagent (Invitrogen, Carlsbad, CA) ac- was developed (Nestor, 1984) were used in the current cording to the manufacturer’s instructions. The cDNA study. The F line has a significantly higher p. muscle were synthesized using Moloney murine leukemia virus weight than the RBC2 line (Lilburn and Nestor, 1991). In (Promega, Madison, WI) reverse transcription based on addition, it has been shown that total HSPG expression in the protocol as described by the manufacturer. The re- pectoralis major muscle was higher in the F line compared verse transcription reaction was performed in a 25-␮L ␮ with the RBC2 line (Liu et al., 2002). However, little infor- volume. An RNA-primer mix [1 L of Oligo(dT)20 (50 mation is known with regard to how the expression level ␮M), 2 ␮g of total RNA, and nuclease-free water up to of individual membrane-associated HSPG may be related 13.5 ␮L] was heated at 65°C for 5 min. This mixture was to skeletal muscle growth characteristics. The current then incubated immediately on ice for 2 min, and 11.5 study was designed to examine the expression patterns ␮L of reaction mix {5 ␮Lof5× First-Strand Buffer [250 of a group of membrane-associated HSPG (SYN 2 through mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, and 4 and GPC1) in both turkey embryonic p. major muscle 50 mM dithiothreitol]; Promega},5␮Lof10mM deoxy- and satellite cells and their relationship with muscle nucleoside triphosphate mix (dATP, dCTP, dGTP, dTTP; growth characteristics using F- and RBC2-line turkeys as Promega), 0.5 ␮L of RNasin (40 U/␮L; Promega), and 1 a model. ␮L of Moloney murine leukemia virus (200 U/␮L)] was 424 LIU ET AL.

Table 1. Primer sequences for real-time PCR

Product Primer1 Sequence2 size SYN 2 5′-CTTGTCGCTGTGGCAGATCGG-3′ (forward) 177 bp 5′-CTGCTGGAGCCTTTTGTGCTGA-3′ (backward) SYN 3 5′-CAGTGACCTGGAAGTCCCAAC-3′ (forward) 181 bp 5′-GCCCGACTCTATCGTGTTGTC-3′ (backward) SYN 4 5′-CTACCCTGGCTCTGGAGACCT-3′ (forward) 234 bp 5′-TCATTGTCCAGCATGGTGTTT-3′ (backward) GPC 1 5′-CCTTTCCCACTCCTCTCCAAA-3′ (forward) 176 bp 5′-GACCTTGGTCGAAGAGCAACT-3′ (backward) GAPDH 5′-GAGGGTAGTGAAGGCTGCTG-3′ (forward) 200 bp 5′-CCACAACACGGTTGCTGTAT-3′ (backward)

1SYN = syndecan; GPC = glypican; GAPDH = glyceraldehydes-3- phosphate dehydrogenase. 2Primer sequences were designed from the following GenBank acces- sion numbers: turkey SYN 2, AY852249; turkey SYN 3, AY852250; turkey SYN 4, AY852251; turkey GPC 1, AY551002; and turkey GAPDH, U94327.

Figure 1. Gel electrophoresis analysis of real-time PCR products. added. The complete reaction mixture was incubated at Real-time PCR products for syndecan (SYN) 2 (lane 2), SYN 3 (lane 3), 55°C for 60 min and then heated at 70°C for 15 min to SYN 4 (lane 4), glypican 1 (lane 5), and glyceraldehyde-3-phosphate dehydrogenase (lane 6) were separated on a 1.5% agarose gel to evaluate inactivate the reverse transcription reaction. The synthe- the amplification specificity of the real-time PCR. Single bands with sized cDNA was diluted with 25 ␮L of nuclease-free water expected product size for each target gene were observed. Lane 1 con- before performing the real-time quantitative PCR. tains the DNA size marker.

Real-Time Quantitative PCR Statistical Analysis The real-time quantitative PCR was performed using DyNAmo Hot Start SYBR Green qPCR kit (MJ Research, The student’s t-test was performed at each sampling Reno, NV). The PCR reaction consisted of 2 ␮L of diluted time to evaluate the differences between means. Differ- reverse transcription reaction, 10 ␮Lof2× master mix, ences were considered significant at P < 0.05. 0.4 ␮L of ROX dye, 0.25 ␮M of each forward and reverse primers, and nuclease-free water up to 20 ␮L. Reaction RESULTS components were assembled in low-profile multi- pleplates (MJ Research) and sealed with Microseal B Ad- Specificity of Real-Time PCR hesive Seals (MJ Research). Primers (Qiagen, Valencia, CA) used for amplification of SYN 2 through 4, GPC 1, In the current study, the real-time quantitative PCR was and glyceraldehyde-3-phosphate dehydrogenase used to measure relative mRNA expression of a group of (GAPDH) were designed from published turkey se- membrane-associated HSPG (SYN 2 through 4 and GPC quences as listed in Table 1. Following reaction assembly, 1) in both embryonic turkey p. major muscle and satellite plates were put into the DNA Engine Opticon 2 real-time cells from the growth-selected F line and its randombred system. The cycling program consisted of denaturation control line, RBC2. Specificity of real-time PCR was evalu- (95°C for 15 min) followed by amplification and quantita- ated by both melting curve analysis and gel electrophore- tion [35 cycles at 94°C for 30 s, at 60°C (for SYN 2 through sis of each sample. The melting curve graphs showed that 4 and GPC 1), or at 55°C (for GAPDH) for 30 s, and 72°C there were only single peaks for SYN 2 through 4, GPC for 30 s with a single fluorescence measurement at the 1, and GAPDH amplification products (data not shown). end of 72°C of each cycle] and a final extension at 72°C Gel electrophoresis revealed single bands of expected for 5 min. The melting curve program was 52 to 95°C, sizes for SYN 2 (177 bp), SYN 3 (181 bp), SYN 4 (234 bp), 0.2°C per read, and a 1-s hold. The final PCR products GPC 1 (176 bp), and GAPDH (200 bp) products amplified were analyzed on 1.5% agarose gel to check amplification from a representative sample (Figure 1). The bands were specificity. Standard curves were constructed for SYN 2 also checked by DNA sequence analysis for specificity. through 4, GPC 1, and GAPDH with serial dilutions of purified PCR products amplified from each gene. The Membrane-Associated HSPG PCR products were purified by agarose gel electrophore- Expression in Turkey P. Major Muscle sis using QIAquick Gel Extraction Kit (Qiagen). All of the sample concentrations fell within the values of standard The SYN 2 mRNA expression remained nearly constant curves. The amount of sample cDNA for each gene was from ED 14 to ED 20 followed by a sharp decline (>5 interpolated from the corresponding standard curve. The fold) from ED 20 to ED 24 in both turkey lines during expression of SYN 2 through 4 and GPC 1 were normal- embryonic p. major muscle development (Figure 2A). The ized to GAPDH expression and calculated as an arbi- F line showed significantly higher SYN 2 expression than trary unit. the RBC2 line at ED 14, 18, 20, and 22. The SYN 3 expres- HEPARAN SULFATE PROTEOGLYCAN EXPRESSION 425

Figure 2. The expression of membrane-associated heparan sulfate proteoglycans in F- and RBC2-line pectoralis major muscle at different embryonic days (ED). The mRNA levels of syndecan (SYN) 2 (A), SYN 3 (B), SYN 4 (C), and glypican 1 (D) were measured by real-time PCR. Error bars represent the standard errors of the means. *P < 0.05. sion gradually decreased from ED 14 to ED 24 with a 10- was no significant difference between the 2 lines in SYN fold decrease at ED 24 compared with ED 14 (Figure 2B). 2 expression observed in either the proliferation or differ- The SYN 4 expression showed a similar pattern to that entiation stages. The SYN 3 expression was constant of SYN 2, with a steady expression level from ED 14 to through both proliferation and differentiation (Figure 3B). ED 22 followed by a sharp decline (>8 fold) from ED 22 Similar to SYN 2, SYN 3 showed no significant differences to ED 24 (Figure 2C). There were no significant differences between the 2 lines in expression. The SYN 4 expression in SYN 3 and SYN 4 expression observed between lines, also remained unchanged over both proliferation and dif- except that the F line had slightly higher SYN 3 and SYN ferentiation stages (Figure 3C). However, during prolifer- 4 expression than did the RBC2 line at ED 22. The GPC ation, the F line showed significantly higher SYN 4 expres- 1 expression gradually decreased from ED 14 to ED 24 sion than the RBC2 line. The GPC 1 expression was upreg- (>10 fold), and no difference in expression was observed ulated (>5 fold) upon the initiation of differentiation, and between the 2 lines (Figure 2D). the increased expression remained throughout the differ- entiation stage in both lines (Figure 3D). Membrane-Associated HSPG Expression in Turkey Satellite Cells DISCUSSION

The mRNA expression of membrane-associated HSPG The results from the current study indicated that each in satellite cells from both the F line and RBC2 line is individual membrane-associated HSPG had distinct ex- shown in Figure 3. The SYN 2 expression was downregu- pression patterns in embryonic p. major muscle tissue lated after differentiation was initiated (Figure 3A). There and satellite cells, suggesting that they may play differen- 426 LIU ET AL.

Figure 3. The expression of membrane-associated heparan sulfate proteoglycans in F- and RBC2-line satellite cells during proliferation (P) and differentiation (F) stages. The mRNA levels of syndecan (SYN) 2 (A), SYN 3 (B), SYN 4 (C), and glypican 1 (D) were measured by real-time PCR. Error bars represent the standard errors of the means. *P < 0.05. tial roles in different stages of muscle growth. The expres- tor (Rapraeger et al., 1991; Yayon et al., 1991). Fibroblast sion of SYN 2 through 4 showed differential expression growth factor 2 is a strong stimulator of muscle cell prolif- in either embryonic p. major muscle tissue or satellite eration and an inhibitor of differentiation (Dollenmeier cells isolated from F-line and RBC2-line male turkeys, et al., 1981). Therefore, it is reasonable to speculate that implying that SYN 2 through 4 may play a pivotal role higher expression of SYN in the F-line p. major muscle in muscle growth characteristics. may enhance FGF2 signaling and, thus, stimulate more Developmental regulated HSPG expression has been muscle cell proliferation. In vitro studies with mouse reported during embryonic p. major muscle development C2C12 muscle cells have shown that SYN 1 (Larraı´n et al., in both F- and RBC2-line turkeys (Liu et al., 2002, 2004). 1998) and SYN 3 (Fuentealba et al., 1999) enhance cell Total HSPG expression was significantly higher from ED responsiveness to FGF2. The increased embryonic muscle 14 through ED 18 in the F line than in the RBC2 line (Liu cell proliferation may generate more myoblasts and result et al., 2002). In agreement with this result, the current in more muscle fibers before hatch because of muscle study showed higher expression of SYN 2 (ED 14, 18, 20, growth through hyperplasia. In fast-growing mice, pro- 22), SYN 3 (ED 22), and SYN 4 (ED 22) in the F line longed myoblast proliferation has been thought to con- than in the RBC2 line during embryonic p. major muscle tribute to muscle fiber hyperplasia at birth (Summers and development. Previously, SYN 1 expression in embryonic Medrano, 1997). p. major muscle was also shown to be higher in the F Expression of SYN 2 and SYN 4 remained constant from line than in the RBC2 line (Liu et al., 2004). It has been ED 14 to ED 22, whereas SYN 3 and GPC 1 expression well documented that HSPG is a low-affinity coreceptor continuously decreased from ED 14 to ED 24. Liu et al. for FGF2 binding to its high-affinity tyrosine kinase recep- (2004) reported that SYN 1 expression remained high HEPARAN SULFATE PROTEOGLYCAN EXPRESSION 427 from ED 18 through ED 24 in both turkey F and RBC2 disparity, the observation that GPC 1 expression was ele- lines. These results suggest that each individual HSPG vated after differentiation was initiated in the current may regulate the embryonic myogenesis program differ- study is consistent with the previous report that showed entially in that they displayed different expression pat- upregulated GPC 1 expression upon the initiation of C2C12 terns during embryonic p. major muscle development. In cell differentiation (Brandan et al., 1996). Based on the contrast to the results from the current study, a previous increased GPC 1 expression during differentiation, it can study showed that only SYN 1, SYN 3, and SYN 4, but be speculated that GPC 1 plays a role during muscle dif- not SYN 2, were detected in mouse embryonic limb mus- ferentiation. cle tissue using a immunostaining method (Cornelison et The precise functions of the individual membrane-asso- al., 2001). The divergence on SYN 2 expression is probably ciated HSPG during myogenesis are not completely un- due to the difference between the detection methods (real- derstood. Both SYN and GPC 1 can regulate FGF2 activi- time PCR vs. immunostaining), the difference between ties (Steinfeld et al., 1996). However, the consequences muscle type (p. major muscle vs. limb muscle), or the on the regulation of muscle growth may not only depend difference between the species (turkey vs. mouse). on the expression levels but also on the biological forms Satellite cells are myogenic cells located between the in which the HSPG exist. Both SYN (Asundi et al., 2003; and plasma membrane of muscle Endo et al., 2003) and GPC 1 (Brandan et al., 1996) can fibers (Mauro, 1961) and are the major contributors to be shed from the cell surface and have both soluble and postnatal muscle growth (Moss and LeBlond, 1971). membrane-associated forms. A transfection study with Therefore, factors that regulate the proliferation and dif- hematopoietic cells suggests that only membrane-associ- ferentiation of satellite cells will affect muscle growth ated HSPG can increase FGF2 binding to its signaling properties. Velleman et al. (2000) reported that F-line sat- receptor, whereas the soluble form of HSPG cannot ellite cells proliferated faster than RBC2-line satellite cells. (Steinfeld et al., 1996). In mouse C2C12 muscle cells, most The current study showed that only SYN 4 expression SYN 1 and SYN 3 exits in membrane-associated forms was higher in F-line satellite cells compared with RBC2- and can enhance FGF2 activities (Larraı´n et al., 1998; line satellite cells during the proliferation stage, and no Fuentealba et al., 1999). Thus, both SYN 1 and SYN 3 may differences in expression were observed for other mem- play important roles in C2C12 myoblast proliferation by brane-associated HSPG in satellite cells. These data sug- enhancing FGF2 signaling. In the current study, elevated gest that SYN 4 may be a key player in regulating satellite GPC 1 expression was observed during satellite cell differ- cell proliferation. In adult mouse skeletal muscle, SYN 3 entiation. However, in mouse C2C12 cells, most GPC 1 was and SYN 4 expression is not detected in quiescent satellite shed from the cell membrane, probably by an endogenous cells; once activated, all satellite cells express SYN 3 and mechanism (Brandan et al., 1996). It has been proposed SYN 4 for at least 96 h (Cornelison et al., 2001), which that the released form of GPC 1 may be incorporated into suggests a possible role of SYN 3 and SYN 4 in satellite the ECM and sequester FGF2 away from its receptors on cell maintenance, activation, and proliferation. Previous plasma membrane (Brandan and Larraı´n, 1998). There- studies also indicated higher SYN 1 expression (Liu et fore, the upregulated GPC 1 expression during satellite al., 2004) and FGF2 expression (Liu et al., 2003) in F- cell differentiation in the current study may result in line satellite cells compared with RBC2-line satellite cells. inhibition of FGF2 signaling, which allows differentiation Because FGF2 is a strong stimulator of cell proliferation, to proceed. it is conceivable that SYN 1 and SYN 4 may be the major In summary, the current study demonstrated that each enhancers of FGF2 signaling in F-line satellite cells, re- individual membrane-associated HSPG was differentially sulting in a higher proliferation rate compared with the expressed in both embryonic tissue and satellite cells, RBC2 line. The faster proliferation of satellite cells from suggesting a distinct role for each individual HSPG in the F line may generate more satellite cells fused to ex- muscle development. Furthermore, given the fact that the isting muscle fibers and consequently enhance posthatch F line had the higher expression of SYN 2, SYN 3, and SYN muscle growth in the F-line turkeys through hypertrophy. 4 in embryonic muscle tissue and the higher expression In vitro studies with mouse C2C12 muscle cells showed of SYN 4 in satellite cells than the RBC2 line, it can be that SYN 1 (Larraı´n et al., 1997), SYN 3 (Fuentealba et al., postulated that these SYN may play an important role in 1999), and SYN 4 (Brandan and Larraı´n, 1998) expression affecting muscle growth properties. was downregulated during skeletal muscle differentia- tion. In contrast, the current study showed that only SYN ACKNOWLEDGMENTS 2 expression was downregulated after differentiation was initiated, whereas SYN 3 and SYN 4 expression remained The authors are grateful to Cindy Coy and Jane Pesall constant during both proliferation and differentiation. for technical assistance. 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