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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Hepatol. Author manuscript; available in PMC Apr 1, 2008.
Published in final edited form as:
PMCID: PMC2263141
NIHMSID: NIHMS39927
Small Proline Rich Proteins (SPRR) Function as SH3 Domain Ligands, Increase Resistance to Injury and are Associated with Epithelial-Mesenchymal Transition (EMT) in cholangiocytes
Anthony J. Demetris, M.D.,1,2* Susan Specht, M.S.,1,2 Isao Nozaki, M.D.,1,2+ John G. Lunz, III,1,2,3 Donna Beer Stolz, PhD.,4 Noriko Murase, M.D.,1,3 and Tong Wu, M.D., PhD.2
1Thomas E Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15213
2Department of Pathology, Division of Transplantation, University of Pittsburgh Medical Center, Pittsburgh, PA 15213
3Department of Surgery, Division of Transplantation, University of Pittsburgh Medical Center, Pittsburgh, PA 15213
4Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
+Isao Nozaki, M.D., National Shikoku Cancer Center Hospital, Department of Surgery, 13 Horinouchi, Matsuyama, JAPAN 790-0007
*Address Correspondence to: A.J. Demetris, M.D., University of Pittsburgh Medical Center, UPMC Montefiore, Room E741 200 Lothrop Street, Pittsburgh, PA 15213, Phone: (412) 647-2067, Fax: (412) 647-2084, E-mail: demetrisaj/at/upmc.edu
Background/Aims
Deficient biliary epithelial cell (BEC) expression of small proline rich protein (SPRR) 2A in IL-6-/- mice is associated with defective biliary barrier function after bile duct ligation. And numerous gene array expression studies show SPRR2A to commonly be among the most highly upregulated genes in many non-squamous, stressed and remodeling barrier epithelia. Since the function of SPRR in these circumstances is unknown, we tested the exploratory hypothesis that BEC SPRR2A expression contributes to BEC barrier function and wound repair.
Methods
The effect of SPRR2A expression was studied in primary mouse BEC cultures; in BEC cell line after forced over expression of SPRR2A; and in human livers removed at the time of liver transplantation.
Results
Forced SPRR2A over-expression showed that it functions as a SH3 domain ligand that increases resistance to oxidative injury and promotes wound restitution by enhancing migration and acquisition of mesenchymal characteristics. Low confluency non-neoplastic mouse BEC cultures show a phenotype similar to the stable transfectants, as did spindle-shaped BEC participating in atypical ductular reactions in primary biliary cirrhosis.
Conclusions
These observations suggest that SPRR2A-related BEC barrier modifications represent a novel, but widely utilized and evolutionarily conserved, response to stress that is worthy of further study.
Keywords: Cholangiocytes, Ductular Reaction, Wound Healing, Oxidative Stress, Repair, SH3 domain, EMT
Previous studies from our laboratory show that IL-6/gp130/STAT3 signaling importantly contributes to BEC barrier function and wound healing (reviewed in (1)), as in the skin (2), and gastrointestinal tract (3). Cellular and molecular mechanisms linking enhanced IL-6/gp130/STAT3 signaling to biliary tree wound healing and barrier function include production of the cytoprotective motogen, Trefoil family factor 3 (TFF3) (4), and increased small proline rich proteins (SPRR) expression (5).
SPRR are encoded by a tandemly arranged four-member gene family (SPRR1-4) contained within a 170-kilobase region of the epidermal differentiation complex (EDC), a cluster of >50 genes located on chromosome 1q21(6) whose products are involved in epidermal differentiation. In this context, SPRR genes are regulated coordinately as part of the EDC and encode for proteins that cross-link other EDC proteins (7). SPRR1-4 are distinguished by the consensus sequence of their proline-rich central repetitive domain (7) with SPRR2 being the most diversified of all the SPRR genes (7).
The highest ratio of non-synonymous to synonymous nucleotide substitution rates on human chromosome 1 occurs between SPRR2A and SPRR2F(8) and the EDC is the most rapidly diverging gene cluster comparing human and chimpanzee genomes (9). SPRR diversity lies primarily within the regulatory regions of the SPRR2 gene family (6), which is thought to enable SPRR2 proteins to precisely modify barriers in response to diverse environmental insults (6).
In the liver, deficient SPRR2A expression in IL-6-/- mice after bile duct ligation is associated with a biliary barrier defect (5). In other tissues, many cDNA microarray studies show that SPRR genes ranked among those most highly upregulated during inflammation/stress, infection, and remodeling involving barrier epithelia from the lung, skin, and intestine (Table 1). SPRR2A and other SPRR family members also rank among the most highly upregulated genes in developing tissues, including the intestine, prostate, colon, and breast, where high SPRR2A expression in the developing adolescent breast bud occurs as it penetrates the mesenchyme (Table 1).
TABLE 1
TABLE 1
cDNA microarray studies showing SPRR genes to be among the most highly upregulated genes under the non-neoplastic contexts listed. The studies are grouped together according to organ.
Since SPRR function(-s) in these stress-related non-squamous contexts are unknown, we tested the exploratory hypothesis that SPRR2A expression was stress and wound repair-related. First, using a biliary epithelial cell line, SG231, we studied the effect of SPRR2A over expression on wound repair and resistance to injury. Second, we studied SPRR2A mRNA and protein expression in primary cultures of non-neoplastic mouse BEC at low and high confluency to simulate “front row” epithelial cells of healing wounds and intact epithelium, respectively. “Front row” cells refers to epithelial cells closest to an open wound that lose close contacts with neighboring cells and undergo shape changes and migration needed for wound closure. Finally, we studied SPRR2A expression in diseased human liver to determine if the alterations observed, in vitro, during high SPRR expression (SG231 forced over expression and low confluency BEC) resembled BEC in diseased human livers.
Cell Cultures and Establishment of Stable Transfection of SG231 Cells
Primary mouse and human BEC were cultured on collagen gels or collagen-coated plates in complete serum-free medium (C-SFM) as described (10). The human bile duct cell line, SG231 (11), was cultured as reported (10). Human SPRR2A cDNA (X53064) was generated by RT-PCR from total RNA from primary human BEC. The SPRR2A cDNA was confirmed by DNA sequencing and inserted into the GFP expressing pTracer mammalian expression vector (Invitrogen, Carlsbad, CA) creating a C-terminal His-V5-tagged SPRR2A. A pTracer plasmid without the insert was used as a control vector. SG231 cells were transfected using FuGENE 6 Transfection Reagent (Roche, Indianapolis, IN), stable transfectants selected with zeocin (1.0 mg/ml; Invitrogen), and pTracer-SPRR2A-His-V5 clones isolated by limiting dilution. Expression was confirmed by Western blot analysis.
SPRR2A expression in transfectants was knocked-down by two SPRR2A siRNAs (ID:203488; ID:42886-40nM; Ambion, Austin, TX) using lipofectamine 2000 (Invitrogen) transfection reagent. Controls were either lipofectamine only or negative control siRNA.
SYBR Green Quantitative Real-time RT-PCR
Gene expression was quantified by SYBR Green real-time RT-PCR using primers described in Table 2. Gene expression was normalized to mouse GAPDH or human β-actin expression.
TABLE 2
TABLE 2
(m = mouse; h = human)
Migration Assay
Using a modified protocol for fibroblasts (12), a linear wound was created with a rubber policeman in confluent BEC monolayers. Images taken at 0h and 24h after wounding were compared and the distance traveled by the cells at the acellular front was measured. Additionally, the number of cells migrating through the pores (8μm) of a transwell (Corning, Lowell, MA) in 24 hours was measured.
Western Blot Analysis
Protein was extracted using Chaps or RIPA buffer with protease inhibitors and Western blotting performed as described (13) (Table 3). Signals were detected using enhanced chemiluminescence reagents (NEN; Life Science Products, Boston, MA) and quantified using Image J software.
Immunoprecipitation Studies
Stable SPRR2A and vector control transfectants were subjected to protein cross-linking using 2mM disuccinimidyl suberate (Pierce Biotechnology, Inc. Rockford, IL) according to the manufacturer’s protocol. Protein was extracted with non-denaturing lysis buffer (1% Triton X-100, 50mM Tris(pH 7.4), 300mM NaCl, 5mM EDTA) containing protease inhibitors. 1mg of protein was immunoprecipitated with 3μg of anti-V5 or anti-C-Src(H-12) antibody (Table 2) at 4°C for 1 hour followed by rotation with 50μl of protein G agarose beads (Santa Cruz Biotechnology) at 4°C overnight. Bead-bound proteins were released by heating to 98°C in gel loading buffer and separated by SDS-PAGE.
Immunohistochemistry
Immunofluorescence and immunoperoxidase staining (Table 3) was performed as described (5).
Flow cytometry
Cell viability after H2O2 exposure was assessed by staining with propidium iodide and flow cytometric analysis on a Coulter EPICS-XL.
TRAP Assay
Total Radical Trapping Antioxidant Potential (TRAP) was determined by a luminometric method (14, 15). The azo compound 2,2’-azo-bis-(2-methyl-propanimidamide)(AAPH; Cayman Chemical, Ann Arbor, MI) undergoes a constant rate of peroxidation, generating free radicals detected by luminol fluorescence. Absorption of AAPH generated radicals is first measured using a known anti-oxidant (Trolox®) as an internal standard. The induction time measures the time that Trolox® prevents the presence of free radicals and fluorescence of luminol. Extension of the Trolox® induction time by cellular lysates reflects their cellular anti-oxidant potential. SPRR2A transfected SG231 cells were lysed in water, sonicated on ice and supernatants collected after centrifugation (10,00xg; 5 minutes). Assay Reaction: 200μl luminol (PerkinElmer, Wellesley, MA), 5μg protein lysate, 10mM AAPH, 100nM Trolox®. The blank contained water instead of cytosol. Graphs (counts per second versus time) were used to determine induction times by extrapolating to 0 CPS using the maximum slope of Trolox® consumption.
Statistical Analysis
Values shown for various tests are the mean ± SD (or SE where noted) with experimental repeats of n ≥ 3. A student’s t-test with α = 0.05 was used to compare two groups.
Association of SPRR2A Expression with Changes in Cell Shape
SPRR2A was stably transfected into the BEC line, SG231. This line was chosen because it was derived from, and maintains, most characteristics of mature intrahepatic BEC, including a distinctly epithelial morphology with clear apical/basal polarization, mucin production, surface micro-villi, and BEC phenotypic markers (11). It also shows responses to cytokines and growth factors similar to mature BEC (16). Stable SPRR2A clones showed 10 to 17 times the level of SPRR2A mRNA/protein expression than normal human BEC (Figure 1). This compares favorably with the ≥ 14-fold increase seen in BEC after bile duct ligation, but significantly less than increases seen in intestinal epithelial cells following surgical or infectious stressors (Table 1).
Figure 1
Figure 1
Stable expression of SPRR2A in SG231 cells results in changes of cell shape and emergence of mesenchymal characteristics. A) SPRR2A mRNA by real-time PCR and B) protein (V5 tag) expression by Western blot in stable transfectants, clone 7 (CL7) and clone (more ...)
Compared to control transfectants, all SPRR2A stable transfectants showed features of front row cells, such as cell enlargement, extension of cytoplasmic processes, and partial disruption of contact with neighboring cells (Figure 1C). Consistent with at least partial EMT occurring in front row cells, E-cadherin and CK-19 protein expression was uniformly decreased in all SPRR2A clones (Figure 1D). Clones (1, 3, and 5) that maintained some epithelioid morphology still showed disruption of close contacts with neighboring cells and significantly decreased E-cadherin and CK19 expression. Dendritic-shaped clones tended to lose E-cadherin and CK19 expression altogether and acquired mesenchymal characteristics. Clone 7, which showed intermediate changes, was chosen for further analysis.
Immunofluorescence staining of control transfectants for E-cadherin, connexin-43, cytokeratin-19, vimentin, F-actin and fibronectin (17) showed an expression pattern typical of epithelial cells (Figure 2A). Clone 7, in contrast, lost many epithelial and gained several mesenchymal cell characteristics (Figure 2A). SPRR2A specific siRNA treatment reversed these effects in clone 7, significantly increasing CK19 expression and improving epithelial sheet morphology (Figure 2B).
Figure 2
Figure 2
Phenotypic characterization of SG231 cells after SPRR2A transfection. A) In the control transfectant (Control), E-cadherin and connexin-43 localized to the cell surface at intercellular borders; cytokeratin-19 localized to the cytoplasm (58); fibronectin (more ...)
Non-confluent BEC cultures also assume a front row, large spreading, phenotype until they reach confluence when a typical cobblestone monolayer is formed. We predicted, therefore, that SPRR2A expression would be increased in non-confluent BEC cultures and decrease upon confluence. Consistent with expectations, SPRR2A expression was 5-fold higher in widely scattered non-confluent BEC cultures and decreased significantly as BEC monolayers reached confluence (Figure 3A). Vimentin and S100A4 mRNA expression were increased at low confluency and decreased with confluence, whereas E-cadherin mRNA expression increased with confluence, except at the final phase when a dense typical cobblestone sheet had already formed. CK19 expression remained relatively stable throughout.
Figure 3
Figure 3
A) SPRR2A, vimentin, S1004A and E-cadherin mRNA expression is affected by cell confluency in primary mouse BEC cultures. BEC were inoculated at increasing cell numbers: 25 (a), 50 (b), 100 (c), 200 (d) X 103 cells/ well and pictures were taken at 48 hours, (more ...)
We next tested whether confluence affected E-cadherin and CK-19 levels in the SPRR2A transfectants and the vector control. E-cadherin and CK-19 levels increased at high confluence, but expression levels in the SPRR2A transfectants always remained significantly less than controls (Figure 3B). Clone 7 never formed typical compact cobblestone sheets, regardless of the level of confluence. Thus, forced SPRR2A expression in SG231 cells appears to maintain them in a “front row” phenotype, overcoming the tendency to form intact epithelial sheets seen in the vector controls.
The Effect of SPRR2A Expression on Cell Migration
Since SPRR2A transfectants mimic “front row” epithelial cells of wounds (18), we tested their ability to effect wound closure in a migration assay, in vitro (4). Despite decreased proliferation (Figure 4A), SPRR2A transfectants showed significantly better restitution/migration that was not affected by preventing proliferation with hydroxyurea (Figure 4B). Inhibition of SPRR2A expression using SPRR2A specific siRNA (Figure 4C) reversed the enhanced migration (Figure 4D). The SPRR2A-expressing clones also had an increased ability to transverse through the pores of a transwell (Figure 4E).
Figure 4
Figure 4
SPRR2A transfectants (clone 7 and 10) proliferate less, but show better migration from the acellular front of a wound than vector control transfectants. A) Cell proliferation was measured 24 hours after seeding in serum supplemented media (SSM) (Day 0). (more ...)
Analysis of Potential SPRR2A Binding Partners
SPRR2A serves as a cross-linking protein during formation of the cornified envelope, but other EDC constituents are not equally upregulated during non-coordinate SPRR2A expression in non-squamous tissues, including BEC (5). This suggests that SPRR2A has other binding partners and functions under these circumstances. Examination of the SPRR2A proline rich sequence revealed putative SH3 domain ligand motifs (Figure 5A).
Figure 5
Figure 5
SPRR2A contains ligands that bind to SH3 domain-containing proteins. A) Mapping of the SH3 ligand motifs in SPRR2A. The seven-residue core SH3 ligand consensus sequence, pXPpXP (where small p = proline-preferred and X tend to be aliphatic), occurs several (more ...)
Speculation about potential binding partners included the SH3 homology domain (Figure 5A) of Src family kinases (SFK) because they critically influence epithelial cell-to-cell junction formation (19). A SH3 domain array analysis confirmed SPPR2A binding to several SH3 domain-containing proteins (Figure 5B). Two of these proteins are SFK (Yes1 and c-Src) (20), which are intimately involved in cell junction formation, as is abl (21), another strong binding partner. Immunoprecipitation studies from SPRR2A transfectants confirmed that SPRR2A-Src (Figure 5C) and SPRR2A-Yes (data not shown) complexes formed spontaneously, in vivo. Finally, immunofluorescence studies showed that the intra-cellular localization of the SPRR2A binding partner, Src, changed after SPRR2A transfection (Figure 5D).
SPRR2A Protects Cells from Injury
BEC participating in ductular reactions, or wound repair, enjoy a survival advantage over hepatocytes and are more resistant than hepatocytes to oxidative stress (22). Since STAT3-dependent BEC SPRR2A expression is seen during stress and remodeling (5) they should also be more resistant to injury.
Cell death after H2O2-induced injury was monitored by propidium iodide staining (Figure 6A) and the release of HMGB1 (Figure 6B) and showed that SPRR2A transfectants were more resistant than vector-transfected controls. SPRR2-associated resistance to injury was reversible by siRNA inhibition of SPRR2A expression (Figure 6B). The SPRR2A transfectants were also more resistant to glycochenodeoxycholate (GCDC)-induced injury (Figure 6C), which is directly relevant to wound healing conditions in the biliary tree.
Figure 6
Figure 6
Non-coordinate SPRR2A expression increases resistance to injury. A) Cell death was measured by flow cytometry for propidium iodide staining of non-viable cells and shows that the SPRR2A transfectants are more resistant to H2O2 exposure than the vector (more ...)
Amino acids, peptides, and proteins have relatively low specific anti-oxidant activity, on a molar basis, but represent an important scavenger of ROS because of relatively high intracellular molar concentrations. The overall cytosolic reducing power can be measured in the Total Radical Trapping Anti-oxidant Potential (TRAP) assay, measuring the time cytosolic extracts can quench the oxidizing potential of AAPH (See Methods). Compared to vector controls the SPRR2A transfectants significantly delayed maximum oxygen uptake by the probe, consistent with a greater reducing power (Figure 6D).
Expression of SPRR2A mRNA and Protein in Diseased Human Livers
We next measured SPRR2A mRNA expression by real-time PCR in normal and in diseased human liver tissue. Figure 7A-B shows that more IL-6 and SPRR2A mRNA are expressed in primary biliary cirrhosis (PBC) than in normal livers. The apparent discrepancy between IL-6 and SPRR2A expression in alcoholic liver disease is likely related to inhibition of STAT3 activity seen in that disorder (23).
Figure 7
Figure 7
A) Expression of IL-6 and B) SPRR2A mRNA by quantitative real time PCR in human liver tissue removed at the time of transplantation. Consistent with a role in response to stress, SPRR2A showed some upregulation in almost all liver diseases, but the upregulation (more ...)
In diseased livers SPRR2 protein localized to BEC (Figure 7C-E), consistent with previous results (5). In PBC, SPRR2 localized to the cytoplasmic periphery in damaged columnar-shaped BEC lining septal bile ducts, which co-expressed SPRR2 and cytokeratin but not vimentin (Figure 7D). Diffuse cytoplasmic SPRR2 localization occurred in some spindle-shaped BEC participating in “atypical” ductular reactions (24), characterized by an anastomosing network of ductules, with poorly defined lumina, lined by flattened cells with scant cytoplasm (24) (Figure 7E). Atypical ductules are encountered most commonly in chronic cholestatic diseases, especially PBC (28). Some atypical spindle-shaped BEC were also focally vimentin and cytokeratin positive (Figure 7E), indicative of a phenotype intermediate between epithelial and mesenchymal cells and similar to the SPRR2A transfectants and low-confluency mouse BEC cultures, in vitro. Immunofluorescence staining of PBC, PSC, and chronic hepatitis livers showed that BEC SPRR2 expression was strongest and most prevalent in PBC, especially in damaged small septal bile ducts and in atypical ductular reactions.
In wounded barrier epithelia, front row cells often undergo partial or complete dissolution of cell-cell contacts and acquire some mesenchymal characteristics. This functions to disaggregate epithelial units and reshape epithelia for movement (17). Epithelia in transition lose polarity, intercellular junctions, and down-regulate cytokeratin filaments in order to rearrange F-actin stress fibers and express filopodia and lamellopodia (17). Several lines of evidence show that SPRR2 expression is associated with this process.
Forced non-coordinate expression of SPRR2A in SG231 cells, in vitro, can lead to a front row phenotype. Since SG231 is a cell line the results should be interpreted with some caution. But non-neoplastic BEC with similar phenotypic characteristics were also observed in low-confluency primary mouse BEC cultures and in atypical ductules in PBC. SPRR2A is also expressed exclusively in the remodeling biliary tree after bile duct ligation (5), and in many injured and remodeling barrier epithelia from the lung, skin, uterus, and intestine after exposure to injury and stress (Table 1). It seems reasonable, therefore, to suggest that SPRR2A upregulation is a stress-related response that helps to protect barrier epithelium from oxidative damage and prepares it for remodeling. It would have been ideal to conditionally block biliary SPRR expression to further investigate SPRR function. But this approach has not been feasible because of redundancy and regulatory diversity in the SPRR gene locus.
Molecular studies show that non-coordinate BEC expression makes SPRR2A available for binding to SH3 domain-containing proteins (25), such as c-Src, c-Yes, and abl. Other binding partners exist. These particular molecules, however, contribute significantly to formation and maintenance of intercellular junctions, shape changes (20, 26), and to acquisition of some mesenchymal characteristics (19, 27).
As seen in the protein binding array, promiscuity of SH3 ligand binding enables numerous potential interactions and facilitates assembly of large multi-molecular complexes. This can lead to context specific effects (28). It is likely, therefore, that SPRR expression might not always produce the same effects, but be dependent on the cell proteome at the time of SPRR expression. This likely accounts for some of the variability seen in our different SPRR2A clones and that Sprr2 is expressed in damaged septal bile ducts and in atypical cholangioles, which differ in their vimentin expression.
Spindle-shaped atypical ductules in PBC can acquire some mesenchymal characteristics similar to the SPRR2A transfectants and low confluency mouse BEC. In embryonic liver ductal plate BEC transiently express vimentin (29) and down-regulate membranous E-cadherin (30) when they “invade” the portal connective tissue to form mature intrahepatic bile ducts. SPRR2A is also highly upregulated when mouse hepatoblasts transform into BEC after exposure to Matrigel (http://www.ncbi.nlm.nih.gov/projects/geo/gds/gds_browse.cgi?gds=970). During injury and repair (1), BEC can transform from polarized cuboidal epithelial cells into spindle-shaped migratory cells that can express vimentin (31). However, since BEC appear to retain some epithelial characteristics during this process, BEC EMT may not be exactly the same as described for kidney tubular epithelial cells (17). Instead, acquisition of mesenchymal characteristics in BEC may be “meta-stable” because they appear to revert to an epithelial phenotype when migration/repair is complete. More study is needed on this topic.
Among liver diseases tested, SPRR2A is most highly expressed in BEC of small atypical ductules from PBC livers, and to a lesser extent in other diseases. It was clear, however, that EMT changes were more common in atypical ductular reactions. It is likely, therefore, that the heterogeneity of BEC in large versus small ducts (32) accounts for this difference. In the uterus, however, SPRR2A is highly upregulated in an estrogen-dependent fashion at the blastocyst implantation site where decreased intercellular junctions, desmoplakin, and cytokeratin are also seen (33). The possibility that estrogen and IL-6 co-dependent BEC barrier modifications might be involved in PBC pathogenesis is also worthy of further study.
This study focuses specifically on BEC but the ubiquity and magnitude of non-coordinate SPRR expression in non-squamous tissues, suggests that it is part of a generic stress/remodeling response. Although not described in mammalian cells, proline utilization is widely recognized as an anti-stress response in plants (34). Proline residues in proline-rich proteins can be converted to either 4- or 5-hydroxy proline by ROS and these changes will not cause cleavage of the polypeptide or damage the amino acid backbone (34).
In conclusion, our studies suggest that SPRR upregulation is a widely utilized stress and remodeling response which contributes to barrier function, wound repair and increase resistance to injury. This may explain why the gene locus is so rapidly diverging (9) and a “hot-spot” for non-synonymous substitutions (8).
Acknowledgments
Supported by National Institutes of Health grant DK49615 (A.J.D.), CA76541 (D.B.S.), and CA 102325 (T.W.).
Abbreviations in this paper
BECBiliary Epithelial Cell
C-SFMComplete Serum-free Medium
EDCEpidermal Differential Complex
EMTEpithelial-mesenchymal Transition
HMGB1High Mobility Group Box 1
PBCPrimary Biliary Cirrhosis
SPRR proteinSmall Proline-rich protein
ROSreactive oxygen species
STAT3Signal Transducer and Activator of Transcription 3

Footnotes
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