Expression and Functional Role of Sprouty-2 in Breast Morphogenesis
Publication Date
April 03, 2013
Journal
PLOS ONE
Authors
Valgardur Sigurdsson, Saevar Ingthorsson, Bylgja Hilmarsdottir, Sigrun M. Gustafsdottir, et al
Volume
8
Issue
4
Pages
e60798
DOI
https://dx.plos.org/10.1371/journal.pone.0060798
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0060798
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/23573284
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616012
Europe PMC
http://europepmc.org/abstract/MED/23573284
Web of Science
000318840100104
Scopus
84875716263
Mendeley
http://www.mendeley.com/research/expression-functional-role-sprouty2-breast-morphogenesis
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Mendeley | Further Information

{"title"=>"Expression and Functional Role of Sprouty-2 in Breast Morphogenesis", "type"=>"journal", "authors"=>[{"first_name"=>"Valgardur", "last_name"=>"Sigurdsson", "scopus_author_id"=>"15842441900"}, {"first_name"=>"Saevar", "last_name"=>"Ingthorsson", "scopus_author_id"=>"26531401200"}, {"first_name"=>"Bylgja", "last_name"=>"Hilmarsdottir", "scopus_author_id"=>"36442398500"}, {"first_name"=>"Sigrun M.", "last_name"=>"Gustafsdottir", "scopus_author_id"=>"57200650285"}, {"first_name"=>"Sigridur Rut", "last_name"=>"Franzdottir", "scopus_author_id"=>"10641873200"}, {"first_name"=>"Ari Jon", "last_name"=>"Arason", "scopus_author_id"=>"36924496300"}, {"first_name"=>"Eirikur", "last_name"=>"Steingrimsson", "scopus_author_id"=>"7003695844"}, {"first_name"=>"Magnus K.", "last_name"=>"Magnusson", "scopus_author_id"=>"7103131151"}, {"first_name"=>"Thorarinn", "last_name"=>"Gudjonsson", "scopus_author_id"=>"8509221100"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"23573284", "doi"=>"10.1371/journal.pone.0060798", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "scopus"=>"2-s2.0-84875716263", "issn"=>"19326203", "pui"=>"368649228", "sgr"=>"84875716263"}, "id"=>"131ba42a-ecef-39ab-9088-0a1dcb4d257c", "abstract"=>"Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.", "link"=>"http://www.mendeley.com/research/expression-functional-role-sprouty2-breast-morphogenesis", "reader_count"=>18, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>4, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>9, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>4, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>9, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>5, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>11}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Ukraine"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1010070"], "description"=>"<p><i>A) Expression of SPRY2 and pEGFR is inversely correlated with cell proliferation.</i> Low expression of SPRY2 is found within the virgin gland with few positive stromal cells (a). Note, increased stromal expression of SPRY2 in pregnant gland accompanied with expression in myoepithelial cell as evidenced by double staining of SPRY2 and the myoepithelial marker CK14 (b). Dramatic increase in SPRY2 expression is seen during lactation (a and b). SPRY2 and EGFR show similar expression pattern at all stages (c) with pEGFR expression seen at terminal buds in pregnant gland. Dramatic increase in pEGFR expression is seen in the lactating gland. Similar expression is found for SPRY2 and pEGFR in lactating gland. Proliferation is increased from virgin to pregnant gland but is reduced during lactation, with only few PCNA positive cells left. Cells counterstained with TOPRO-3, Bar = 100 µm. <i>B) SPRY2 expression is highest during lactation accompanied by activation of Erk/MAPK pathway.</i> Western blot demonstrated the expression differences of SPRY2 in virgin, pregnant and lactating glands. There is over 38 fold increase in SPRY2 expression during lactation compared to virgin state. Total ERK and pERK is also significantly increased during lactation. Actin was used as a loading control. <i>C) SPRY2 activity is peaking during pregnancy in the mouse mammary gland.</i> Using proximity ligation assay it was shown that phosphorylated SPRY2 was significantly more expressed during pregnancy compared to virgin and lactating gland. Bar = 25 µm.</p>", "links"=>[], "tags"=>["spry2", "lactating", "mammary"], "article_id"=>670517, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060798.g002", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_SPRY2_in_virgin_pregnant_and_lactating_mouse_mammary_gland_/670517", "title"=>"Expression of SPRY2 in virgin, pregnant and lactating mouse mammary gland.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-03 00:08:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010087"], "description"=>"<p><i>A) D492 cells show significant knockdown of SPRY2.</i> D492 were transfected with non-silencing (NS) shRNA and different version of knockdown (KD) shRNA against SPRY2. KD3 showed most efficient knockdown (70%) measured by western blot. KD3A is a single cell cloned subline from KD3. <i>B) D492SPRY2<sup>-KD3</sup> retains an epithelial phenotype in monolayer culture.</i> No phenotypic differences were observed in monolayer of D492<sup>NS</sup> and <i>D492<sup>SPRY2-KD3</sup></i> (upper row). Transfection efficacy was evaluated by GFP (lower row). <i>C) D492<sup>SPRY2-KD</sup> cells have acquired increased migration potential.</i> When plated on porous transwell filter <i>D492<sup>SPRY2-KD3</sup></i> showed increased migration compared to D492<sup>NS</sup>. Single cell derived clone KD3A from KD3 had the highest migration potential. <i>D) SPRY2 knockdown has no effect on cell proliferation</i>. Monolayer proliferation of D492<sup>NS</sup>, D492<sup>SPRY2-KD3</sup> and D492<sup>SPR2-KD3A</sup> was evaluated at different time points, as indicated. There was no remarkable difference in the proliferation rate of the NS and KD cells, although at day 4 D492<sup>SPRY2-KD3A</sup> seemed to proliferate slightly less.</p>", "links"=>[], "tags"=>["knockdown", "d492", "epithelial"], "article_id"=>670534, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060798.g004", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SPRY2_Knockdown_in_D492_breast_epithelial_stem_cell_line_/670534", "title"=>"SPRY2 Knockdown in D492 breast epithelial stem cell line.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-03 00:08:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010096"], "description"=>"<p><i>A) Endothelial cells stimulate growth of D492 cells.</i> When plated in 3D rBM culture with breast endothelial cells (BRENCs), D492 cells can form complex branching colonies from as little as 100–1000 cells compared to 7×10<sup>3</sup> –10<sup>4</sup> in 3D monoculture. <i>B) D492-derived branching structures form bi-layered epithelium with BRENCs positioned extralobular.</i> The branching colonies are bi-layered and polarized structures as evidenced by the expression of the myoepithelial marker CK14 on the outer side and the luminal epithelial marker CK19 on the inner side (upper figure). In co-cultures endothelial cells stay as single cells positioned outside the branching structures as seen with CD31 staining (lower figure, arrows). Bar  =  100 µm. Sections counterstained with TOPRO-3 nuclear stain. <i>C) Phenotypes of D492 in co-culture with BRENCs.</i> In co-culture with endothelial cells D492 cells form both branching- and spindle-like colonies. The branching colonies show strong expression of E-cadherin, while spindle like colonies have undergone EMT as evidenced by cadherin switch from E- to N-cadherin. Staining for the proliferation marker ki67 shows that both the branching and spindle like colonies are viable and growing. Bar  =  100 µm. Sections counterstained with TOPRO-3 nuclear stain. <i>D) Spry2-KD cells show an increase in the spindle-like morphology.</i> While D492<sup>NS</sup> cells form about 40% spindle-like colonies there is a significant increase in the D492<sup>Spry2-KD3</sup> cells up to 65%. The D492<sup>Spry2-KD3A</sup> form almost exclusively spindle-like colonies in co-culture with endothelial cells.</p>", "links"=>[], "tags"=>["disturbed", "spry2", "kd", "cells", "co-cultured", "endothelial"], "article_id"=>670543, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060798.g006", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Epithelial_integrity_is_disturbed_in_SPRY2_KD_cells_when_co_cultured_with_endothelial_cells_/670543", "title"=>"Epithelial integrity is disturbed in SPRY2 KD cells when co-cultured with endothelial cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-03 00:09:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010097", "https://ndownloader.figshare.com/files/1010098"], "description"=>"<div><p>Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting <i>SPRY2</i>. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.</p> </div>", "links"=>[], "tags"=>["sprouty-2"], "article_id"=>670544, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0060798.s001", "https://dx.doi.org/10.1371/journal.pone.0060798.s002"], "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_and_Functional_Role_of_Sprouty_2_in_Breast_Morphogenesis_/670544", "title"=>"Expression and Functional Role of Sprouty-2 in Breast Morphogenesis", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-04-03 00:09:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010092"], "description"=>"<p><i>A) SPRY2-KD resulted in increased branching colonies in 3D culture.</i> Stereoscopic images of representative 3D rBM gels for D492<sup>NS</sup>, D492<sup>SPRY22-KD3</sup> and D492<sup>SPRY2-KD3A</sup> showing increased branching upon SPR2 knockdown. Bar  =  2mm. <i>B) Types of morphogenesis in 3D culture</i>. Epithelial colonies were divided into three morphotypes: simple branching, complex branching and other (mostly solid round). Representative images of simple- and complex branching are shown. <i>C) 3D morphogenesis of D492<sup>NS,</sup> D492<sup>SPRY2-KD3</sup> and D492<sup>SPRY2-KD3A</sup> cells.</i> In a setup with 10<sup>4</sup> cells both D492<sup>SPRY2-KD3</sup> and D492<sup>SPRY2-KD3A</sup> cells showed an increase in simple- and complex branching pattern. Bar  = 100 µm. <i>D) Large complex colonies in 3D rBM culture.</i> Complex branching colonies over 250 µm were counted. This showed a 2 fold increase in size of the SPRY2 KD cells. <i>E) 3D morphogenesis of D492<sup>NS,</sup> D492<sup>SPRY2-KD3</sup> and D492<sup>SPRY2-KD3A</sup> cells with variable amount of cells.</i> In a setup using 1.3×10<sup>4</sup>, 10<sup>4</sup> and 7×10<sup>3</sup> cells, the SPR2-KD cells showed superior branching abilities compared to NS cells. <i>F) SPRY2 expression in branching colonies from D492<sup>NS</sup> and D492<sup>SPRY2-KD3</sup></i>. As seen before SPRY2 was located at branching tips in NS cells. SPRY2 KD cells showed reduced expression of SPRY2 with some areas of diffuse staining. Bar  = 100 µm.</p>", "links"=>[], "tags"=>["knockdown", "d492", "branching"], "article_id"=>670539, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060798.g005", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SPRY2_Knockdown_in_D492_promotes_increased_branching_morphogenesis_/670539", "title"=>"SPRY2 Knockdown in D492 promotes increased branching morphogenesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-03 00:08:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010074"], "description"=>"<p><i>A) D492 cells generate branching structures when cultured in rBM.</i> When seeded in rBM D492 cells generate TDLU-like structures. By generating in vitro TDLU-like structures it is possible to follow individual steps in the branching morphogenesis process. Until day 8 or 9 cells grow as single colonies. First sign of initial budding occurs at day 10 and 11 (yellow arrows) followed by duct elongation and bifurcation (blue and red arrows), respectively. <i>B) SPRY2 expression shows a dramatic shift during TDLU formation in 3D culture.</i> Colonies were isolated from 3D cultures at different time points as indicated. Initially at day 8 there is relative high expression of SPRY2 mRNA but its expression is reduced during initial budding but increases again during duct elongation and further bifurcation of complex branching. Western blot confirms that SPRY2 levels increase up to day 16 and remain high while pEGFR is slightly decreasing for day 16 to day 19. Actin was used as a loading control. <i>C) pEGFR and SPRY2 are expressed at the growing tips of TDLU-like structures.</i> D492-derived TDLU-like structures generated in 3D culture were stained with antibodies against SPRY2, EGFR, pEGFR, β4-integrin and F-actin. pEGFR was predominantly expressed at the branching tips while total EGFR had a more general distribution. SPRY2 was also expressed at branching tips but not in clefts. Co-staining of SPRY2 and EGFR show strong expression at the branching tips (arrows). F-actin staining gives a general outlook of a branching colony while β4-integrin outlines their connection to the surrounding rBM matrix. Phosphorylated SPRY2 (right) was analyzed using proximity ligation assay as described above. pSPRY2 was predominantly expressed at the branching tips showing similar pattern as total SPRY2. Cells were counterstained with TOPRO-3 nuclear stain. Bar = 100 µm.</p>", "links"=>[], "tags"=>["correlated", "points", "branching", "morphogenesis", "d492"], "article_id"=>670521, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060798.g003", "stats"=>{"downloads"=>4, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SPRY2_expression_is_correlated_with_critical_points_in_branching_morphogenesis_of_D492_breast_stem_cell_line_/670521", "title"=>"SPRY2 expression is correlated with critical points in branching morphogenesis of D492 breast stem cell line.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-03 00:08:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010067"], "description"=>"<p>Expression of SPRY2 was evaluated in normal human breast tissue derived from reduction mammoplasty biopsies. <i>A) Expression of SPRY2 is most prominent in the luminal epithelial cells.</i> SPRY2 expression was predominantly found within the epithelial compartment of duct and lobuli as evidenced by immunohistochemistry and in situ hybridization. SPRY2 was predominantly expressed in luminal epithelial cells both in ducts and lobuli. SPRY2 was co-stained for K14 (myoepithelial cells) and K18 (luminal epithelial cells). Note the co-expression of SPRY2 and K18 in luminal epithelial cells. SPRY2 expression was also presence in the stroma, most likely in endothelial cells (arrows). Sections were counterstained with TOPRO-3. Bar  = 100 µm. <i>B) Expression differences of SPRY2 in luminal- and myoepithelial cells.</i> Real time PCR was used to quantify expression difference of <i>SPRY2</i> between luminal- and myoepithelial cells. <i>SPRY2</i> expression was generally low in myoepithelial cells compared to luminal epithelial cells that expressed up to 58 fold more <i>SPRY2</i>. Measurement was done in paired luminal and myoepithelial cells from three different biopsies.</p>", "links"=>[], "tags"=>["spry2", "lobules", "ducts"], "article_id"=>670514, "categories"=>["Cancer", "Biochemistry", "Developmental Biology"], "users"=>["Valgardur Sigurdsson", "Saevar Ingthorsson", "Bylgja Hilmarsdottir", "Sigrun M. Gustafsdottir", "Sigridur Rut Franzdottir", "Ari Jon Arason", "Eiríkur Steingrímsson", "Magnus K. Magnusson", "Thorarinn Gudjonsson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060798.g001", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_SPRY2_in_lobules_and_ducts_in_the_normal_human_breast_gland_/670514", "title"=>"Expression of SPRY2 in lobules and ducts in the normal human breast gland.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-03 00:08:34"}

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Relative Metric

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