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Identification of a Binding Partner for the Endothelial Cell Surface Proteins TEM7 and TEM7R

¹ The Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center and Program in Human Genetics and Molecular Biology, Johns Hopkins Medical Institutions, Baltimore, Maryland; ² Department of Pathology, University of South Carolina School of Medicine, South Carolina Cancer Center, Division of Basic Research, Columbia, South Carolina; ³ Genzyme Molecular Oncology, Framingham, Massachusetts; ⁴ Imgenex Corporation, San Diego, California; and ⁵ Tumor Angiogenesis Section, Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, Maryland

	ABSTRACT

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Tumor endothelial marker 7 (TEM7) was recently identified as an mRNA transcript overexpressed in the blood vessels of human solid tumors. Here, we identify several new variants of TEM7, derived by alternative splicing, that are predicted to be intracellular (TEM7-I), secreted (TEM7-S), or on the cell surface membrane (TEM7-M) of tumor endothelium. Using new antibodies against the TEM7 protein, we confirmed the predicted expression of TEM7 on the cell surface and demonstrated that TEM7-M protein, like its mRNA, is overexpressed on the endothelium of various tumor types. We then used an affinity purification strategy to search for TEM7-binding proteins and identified cortactin as a protein capable of binding to the extracellular region of both TEM7 and its closest homologue, TEM7-related (TEM7R), which is also expressed in tumor endothelium. The binding domain of cortactin was mapped to a unique nine-amino acid region in its plexin-like domain. These studies establish the overexpression of TEM7 protein in tumor endothelium and provide new opportunities for the delivery of therapeutic and imaging agents to the vessels of solid tumors.

	Introduction

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Targeting the endothelial cells that line tumor blood vessels is a promising new strategy for the treatment of cancer (1) . However, realization of the full potential of a vascular-directed approach will require the exploitation of new targets that are expressed predominantly on tumor endothelium (2) . In a systematic attempt to uncover such targets, we recently used serial analysis of gene expression on endothelial cells isolated from human normal or malignant colorectal tissues. These studies led to the identification of several novel tumor endothelial markers (TEMs), the most abundant of which was called TEM7 (3) . In situ hybridization studies validated the expression of TEM7 in the endothelium of colorectal cancer and demonstrated that TEM7 mRNA was also abundantly expressed in the endothelium of a variety of other human cancer types including, breast, lung, and brain tumors. Based on the full-length nucleotide sequence, TEM7 seems to be a typical type I transmembrane protein containing a signal peptide followed by a nidogen-like domain and a single hydrophobic domain (Fig. 1A) . The only other protein that seems to share significant homology to TEM7 is TEM7-related (TEM7R), another putative cell surface protein that also contains a nidogen-like domain. In situ hybridization revealed that TEM7R, like TEM7, was abundantly expressed in the endothelium of malignant colorectal cancer but was absent or rare in normal colonic mucosa (4) . By developing antibodies against human TEM7, we now demonstrate that TEM7 protein, like its mRNA, is elevated in tumor tissues and is expressed predominantly by the endothelial cells of tumor-infiltrating blood vessels. To provide potential insights into the structure and function of this endothelial protein, we then attempted to identify TEM7-binding partners using affinity chromatography.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Analysis of TEM7 structure and expression. A, schematic of TEM7 variants. SP, signal peptide; NIDO, region sharing similarity to nidogen G1 domain; PSI, domain found in plexins, semaphorins, and integrins; TM, transmembrane domain. The region where IM193 and IM568 monoclonal antibodies (mAb) bind TEM7-M is indicated. TEM7-S represents the structure of both TEM7S1 and TEM7S2, which only differ slightly in amino acid composition near their COOH terminus. To search for TEM-binding proteins, AP was fused to the extracellular domain of TEM7 (APT7) or TEM7R (APT7R). B, intron/exon structure of the TEM7 gene. Exon 11 has two alternative splice acceptor sites; splice acceptor 1 (S1) gives rise to E11A and splice acceptor 2 (S2) gives rise to E11B. *, exon 10, exon 11A, and exon 15 all have stop codons that disrupt the coding sequence (black in inset) of the different TEM7 variants. The secreted form of TEM7 uses the stop codon of either exon 10 (TEM7-S2) or 11A (TEM7-S1), whereas the intracellular form (TEM7-I) uses exon 11A. The previously described membrane-bound form (TEM7-M) uses exon 1B to initiate transcription, exon 14 to produce a transmembrane domain, and a stop codon in exon 15. C, immunoprecipitation of TEM7. Left panel. A Mr 85,000 protein was immunoprecipitated (IP) from TEM7-M-transfected 293 cells (293/T7) but not control 293 cells using either a monoclonal antibody (IM193) or a polyclonal antibody (pAb) against TEM7. IM193 antibody was used for immunoblotting (IB). Right panel. Proteins immunoprecipitated with IM193 were untreated or treated with N-glycosidase-F or a mixture of glycosidases and then immunoblotted with IM193 monoclonal antibody. D. TEM7-M protein is elevated in tumor tissues. Left panel. Immunoblotting with IM193 antibody revealed a triplet pattern that was elevated in tumors compared with matched normal tissues from five colorectal cancer patients (two representative cases are shown). A triplet was also elevated in VX2 liver tumors of rabbits compared with normal liver tissue. Note that the bottom band of the triplet (Mr 85,000) migrates at a position similar to that observed in 293 cells transfected with TEM7-M.

	Materials and Methods

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Immunostaining.
Immunohistochemistry on paraffin sections using IM193 antibody and immunofluorescence on fresh-frozen sections using IM568 antibody were performed as described previously (5) .

Immunoelectron Microscopy.
Fresh colorectal cancer specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldeyde in PBS (pH 7.2) overnight at 4°C. Samples were rinsed in PBS, incubated with 0.25% tannic acid (Mallinckrodt, St. Louis, MO) in PBS for 1 hour and rinsed again, and un-cross-linked glutaraldeyde was reduced with 50 mmol/L NH₄Cl in PBS. Samples were washed in 0.1 mol/L maleate buffer before en bloc staining with 2% uranyl acetate in 0.1 mol/L maleate buffer. After a graded ethanol series, dehydrated samples were infiltrated and embedded with L.R. white resin. Samples were polymerized in tightly sealed gelatin capsules for 2 days at 40°C. Seventy- to 80-nm-thick sections were cut and picked up on Formvar-coated nickel grids. After incubation with IM568 monoclonal antibody and 6-nm gold-conjugated antimouse secondary antibody (The Jackson Laboratory, Bar Harbor, ME), sections were post-fixed with 2% glutaraldehyde and stained with uranyl acetate followed by lead citrate. All grids were viewed and photographed on a Philips CM 120 TEM (Philips, Eindhoven, The Netherlands) operating at 80 Kv.

Immunoblotting.
Normal human colonic mucosa, colorectal tumors, normal rabbit hepatic tissue, and VX2 liver tumors were snap frozen immediately after surgical resection and stored at –80°C before use. Tissues were homogenized in TNT buffer [50 mmol/L Tris (pH 7.5), 75 mmol/L NaCl, and 1% Triton X-100 containing a mixture of protease inhibitors (Roche Diagnostics, Mannheim, Germany)] and clarified by centrifugation. Protein extracts from tissues or lysed cells were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Immunoblots were probed with an anti-TEM7 antibody (IM193), anti-myc antibody (Sigma, St. Louis, MO), or anticortactin antibody (Upstate Biotechnology, Lake Placid, NY) followed by an horseradish peroxidase-conjugated antimouse secondary antibody (The Jackson Laboratory) and visualized using the ECL plus system (Amersham Biosciences, Uppsala, Sweden) according to the supplier’s instructions.

Immunoprecipitation.
TNT extracts of mouse brain tissues (Pelfreez Biologicals, Brown Deer, WI), 293 cells, or 293 cells transfected with TEM7-M were incubated overnight with the antibodies described above or with nonspecific rabbit or mouse IgG as controls. Precipitated proteins were eluted from protein A or G-agarose beads (Roche Diagnostics) and detected by immunoblotting with anti-TEM7 (IM193) or anticortactin (Upstate Biotechnology) monoclonal antibodies. For deglycosylation, precipitated proteins were treated with N-glycosidase-F alone or a mixture containing N-glycosidase-F, endo-O-glycosidase, -2 (3 ,6 ,8 ,9) -neuraminidase, ß (1 ,4) -galactosidase and ß-N-acetylglycosaminidase (Sigma).

Alkaline Phosphatase-Tumor Endothelial Marker 7/7R and Cortactin Deletion Constructs.
cDNAs encoding the extracellular regions of TEM7-M (amino acids 1–423) or TEM7R (amino acids 1–450) were PCR-amplified and directionally cloned into the alkaline phosphatase (AP)-Tag5 vector (Genhunter, Nashville, TN) by incorporating the restriction enzyme sites NheI and HindIII (TEM7-M) or NheI and BglII (TEM7R) into the PCR forward and reverse primers, respectively. The cortactin deletion constructs were made by PCR amplification from full-length human cortactin cDNA (accession no. BC008799). A Sfi-1 restriction site was placed in the forward primer, and an EcoR1 site was placed in the reverse primer for directional cloning into pCMV-Myc (Becton Dickinson, San Jose, CA). All vectors generated by PCR were sequence verified to ensure they were mutation-free.

AP-Tumor Endothelial Marker 7/7R-Binding Assay.
Extracts of 293 cells expressing cortactin deletion constructs, crude brain extracts, or brain extracts immunoprecipitated with anticortactin antibodies were separated by SDS-PAGE and transferred to a PDVF membrane (Millipore). Peptides of cortactin were spotted directly onto nitrocellulose. Membranes were probed with supernatants from 293 transfectants expressing either AP alone (control), AP-TEM7, or AP-TEM7R and then visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Sigma). For added sensitivity, in some experiments, anti-AP antibody (Sigma) was added, followed by horseradish peroxidase-conjugated antimouse secondary antibody (The Jackson Laboratory). Blots were visualized on X-ray film using an ECL detection system (Amersham Biosciences).

Biochemical Purification of Cortactin.
Using the AP-TEM7R-binding assay as a screen to follow activity, we fractionated protein extracts from 100 mouse brains (Pelfreez Biologicals) using anion exchange (Bio-Scale Q20 column; Bio-Rad, Hercules, CA) followed by cation exchange (Bio-Scale S20 column; Bio-Rad) chromatography. This was followed by affinity chromatography using anti-AP beads (Sigma) that had been armed with the AP-TEM7R fusion protein. Finally, purified extract was loaded onto an SDS-denaturing gel and stained with Coomassie Blue, and two prominent protein bands that migrated at about M_r 75,000 and 80,000 were gel isolated. After tryptic digestion and analysis by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry (Microchemistry and Proteomics Analysis Facility, Harvard University, Boston, MA), each protein product was identified as cortactin.

	Results

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Characterization of Tumor Endothelial Marker 7 Gene Products.
While attempting to clone the full-length human TEM7 gene by PCR amplification of cDNA obtained from tumor-derived endothelial cells (3) , we noticed additional PCR products that were larger than expected. Sequencing of these products revealed that various forms of TEM7 exist in tumor endothelium, each derived by alternative splicing (Fig. 1A ; Table 1 ). Comprehensive PCR analyses and comparisons with expressed sequence tag and genomic sequence allowed us to compile the intron/exon structure of TEM7 shown in Fig. 1B . In one of the transcripts identified, an alternative splice acceptor site resulted in a new exon (exon11A) encoding a stop codon before the transmembrane domain (Fig. 1B) . The predicted protein product is a secreted TEM7 molecule (TEM7-S1) with an intact nidogen-like domain. Likewise, another novel exon, exon 10, was identified and found to contain a premature stop codon producing a second secreted form of TEM7 (TEM7-S2; see Table 1 ). We also noticed new sequences in the expressed sequence tag repository, which suggested that an alternative exon 1 (called E1A) might exist upstream of the original exon 1 (called E1B). Indeed, using PCR primers from sequences within this exon, we were able to verify its expression in cDNA isolated from tumor-derived endothelium. Sequencing of the amplified cDNA revealed that exon 1A is used in conjunction with exon E11A such that the predicted product lacks both a signal peptide and transmembrane domain and is expected to reside intracellularly (TEM7-I; see Table 1 ). To differentiate the various forms of TEM7, we now refer to the original membrane bound form as TEM7-M.

To determine whether expression of the TEM7 protein, like its mRNA, was elevated in colorectal tissues, we prepared protein extracts from normal colonic mucosa or colorectal tumors of five patients. Immunoblotting with TEM7 antibodies revealed elevated expression of the expected M_r 85,000 product as well as additional M_r 90,000 and 95,000 products in each of the tumor tissues tested (Fig. 1D ; data not shown). The reason for the two larger bands observed in each of the tumor samples is not clear, but could be due to alternative splicing, posttranslational glycosylation, or ubiquitination. Although the relative abundance of each of the products in the M_r 85,000/90,000/95,000 triplet varied between samples, all three were readily immunoprecipitated from tumor extracts with either the polyclonal or the monoclonal anti-TEM7 antibodies.

Cellular Localization of Tumor Endothelial Marker 7 Protein.
To identify the cellular source of the TEM7 protein observed in the human tumor extracts, we performed a histologic survey of various tumor types. Immunohistochemistry revealed a vessel-like pattern of TEM7 staining in colon, esophagus, lung, and bladder cancers, whereas staining was absent or barely detectable in the normal control tissues (Fig. 2A ; data not shown). To determine whether the endothelial cells were responsible for the vessel-like patterns of staining, we performed colocalization studies with an antibody to the pan-endothelial marker von Willebrand factor. As shown in Fig. 2B , TEM7 immunofluorescence colocalized with von Willebrand factor in tumor endothelium. Additional studies using transmission electron microscopy revealed that TEM7-M was predominantly expressed at the tight junctions between endothelial cells, although some expression at the luminal surface was also detected (Fig. 2C) .

View larger version (97K): [in this window] [in a new window]   Fig. 2. TEM7 expression in tumor vessels. A, Immunohistochemical staining of TEM7-M in normal and tumor tissues of the colon, lung, and esophagus. IM193 antibody was used to detect TEM7-M, and an anti-von Willebrand factor (vWF) antibody was used as a control for vessel staining. After the addition of a horseradish peroxidase-labeled tertiary antibody, horseradish peroxidase was visualized with 3,3'-diaminobenzidine (brown). Bar = 50 µm. B, immunofluorescence staining of TEM7 in colon cancer. Colocalization of TEM7-M staining with the pan-endothelial marker von Willebrand factor demonstrates that the endothelial cells are the predominant cell type responsible for the vessel staining observed by immunohistochemistry. Bar = 50 µm. C, TEM7 staining by transmission electron microscopy. Using IM568 antibody to detect TEM7-M, staining was observed predominantly in the tight junctions between endothelial cells (EC), but a lower level of expression on the luminal surface of tumor endothelium was also observed (arrowheads). Bar = 200 nm.

Identification of a Tumor Endothelial Marker 7/7R-Binding Protein.
To identify TEM7-binding partners, we constructed a fusion protein in which the extracellular domain of TEM7 was joined to AP (AP-T7; Fig. 1A ). Because TEM7R mRNA has been shown to be highly expressed in tumor endothelium, we also fused its extracellular region to AP (AP-T7R). The AP-TEM vectors were transfected into mammalian 293 cells, and the secreted fusion proteins used to probe various tissue extracts using a modified Western blotting assay. Protein extracts were resolved on denaturing gels, transferred to a membrane, and blotted using AP-T7/T7R fusion proteins. When various tissue extracts were tested in this assay using AP alone, no binding was detected. However, when probed with either AP-T7 or AP-T7R, a prominent doublet of M_r 80,000 and 85,000 was observed in every mouse and human tissue examined. Brain tissue seemed to be unique in that it contained a M_r 75,000 product in addition to the M_r 80,000/85,000 doublet (Fig. 3A) . We chose to use mouse brain for biochemical purification because the products recognized by the AP-T7 and AP-T7R fusion proteins were particularly abundant in this tissue. Although similar patterns were observed with both AP-T7 and AP-T7R probes, the latter yielded slightly stronger signals and was therefore used for subsequent screening assays.

View larger version (41K): [in this window] [in a new window]   Fig. 3. TEM7 and TEM7R bind cortactin. A, the T7R-AP binding assay (BA) used to identify TEM7/7R interacting proteins. Note that three bands of approximately Mr 75,000, 80,000, and 85,000 were observed in crude extracts prepared from mouse brain tissue (left panel). Brain extracts were immunoprecipitated with either nonspecific IgG control antibody (IP-IgG) or an anticortactin monoclonal antibody (IP-Cor). Immunoprecipitated proteins the expected sizes of cortactin were readily detected when probed with AP-TEM7R fusion protein (right panel). B. TEM7 binds cortactin. Precipitated proteins were detected by immunoblotting with either anti-TEM7 (-T7) or anticortactin (-Cor) antibodies. Extracts of 293 cells transfected with TEM7-M (293/T7) were immunoprecipitated with either nonspecific IgG antibody (IP-IgG) or anti-TEM7 monoclonal antibody (IP-T7; left panel). Note that endogenous cortactin in 293/T7 cells coprecipitates with TEM7 and migrates as a doublet of approximately Mr 80,000 and 85,000. Endogenous TEM7 and cortactin interact in tumor extracts (right panel). Cortactin and TEM7 were both found to be expressed endogenously in extracts derived from rabbit tumor tissue (tumor lysate). The Mr 85,000 TEM7 product appeared in cortactin (IP-Cor) but not control (IP-IgG) immunoprecipitates of these extracts. C, cortactin deletion constructs used to map the TEM7/7R-binding domain. The cortactin repeat region (gray) and the SH3 domain (black) flank the plexin-like domain (white) that binds TEM7/T7R. Expression of each deletion construct was verified by immunoblotting with anti-myc antibodies. Binding of TEM7R to each of the cortactin deletions was measured using AP-T7R as a probe in the AP-binding assay. D. Thirteen amino acid peptides spanning the region of cortactin responsible for binding TEM7/7R were spotted onto a membrane and probed with AP-TEM7R. The three peptides that gave the strongest signal are shown along with their minimal consensus (red).

To verify the interaction between endogenous cortactin and TEM7/7R, we immunoprecipitated cortactin from mouse brain extracts using anticortactin monoclonal antibody and then probed the blot using the AP-TEM7R fusion protein. As shown in Fig. 3A , AP-TEM7R binds specifically to proteins the expected size of cortactin. To determine whether TEM7, like TEM7R, could also bind cortactin, the IM193 monoclonal antibody was used to immunoprecipitate TEM7 from 293 cells expressing exogenous TEM7. A strong band of the expected size for endogenous cortactin was observed in these immunoprecipitates (Fig. 3B) . Importantly, endogenous TEM7 also coprecipitated with cortactin in lysates derived from rabbit tumor tissues, although only the M_r 85,000 product of the triplet was observed (Fig. 3B) . Thus, cortactin seems to bind both TEM7 and TEM7R.

To map the region of cortactin responsible for binding TEM7 and TEM7R, we generated a series of myc-tagged deletion constructs (Fig. 3C) . After exogenous expression in 293 cells, each of the cortactin deletions could be readily detected using an anti-myc antibody (Fig. 3C) . Probing the same deletion-containing extracts with the TEM7R-AP fusion protein revealed that the minimal sequence required for binding was in the plexin-like domain of cortactin immediately adjacent to the SH3 domain. Because the minimal region required for binding was still more than 100 amino acids, we generated a series of overlapping 20-amino acid peptides encompassing this entire region. When each of these peptides were spotted onto nitrocellulose and blotted with the AP-TEM7R fusion protein, a minimal nine-amino acid consensus sequence that seemed to be critical for binding was identified (Fig. 3D) .

	Discussion

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Selective delivery of agents to tumor endothelium is a major goal of current anti-angiogenic treatment strategies for cancer. An ideal marker for such selective targeting would be highly expressed in tumor endothelium but absent or exceedingly rare in all nontumor endothelium of adults. To date, few, if any, markers have been identified that meet such strict criteria. TEM7 is of particular interest in this regard, because it was the most abundant novel transcript identified in an unbiased screen for transcripts differentially expressed in tumor endothelial cells and it was predicted to reside at the cell surface. In the current work, we report the development of TEM7 antibodies and demonstrate that the TEM7 protein, like its mRNA, is overexpressed in human tumor endothelium. We also demonstrate that TEM7-M is glycosylated and present at the cell surface, although most of it seems to lie in the adhesive junctions between endothelial cells. Whether TEM7/TEM7R can be targeted in vivo remains to be shown.

In an attempt to identify binding partners for TEM7, we adopted an affinity purification approach that allowed us to identify cortactin as a protein capable of binding both TEM7 and TEM7R. The minimal region of cortactin required for TEM7 binding was a nine-amino acid sequence located immediately adjacent to the SH3 domain. It is conceivable that this nine-amino acid sequence will allow construction of small molecular weight compounds (peptides or analogs) that can target tumor endothelium for diagnostic or therapeutic purposes.

The identification of a putative intracellular TEM7 variant suggests that a physiologic interaction between TEM7 and cortactin could occur intracellularly. In support of this view, TEM7-I contains a nidogen-like domain that may be involved in mediating protein-protein interactions (9) , and both cortactin and TEM7-I are expressed in tumor endothelium. Regardless of whether cortactin is a physiologic ligand of TEM7 and TEM7R, its nine-amino acid binding domain may be a useful tool for directing agents to tumor endothelium.

The identification of splice variants of TEM7 that are likely to be secreted is also of interest. For example, there is an urgent need for serum markers that can directly measure the response to anti-angiogenic therapies. It will therefore be interesting to develop enzyme-linked immunosorbent assays for detection of circulating TEM7-S1 and TEM7-S2 proteins in patients with cancer and other diseases involving abnormal angiogenesis. TEM7-S1 and TEM7-S2 levels could provide useful surrogate markers of angiogenesis but will require additional studies and the development of monoclonal antibodies that target the extracellular domain of TEM7-S.

	ACKNOWLEDGMENTS

 
We thank Leslie Meszler (Johns Hopkins Oncology Cell Imaging Facility) and Michael Delannoy (Johns Hopkins University School of Medicine Microscope Facility) for expert help with the microscopic imaging.

	FOOTNOTES

 
Grant support: National Cancer Institute, Department of Health and Human Services (B. St. Croix), the Miracle Foundation (B. Vogelstein), Genzyme Molecular Oncology (Genzyme; K. Kinzler), and National Institutes of Health grant CA57345 (K. Kinzler).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. Nanda and P. Buckhaults contributed equally to this work. A. Nanda is a student in the Medical Scientist Training Program and the Program in Human Genetics. Under a licensing agreement between the Johns Hopkins University and Genzyme, technologies related to serial analysis of gene expression and the TEMs were licensed to Genzyme for commercial purposes, and B. Vogelstein, K. Kinzler, and B. St. Croix are entitled to a share of the royalties received by the university from the sales of the licensed technologies. The serial analysis of gene expression technology is freely available to academia for research purposes. K. Kinzler is a consultant to Genzyme. The university and researchers (B. Vogelstein and K. Kinzler) own Genzyme stock, which is subject to certain restrictions under university policy. The terms of these arrangements are being managed by the university in accordance with its conflict of interest policies.

Requests for reprints: Brad St. Croix, Tumor Angiogenesis Section, Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, MD 21702. E-mail: stcroix{at}ncifcrf.gov

Received 7/30/04. Revised 9/27/04. Accepted 10/11/04.

	REFERENCES

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