Desmoglein-1, differentiation, and

Christoph M. Hammers, John R. Stanley

J Clin Invest. 2013;123(4):1419-1422. https://doi.org/10.1172/JCI69071.

Commentary

Desmoglein-1 (DSG1), a desmosomal , maintains the structure of through its adhesive function. However, heterozygous mutations in DSG1 in humans result in abnormal differentiation, as does downregulation of DSG1 in human skin organ culture, suggesting that it may have important signaling functions. In this issue of the JCI, Harmon et al. elucidate how the binding of the DSG1 cytoplasmic tail to the scaffolding protein Erbin decreases signaling through the Ras-Raf pathway to promote stratification and differentiation of in the epidermis.

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Acknowledgments 5. Naik SU, et al. Pharmacological activation of liver 14. Martel C, et al. Lymphatic vasculature mediates X receptors promotes reverse cholesterol transport macrophage reverse cholesterol transport in mice. The Fernández-Hernando Lab is supported in vivo. Circulation. 2006;113(1):90–97. J Clin Invest. 2013;123(4):1571–1579. by grants from the NIH (R01HL107953 6. Rayner KJ, et al. MiR-33 contributes to the regu- 15. Karkkainen MJ, et al. A model for therapy of and R01HL106063). lation of cholesterol homeostasis. Science. 2010; human hereditary . Proc Natl Acad Sci 328(5985):1570–1573. U S A. 2001;98(22):12677–12682. 7. Feig JE, et al. LXR promotes the maximal egress of 16. Reis ED, et al. Dramatic remodeling of advanced Address correspondence to: Carlos Fernán- monocyte-derived cells from mouse aortic plaques atherosclerotic plaques of the apolipoprotein E-de- dez-Hernando, 522 First Avenue, Smilow during atherosclerosis regression. J Clin Invest. 2010; ficient mouse in a novel transplantation model. 703, New York, New York 10016, USA. 120(12):4415–4424. J Vasc Surg. 2001;34(3):541–547. 8. Joseph SB, et al. Synthetic LXR ligand inhibits the 17. Llodra J, Angeli V, Liu J, Trogan E, Fisher EA, Ran- Phone: 212.263.4122; Fax: 212.263.4129; development of atherosclerosis in mice. Proc Natl dolph GJ. Emigration of monocyte-derived cells E-mail: carlos.fernandez-hernando@ Acad Sci U S A. 2002;99(11):7604–7609. from atherosclerotic lesions characterizes regres- nyumc.org. 9. Rayner KJ, et al. Antagonism of miR-33 in mice pro- sive, but not progressive, plaques. Proc Natl Acad Sci motes reverse cholesterol transport and regression of U S A. 2004;101(32):11779–11784. atherosclerosis. J Clin Invest. 2011;121(7):2921–2931. 18. Potteaux S, et al. Suppressed monocyte recruit- 1. Castelli WP, et al. HDL cholesterol and other lip- 10. Verschuren L, et al. LXR agonist suppresses ath- ment drives macrophage removal from atheroscle- ids in coronary heart disease. The cooperative erosclerotic lesion growth and promotes lesion rotic plaques of Apoe–/– mice during disease regres- lipoprotein phenotyping study. Circulation. 1977; regression in apoE*3Leiden mice: time course and sion. J Clin Invest. 2011;121(5):2025–2036. 55(5):767–772. mechanisms. J Lipid Res. 2009;50(2):301–311. 19. Iqbal J, Hussain MM. Intestinal lipid absorption. Am 2. Khera AV, et al. Cholesterol efflux capacity, 11. Boden WE, et al. Niacin in patients with low HDL J Physiol Endocrinol Metab. 2009;296(6):E1183–E1194. high-density lipoprotein function, and atheroscle- cholesterol levels receiving intensive statin therapy. 20. Harvey NL, et al. Lymphatic vascular defects pro- rosis. N Engl J Med. 2011;364(2):127–135. N Engl J Med. 2011;365(24):2255–2267. moted by Prox1 haploinsufficiency cause adult-on- 3. Rader DJ, Alexander ET, Weibel GL, Billheimer J, 12. Schwartz GG, et al. Rationale and design of the set obesity. Nat Genet. 2005;37(10):1072–1081. Rothblat GH. The role of reverse cholesterol trans- dal-OUTCOMES trial: efficacy and safety of dalce- 21. Nanjee MN, Cooke CJ, Wong JS, Hamilton RL, port in animals and humans and relationship to ath- trapib in patients with recent acute coronary syn- Olszewski WL, Miller NE. Composition and ultra- erosclerosis. J Lipid Res. 2009;50(suppl):S189–S194. drome. Am Heart J. 2009;158(6):896–901 e893. structure of size subclasses of normal human 4. Wang X, et al. Macrophage ABCA1 and ABCG1, but 13. Voight BF, et al. Plasma HDL cholesterol and risk peripheral lymph lipoproteins: quantification of not SR-BI, promote macrophage reverse cholesterol of myocardial infarction: a mendelian randomisa- cholesterol uptake by HDL in tissue fluids. J Lipid transport in vivo. J Clin Invest. 2007;117(8):2216–2224. tion study. Lancet. 2012;380(9841):572–580. Res. 2001;42(4):639–648. Desmoglein-1, differentiation, and disease Christoph M. Hammers and John R. Stanley

Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Desmoglein-1 (DSG1), a desmosomal protein, maintains the structure of was found in all -possessing tis- epidermis through its adhesive function. However, heterozygous mutations sues, including nonepithelial myocardium, in DSG1 in humans result in abnormal differentiation, as does downreg- expression of the family members DSG1 ulation of DSG1 in human skin organ culture, suggesting that it may have and DSG3 was mainly limited to the epider- important signaling functions. In this issue of the JCI, Harmon et al. eluci- mis and mucosa (2). date how the binding of the DSG1 cytoplasmic tail to the scaffolding protein Molecular cloning also indicated that Erbin decreases signaling through the Ras-Raf pathway to promote stratifi- the desmoglein cytoplasmic tail had one cation and differentiation of keratinocytes in the epidermis. segment with homology to that of classical . This segment binds the arma- Introduction tural molecules. In this Commentary, we dillo protein , an interaction In an era of research budget cuts and tight- will illustrate this observation by tracing thought to be important in desmosome ened paylines, the significance and trans- recent studies centered on desmoglein-1 assembly through cobinding of plakoglo- lational potential of grant proposals are (DSG1) and its associated . bin (and perhaps other armadillo ) sometimes equated with the prevalence to , the major desmosome of the disease being studied and/or the The discovery of DSG1 plaque protein (refs. 3–5 and Figure 1). obvious clinical application of the pro- The family of was character- Desmogleins also have unique intracellular posal. However, it is important to remem- ized in the early 1990s through biochemical segments (6), the functions of which have ber that we have learned a tremendous isolation, monoclonal production, now been partially elucidated by Harmon amount about the normal physiology and and then expression cloning, which showed et al. (7) in this issue of the JCI. pathophysiology of organs through the they were in the supergene family of cad- study of rare diseases and through stud- herins. Because desmogleins were present The rare autoimmune blistering ies of the basic of seemingly in the electron lucent center of the desmo- skin disease foliaceus boring (that is, uncommunicative) struc- some, a structure, and were in elucidates the adhesive function the family of adhesion molecules, of DSG1 they were presumed to function in cell-cell At about the same time that desmog- Conflict of interest: The authors have declared that no conflict of interest exists. adhesion (1). In fact, “desmoglein” is com- leins were being characterized, the anti- Citation for this article: J Clin Invest. 2013; posed of the Greek words “desmos” for “tie” gen of a rare autoimmune blistering skin 123(4):1419–1422. doi:10.1172/JCI69071. and “glein” for “glue-like.” Whereas DSG2 disease, pemphigus foliaceus (PF), was

The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 4 April 2013 1419 commentaries

Figure 1 structure, adhesion, and sig- naling modulated by Erbin and/or DSG1. The ErbB family includes four receptors with tyrosine kinase activity (ErbB1 [also known as EGFR], ErbB2, ErbB3, ErbB4), which form homodimers and/or heterodimers upon ligand binding. Autophosphorylation leads to direct activation of the PI3K or phospholi- pase C-γ pathway (not shown). Via adaptor proteins (e.g., Grb2), the guanyl nucleotide exchange factor son of sevenless (SOS) allows exchange of GDP for GTP on Ras and thus activation of this small GTPase. The scaffolding protein SHOC2 accelerates formation of Ras/Raf complexes and leads, in absence of DSG1 and/or Erbin, to acti- vation of the Raf/MEK/ERK pathway, which inhibits differentiation of keratinocytes. In the presence of DSG1, the scaffolding protein Erbin skews the fate of keratinocytes toward differentiation by binding SHOC2 and inhib- iting formation of Ras/Raf complexes. Erbin binds to the cytoplasmic tail of DSG1 but not to the intracellular cadherin-like sequence (ICS) that binds plakoglobin (Pg), a protein important for desmosome integrity and func- tion. ET from Staphylococcus aureus cleaves DSG1 between extracellular domain 3 (EC3) and EC4. Anti-DSG1 (αDSG1 ab) mediating PF predominantly target N-ter- minal extracellular domains, causing loss of cell-cell adhesion. DP, desmoplakin; EC1– EC4, extracellular domains of DSG1; EA, extracellular anchor (also known as EC5); TM, transmembrane domain; IA, intracellular anchor; PL, proline-rich linker; RUD, repeat unit domains; TD, terminal domain; DSC1, -1.

described as a of about the dren (8). These infectious diseases, known However, throughout this work, which same molecular weight as that of DSG1. to be associated with the production of has been so important for understanding Immunochemical studies indicated that a staphylococcal toxin called exfoliative the structure of the epidermis and diseases the autoantigen was indeed DSG1 (8), and toxin (ET), are characterized by blisters that perturb it, DSG1 has been considered further studies have shown that the auto- in the superficial epidermis due to loss of basically as a structural adhesion molecule. , even as monovalent cloned cell-cell adhesion with identical histology single chain variable fragments from to that of PF. The crystal structure of ET DSG1 is more than a structural patients (9), can cause loss of cell adhesion suggested that it was a serine protease with molecule — hints from cell biologic in the superficial epidermis where DSG1 a specific receptor; however, that receptor studies of desmogleins is highly expressed. Thus, the study of a remained unknown for many years. The and a rare disease rare dermatologic disease demonstrated data that indicated that anti-DSG1 anti- Perturbations of desmoglein expression in the critical function of DSG1 in cell adhe- bodies cause loss of cell adhesion in PF sug- the epidermis have been shown to do more sion and maintenance of the structure of gested that the specific receptor for ET was than affect cell adhesion. For example, the normal epidermis. likely DSG1 as well, because loss of func- misexpression of DSG3 in the superficial The work on PF and DSG1 provided tion of DSG1 either by antibody binding epidermis not only allows increased adhe- insight into the pathophysiology of a much or enzymatic cleavage resulted in the same sion in those layers (10) but also affects more common disease, bullous impetigo, phenotypic blister. Indeed, that conclusion differentiation, resulting in an abnormal as well as the related disease staphylococ- was confirmed when it was shown that ET stratum corneum, resembling that of cal scalded skin syndrome, both of which cleaved DSG1 (but not DSG3 or E-cad- mucous membranes, that allows increased are significant causes of morbidity in chil- herin) at one specific peptide bond (8). transepidermal water loss (11). Similarly,

1420 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 4 April 2013 commentaries misexpression of DSG2 in epidermis restored differentiation. The authors then Finally, may be important results in increased proliferation in the hypothesized that peripheral localization in tumor suppression, and modulation of suprabasilar layers (12). of Erbin may bring it into contact with signaling through desmosomal cadherin DSG1 is uniquely positioned, just above SHOC2, inhibiting the ability of SHOC2 binding proteins, including Erbin, may the basal layer, to be a candidate to direct to form Ras-Raf complexes upstream of be critical to that function (16, 21). For stratification and/or differentiation of ERK (17). Indeed, Harmon et al. found example, the most common malignant epidermis. A recent elegant study in which that only when DSG1 brings Erbin to the neoplasm of skin, basal cell carcinoma, DSG1 was downregulated in skin organ does it interfere with the shows diminished cell surface DSG1 and culture confirmed the importance of binding of Ras to the SHOC2 protein (7). cytoplasmic localization of Erbin (16, 22), DSG1 for directing those functions (13). To explain these findings, Harmon et al. suggesting that the mechanism proposed In this study, DSG1 knockdown activated proposed an elegant model in which, in by Harmon et al. might be active in main- EGF receptor/ErbB2 (EGFR/ErbB2) sig- the presence of DSG1, SHOC2-Erbin com- taining its lack of differentiation. naling. Specific blockade of activation of plexes form at the expense of SHOC2-Ras These and other interesting issues await ERK1/2, downstream of one of the arms of complexes, thus decreasing ERK activation further study. However, it is very clear this activated receptor, restored epidermal and driving differentiation of keratinocytes that desmosomes and desmogleins are differentiation in DSG1-knockdown cells. (Figure 1). With a very creative approach, not only structural regulators of adhe- DSG1-mediated adhesion was not needed the authors then confirmed that their sion in the epidermis but also are key for this effect and neither was plakoglobin model was relevant to the pathophysiol- regulators of that in binding, but localization at the cell mem- ogy of SPPK. Proximity ligation analysis turn affect differentiation and epidermal brane was necessary. demonstrated that patients with SPPK homeostasis as well as being involved in Consideration of the rare genetic dis- (with genetically decreased DSG1 expres- carcinogenesis. These insights were origi- ease striate sion), in contrast to healthy controls, have nally gained from studies of rare diseases (SPPK; OMIM 148700), which is caused increased SHOC2-Ras complexes, com- and from creative cell biological studies by a heterozygous mutation in the DSG1 pared with SHOC2-Erbin complexes. As that looked beyond the structural role gene (14), lends further support to the hypothesized, this leads to increased pERK of desmosomal proteins. Applying these notion that DSG1 affects not only adhe- levels and decreased expression of differen- new insights will suggest promising new sion but also differentiation. Patients with tiation markers (7). targets for therapy of rare and more com- SPPK show a striate (or more diffuse) kera- If SPPK is due to elevated Ras activity, one mon skin diseases. toderma of the palms and soles. Histolog- would predict that other genetic diseases in ical and ultrastructural analyses reveal not which Ras pathways are activated should Acknowledgments only widening of cell-cell spaces and small show similar clinical findings on the palms This work was supported by a grant from desmosomes (consistent with decreased and soles. In fact, they do: in the group of the NIAMS, NIH (R01-AR052672) to J.R. adhesion) but also marked thickening of rare genetic diseases of the Ras/MEK/ERK Stanley and by a research scholarship from the palmoplantar epidermis with abnor- pathway (sometimes called RASopathies), the DFG (HA6736/1-1) to C.M. Hammers. mal differentiation (15). palmar and plantar keratoderma is found in about 76% of patients with Costello syn- Address correspondence to: John R. Stan- Molecular mechanisms drome and in about 36% of patients with ley, Department of Dermatology, Uni- of DSG1-mediated modulation cardio-facio-cutaneous syndrome (which versity of Pennsylvania, 415 Curie Blvd., of keratinocyte differentiation overlaps genetically and phenotypically 211 CRB, Philadelphia, Pennsylvania In order to address the molecular mech- with Noonan’s syndrome) (18, 19). 19104, USA. Phone: 215.898.3240; Fax: anisms of how DSG1 might modu- 215.573.2033; E-mail: [email protected]. late differentiation, Harmon et al. (7) Loose ends upenn.edu. hypothesized that heretofore uncharac- Although Harmon et al. present a beau- terized binding partners of the desmog- tiful story that explains some aspects of 1. Steinberg MS, Shida H, Giudice GJ, Shida M, Patel lein-unique domains were involved. Using epidermal homeostasis and the molec- NH, Blaschuk OW. On the molecular organization, diversity and functions of desmosomal proteins. yeast two-hybrid screening, they found ular pathophysiology of SPPK, there are Ciba Found Symp. 1987;125:3–25. that the cytoplasmic domains of DSG1 many questions that remain. In addition 2. Schafer S, Koch PJ, Franke WW. Identification of carboxy-terminal to the plakoglobin-bind- to DSG1, there are several desmogleins (as the ubiquitous human desmoglein, Dsg2, and the expression catalogue of a subfamily of desmosomal ing domain bound Erbin, a molecular well as other cadherin superfamily mem- cadherins. Exp Cell Res. 1994;211(2):391–399. scaffolding protein known to redistribute bers) expressed in epidermis and mucous 3. Bornslaeger EA, et al. 1 interferes with to the cell surface of keratinocytes during membranes at various levels. How do the plakoglobin binding to desmoplakin, yet together differentiation (16). Harmon et al. con- tails of these molecules regulate epidermal with plakoglobin promotes clustering of desmoso- mal plaque complexes at cell-cell borders. J Cell Sci. firmed this redistribution and showed homeostasis? What are their binding part- 2001;114(pt 4):727–738. that Erbin colocalized with DSG1 in the ners? How do other binding partners of 4. Korman NJ, Eyre RW, Klaus-Kovtun V, Stanley JR. differentiating epidermis. Genetic down- DSG1, for example, plakoglobin and other Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus regulation of Erbin decreased expression and armadillo proteins, affect sig- foliaceus and . N Engl J Med. of various keratinocyte differentiation naling and differentiation? In addition, it 1989;321(10):631–635. markers in organ culture and at the same is unclear how mutations in or in 5. Kowalczyk AP, et al. The head domain of plako­ philin-1 binds to desmoplakin and enhances its time increased pERK; conversely, pharma- other desmosomal components, such as recruitment to desmosomes. Implications for cutane- cologic blockade of ERK phosphorylation desmoplakin, cause SPPK (20). ous disease. J Biol Chem. 1999;274(26):18145–18148.

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6. Koch PJ, Franke WW. Desmosomal cadherins: 12. Brennan D, et al. Suprabasal Dsg2 expression in 17. Matsunaga-Udagawa R, et al. The another growing multigene family of adhesion transgenic mouse skin confers a hyperproliferative Shoc2/SUR-8 accelerates the interaction of Ras and molecules. Curr Opin Cell Biol. 1994;6(5):682–687. and apoptosis-resistant phenotype to keratino- Raf. J Biol Chem. 2010;285(10):7818–7826. 7. Harmon RM, et al. Desmoglein-1/Erbin inter- cytes. J Cell Sci. 2007;120(pt 5):758–771. 18. Siegel DH, Mann JA, Krol AL, Rauen KA. Derma- action suppresses ERK activation to support 13. Getsios S, et al. Desmoglein 1-dependent suppres- tological phenotype in Costello syndrome: conse- epidermal differentiation. J Clin Invest. 2013; sion of EGFR signaling promotes epidermal dif- quences of Ras dysregulation in development. Br J 123(4):1556–1570. ferentiation and . J Cell Biol. 2009; Dermatol. 2012;166(3):601–607. 8. Stanley JR, Amagai M. Pemphigus, bullous impe- 185(7):1243–1258. 19. Nava C, et al. Cardio-facio-cutaneous and Noonan tigo, and staphylococcal scalded skin syndrome. 14. Rickman L, et al. N-terminal deletion in a desmoso- syndromes due to mutations in the RAS/MAPK N Engl J Med. 2006;355(17):1800–1810. mal cadherin causes the autosomal dominant skin signalling pathway: genotype-phenotype relation- 9. Ishii K, Lin C, Siegel DL, Stanley JR. Isolation disease striate palmoplantar keratoderma. Hum ships and overlap with Costello syndrome. J Med of pathogenic monoclonal anti-desmoglein 1 Mol Genet. 1999;8(6):971–976. Genet. 2007;44(12):763–771. human antibodies by phage display of pemphigus 15. Wan H, et al. Striate palmoplantar keratoderma 20. McGrath JA. Inherited disorders of desmosomes. foliaceus autoantibodies. J Invest Dermatol. 2008; arising from desmoplakin and desmoglein 1 muta- Australas J Dermatol. 2005;46(4):221–229. 128(4):939–948. tions is associated with contrasting perturbations 21. Dusek RL, Attardi LD. Desmosomes: new perpetra- 10. Wu H, et al. Protection of neonates against pemphi- of desmosomes and the filament network. tors in tumour suppression. Nat Rev Cancer. 2011; gus foliaceus by desmoglein 3. N Engl J Med. 2000; Br J Dermatol. 2004;150(5):878–891. 11(5):317–323. 343(1):31–35. 16. Lebeau S, et al. Comparative analysis of the expres- 22. Tada H, Hatoko M, Tanaka A, Kuwahara M, 11. Elias PM, et al. Desmoglein isoform distribution sion of ERBIN and Erb-B2 in normal human skin Muramatsu T. Expression of desmoglein I and pla- affects stratum corneum structure and function. and cutaneous carcinomas. Br J Dermatol. 2005; koglobin in skin carcinomas. J Cutan Pathol. 2000; J Cell Biol. 2001;153(2):243–249. 152(6):1248–1255. 27(1):24–29. WNT signaling in stem cell differentiation and tumor formation Hong Ouyang,1,2 Yehong Zhuo,3 and Kang Zhang1,2,3,4

1Molecular Medicine Research Center and Department of Ophthalmology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, People’s Republic of China. 2Department of Ophthalmology and Shiley Eye Center and Institute for Genomic Medicine, UCSD, San Diego, California, USA. 3State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People’s Republic of China. 4Veterans Administration Healthcare System, San Diego, California, USA.

Embryonic stem cells (ESCs) hold great therapeutic promise for the regener- degeneration improved visual acuity, with ation of functional cell types and clinical applications. However, tumorigenic no signs of hyperproliferation or tumorige- potential of stem cells in a transplanted host remains a major obstacle. In nicity after 4 months (4). These examples this issue of the JCI, Cui and colleagues identified TCF7-mediated canonical illustrate purity, stability, and proper local- WNT signaling as a critical determinant of both the tumorigenicity and ther- ization of transplanted cells in vivo and apeutic function of ESC-derived retinal progenitor cells (ESC-RPCs). Their prompted the development of numerous findings suggested that addressing key extracellular signaling and related differentiation protocols. Still, the risk intrinsic factors will be essential for the successful use of ESC-derived pro- of tumor formation remains a barrier, genitor transplantation. because there are no parameters to quan- tify safety factors and because the appro- Photoreceptor degeneration underlies side effects. It has been previously demon- priate stage of differentiation at which major causes of blindness, including macu- strated that ESCs or ESC-derived progen- ESC-derived progenitors should be used lar degeneration and retinitis pigmentosa, itors spontaneously form tumors upon has not been well evaluated. In this issue, affecting tens of millions of people world- transplantation in vivo, even when the Cui and colleagues attempted to address wide. In theory, vision of affected patients cells are predifferentiated or presorted (2). the above concerns by identifying major may improve if the diseased cells (rods and Despite this issue, successful cell replace- extracellular signaling and intrinsic factors cones) are replaced with new, healthy cells ment of human ESC–derived (hESC-de- controlling tumorigenicity and therapeutic that form appropriate connections with the rived) retinal cells for vision restoration in potential of ESC-derived retinal progenitor host retina. Embryonic stem cells (ESCs) animal models of photoreceptor degener- cells (ESC-RPCs) (5). possess unlimited self-renewal capabilities ation has been reported recently. Photore- and the ability to differentiate into any ceptor precursors derived from hESCs have ESC tumorigenic potential adult cell type (1). These unique features been shown to migrate into Crx-deficient The best proof of pluripotency of ESCs is make ESC-based therapy appealing for the mouse retina following intraocular injec- their ability to form teratomas in which treatment of various degenerative disor- tion, express appropriate markers for both the stem cells differentiate to various tis- ders but may also cause unwanted serious rod and cone photoreceptors, and subse- sue types of the embryo in a disordered quently restore some light responses (3). In fashion following transplantation into another study, subretinal transplantation immunosuppressed mice. Teratomas Conflict of interest: The authors have declared that no conflict of interest exists. of hESC-derived retinal pigment epithe- usually contain all three germ layers and Citation for this article: J Clin Invest. 2013; lium in patients with Stargardt’s macular have typical tumor characteristics (6). 123(4):1422–1424. doi:10.1172/JCI69324. dystrophy and dry age-related macular In other words, ESCs are naturally tum-

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