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1.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-34281202

RESUMEN

Autologous chondrocyte implantation (ACI) is a cell therapy for the treatment of focal cartilage defects. The ACI product that is currently approved for use in the European Union (EU) consists of spheroids of autologous matrix-associated chondrocytes. These spheroids are spherical aggregates of ex vivo expanded human autologous chondrocytes and their self-synthesized extracellular matrix. The aim is to provide an overview of the preclinical and nonclinical studies that have been performed to ensure reproducible quality, safety, and efficacy of the cell therapy, and to evaluate the clinical data on ACI with spheroids. A systematic review was performed to include all English publications on self-aggregated spheroids of chondrocytes cultured in autologous serum without other supplements. A total of 20 publications were included, 7 pre- and nonclinical and 13 clinical research publications. The pre- and nonclinical research publications describe the development from concept to in vivo efficacy and quality- and safety-related aspects such as biodistribution, tumorigenicity, genetic stability, and potency. The evaluation of clinical research shows short- to mid-term safety and efficacy for the ACI with spheroid-based treatment of cartilage defects in both randomized clinical trials with selected patients, as well as in routine treatment providing real-world data in more complex patients.


Asunto(s)
Condrocitos/trasplante , Animales , Enfermedades de los Cartílagos/cirugía , Enfermedades de los Cartílagos/terapia , Cartílago Articular/cirugía , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Condrocitos/citología , Condrocitos/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Traumatismos de la Rodilla/cirugía , Traumatismos de la Rodilla/terapia , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Procedimientos Ortopédicos/métodos , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patología , Osteoartritis de la Rodilla/terapia , Esferoides Celulares , Trasplante Autólogo
2.
J Am Soc Nephrol ; 27(9): 2658-69, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-26823548

RESUMEN

NF-κB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type-specific functions of NF-κB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-κB signaling in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. NF-κB reporter activity and nuclear localization of phosphorylated NF-κB subunit p65 analyses in mice revealed that IRI induced widespread NF-κB activation in renal tubular epithelia and in interstitial cells that peaked 2-3 days after injury. To genetically antagonize tubular epithelial NF-κB activity, we generated mice expressing the human NF-κB super-repressor IκBαΔN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-κB-dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IκBαΔN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-κB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response.


Asunto(s)
Lesión Renal Aguda/etiología , FN-kappa B/fisiología , Animales , Apoptosis , Modelos Animales de Enfermedad , Túbulos Renales , Masculino , Ratones , Daño por Reperfusión , Transducción de Señal , Urotelio
3.
J Transl Med ; 14(1): 317, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27846904

RESUMEN

BACKGROUND: Cell-based therapies such as autologous chondrocyte implantation are promising therapeutic approaches to treat cartilage defects to prevent further cartilage degeneration. To assure consistent quality of cell-based therapeutics, it is important to be able to predict the biological activity of such products. This requires the development of a potency assay, which assesses a characteristic of the cell transplant before implantation that can predict its cartilage regeneration capacity after implantation. In this study, an ex vivo human cartilage repair model was developed as quality assessment tool for potency and applied to co.don's chondrosphere product, a matrix-associated autologous chondrocyte implant (chondrocyte spheroids) that is in clinical use in Germany. METHODS: Chondrocyte spheroids were generated from 14 donors, and implanted into a subchondral cartilage defect that was manually generated in human articular cartilage tissue. Implanted spheroids and cartilage tissue were co-cultured ex vivo for 12 weeks to allow regeneration processes to form new tissue within the cartilage defect. Before implantation, spheroid characteristics like glycosaminoglycan production and gene and protein expression of chondrogenic markers were assessed for each donor sample and compared to determine donor-dependent variation. RESULTS: After the co-cultivation, histological analyses showed the formation of repair tissue within the cartilage defect, which varied in amount for the different donors. In the repair tissue, aggrecan protein was expressed and extra-cellular matrix cartilage fibers were present, both indicative for a cartilage hyaline-like character of the repair tissue. The amount of formed repair tissue was used as a read-out for regeneration capacity and was correlated with the spheroid characteristics determined before implantation. A positive correlation was found between high level of aggrecan protein expression in spheroids before implantation and a higher regeneration potential after implantation, reflected by more newly formed repair tissue. CONCLUSION: This demonstrated that aggrecan protein expression levels in spheroids before implantation can potentially be used as surrogate potency assay for the cartilage cell transplant to predict its regenerative capacity after implantation in human patients.


Asunto(s)
Cartílago Articular/patología , Trasplante de Células , Condrocitos/trasplante , Cicatrización de Heridas , Biomarcadores/metabolismo , Técnicas de Cocultivo , Regulación de la Expresión Génica , Glicosaminoglicanos/metabolismo , Humanos , Implantes Experimentales , Modelos Lineales , Modelos Biológicos , Regeneración , Esferoides Celulares/trasplante , Donantes de Tejidos
4.
Curr Biol ; 12(22): 1941-5, 2002 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-12445388

RESUMEN

Wnt signaling functions repeatedly during embryonic development to induce different but specific responses. What molecular mechanisms ensure that Wnt signaling triggers the correct tissue-specific response in different tissues? Early Xenopus development is an ideal model for addressing this fundamental question, since there is a dramatic change in the response to Wnt signaling at the onset of zygotic gene transcription: Wnt signaling components encoded by maternal mRNA establish the dorsal embryonic axis; zygotically expressed Xwnt-8 causes almost the opposite, by promoting ventral and lateral and restricting dorsal mesodermal development. Although Wnt signaling can function through different signal transduction cascades, the same beta-catenin-dependent, canonical Wnt signal transduction pathway mediates Wnt signaling at both stages of Xenopus development. Here we show that, while the function of the transcription factor XTcf-3 is required for early Wnt signaling to establish the dorsal embryonic axis, closely related XLef-1 is required for Wnt signaling to pattern the mesoderm after the onset of zygotic transcription. Our results show for the first time that different transcription factors of the Lef/Tcf family function in different tissues to bring about tissue-specific responses downstream of canonical Wnt signaling.


Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/metabolismo , Morfogénesis/fisiología , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas de Pez Cebra , Animales , Embrión no Mamífero/fisiología , Femenino , Impresión Genómica , Factor de Unión 1 al Potenciador Linfoide , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , ARN Mensajero/genética , Factores de Transcripción TCF , Factor de Transcripción 3 , Proteína 1 Similar al Factor de Transcripción 7 , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis , Cigoto/fisiología
5.
Int J Dev Biol ; 61(1-2): 29-34, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28287244

RESUMEN

In Xenopus gastrula stage embryos, four isoforms of Tcf1 (B, C, D and E) are present with high amino acid sequence conservation compared to fish, mice and human. We studied possible functional differences between these Tcf1 isoforms during early Xenopus development. After overexpression of single Tcf1 isoforms, two distinct phenotypes were observed. Overexpression of the B or D isoforms of Tcf1, which both lack a C-clamp, enhances early canonical Wnt signaling and induces ectopic dorsal mesoderm at the expense of ventrolateral mesoderm prior to gastrulation, causing severe antero-dorzalization of embryos. Overexpression of the E-isoform, which contains a complete C-clamp, does not induce ectopic dorsal mesoderm, but rather leads to severe caudal truncation. Overexpression of the C-isoform, which contains a partial C-clamp, induces a similar phenotype. Mutation of a single amino acid in the C-clamp, known to produce a hypomorphic mutant in D. melanogaster, led to a gain of function in inducing ectopic organizer tissue, as observed after overexpression of the B or D isoforms of Tcf1. Depletion of the C-clamp exon from the zygotic mRNA pool, by injection of a morpholino oligo that targets the splice acceptor site of the exon containing the C-clamp, caused a severe shortening of the AP-axis. Furthermore, embryos showed poor development of the CNS, paraxial mesoderm and primary blood vessels. In situ hybridization analysis showed that Lef1 expression was downregulated at the mid-hindbrain boundary, in the otic vesicles and the branchial arches. The results indicate that in post-gastrula stage Xenopus embryos, the E-tail of Tcf1 is required for expression of Lef1 and for blood vessel formation.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Neovascularización Fisiológica/fisiología , Isoformas de Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Animales , Gastrulación/fisiología , Factor Nuclear 1-alfa del Hepatocito/genética , Mesodermo/metabolismo , Isoformas de Proteínas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus/metabolismo , Proteínas de Xenopus/genética
6.
Gene Expr Patterns ; 3(2): 123-6, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12711535

RESUMEN

We report the cloning and expression of Xenopus Tcf-1. The amino acid sequence of Tcf-1 of Xenopus laevis and Xenopus tropicalis is closely related to that of chicken, mouse and man. Thus, the family of Tcf/Lef proteins in the amphibian Xenopus comprises four members as in higher vertebrates. RT-PCR analysis revealed that Tcf-1 RNA encoding a beta-catenin binding isoform is maternally present as well as throughout early development. Different transcripts are expressed by alternative splicing. In cleavage and blastula stage embryos, Tcf-1 RNA is present at high levels in the animal hemisphere. During gastrulation Tcf-1 is differentially expressed with high levels in the animal cap and most of the marginal zone except for a narrow domain around the blastopore. At neurula stages expression is predominant in the neural plate. At tailbud stages expression is localized in specific areas of the brain, in the eyes, the otic vesicle, branchial arches and head mesenchyme, somites, tailbud, pronephros and pronephric duct.


Asunto(s)
Proteínas de Unión al ADN/genética , Embrión no Mamífero/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Factor de Unión 1 al Potenciador Linfoide , Datos de Secuencia Molecular , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 1 de Transcripción de Linfocitos T , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Xenopus , Proteínas de Xenopus , beta Catenina
7.
Int J Dev Biol ; 53(1): 81-9, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19123129

RESUMEN

Tcf/Lef HMG box transcription factors are nuclear effectors of the canonical Wnt signaling pathway, which function in cell fate specification. Lef1 is required for the development of tissues and organs that depend on epithelial mesenchymal interactions. Here, we report the effects of lef1 loss of function on early development in X. tropicalis. Depletion of lef1 affects gene expression already during gastrulation and results in abnormal differentiation of cells derived from ectoderm and mesoderm. At tail bud stages, the epidermis was devoid of ciliated cells and derivatives of the neural crest, e.g. melanocytes and cephalic ganglia were absent. In the Central Nervous System, nerve fibers were absent or underdeveloped. The development of the paraxial mesoderm was affected; intersomitic boundaries were not distinct and development of the hypaxial musculature was impaired. The development of the pronephros and pronephric ducts was disturbed. Most striking was the absence of blood flow in lef1 depleted embryos. Analysis of blood vessel marker genes demonstrated that lef1 is required for the development of the major blood vessels and the heart.


Asunto(s)
Ectodermo/embriología , Ectodermo/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Factores de Transcripción TCF/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Animales , Tipificación del Cuerpo , Diferenciación Celular , Vasos Coronarios , Ectodermo/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Exones/genética , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Mesodermo/citología , Miocardio/metabolismo , Especificidad de Órganos , Fenotipo , Factores de Transcripción TCF/genética , Xenopus/genética
8.
Science ; 312(5775): 921-4, 2006 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-16645052

RESUMEN

Wnt proteins function as morphogens that can form long-range concentration gradients to pattern developing tissues. Here, we show that the retromer, a multiprotein complex involved in intracellular protein trafficking, is required for long-range signaling of the Caenorhabditis elegans Wnt ortholog EGL-20. The retromer functions in EGL-20-producing cells to allow the formation of an EGL-20 gradient along the anteroposterior axis. This function is evolutionarily conserved, because Wnt target gene expression is also impaired in the absence of the retromer complex in vertebrates. These results demonstrate that the ability of Wnt to regulate long-range patterning events is dependent on a critical and conserved function of the retromer complex within Wnt-producing cells.


Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Glicoproteínas/fisiología , Complejos Multiproteicos/fisiología , Transducción de Señal , Proteínas Wnt/fisiología , Animales , Tipificación del Cuerpo , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Expresión Génica , Glicoproteínas/análisis , Glicoproteínas/genética , Humanos , Mutación , Neuronas/citología , Neuronas/fisiología , Interferencia de ARN , Transgenes , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiología , Xenopus
9.
Proc Natl Acad Sci U S A ; 103(14): 5397-402, 2006 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-16567647

RESUMEN

The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporal-specific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. We have recently shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. In the present study, we show that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. We find that PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. Our data establish PR130 as a modulator of the Wnt-signaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit.


Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Línea Celular , Humanos , Hibridación in Situ , Proteína Fosfatasa 2 , Xenopus/embriología
10.
Biochem Biophys Res Commun ; 349(2): 855-62, 2006 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-16950205

RESUMEN

Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays > 60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.


Asunto(s)
Conexinas/química , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Conexina 26 , Conexina 30 , Conexinas/genética , Conexinas/metabolismo , Uniones Comunicantes , Regulación del Desarrollo de la Expresión Génica , Humanos , Mesodermo/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/metabolismo , Proteína beta1 de Unión Comunicante
11.
Genes Dev ; 19(3): 376-86, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15687260

RESUMEN

The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required for early segmentation. In addition, Naked cuticle is proposed to function as a switch, acting to restrict classical Wnt signaling and to activate a second Wnt signaling pathway that controls planar cell polarity during gastrulation movements in vertebrates. Little is known about the biochemical function of Naked cuticle or its regulation. Here we report that PR72, a Protein Phosphatase type 2A regulatory subunit of unknown function, interacts both physically and functionally with Naked cuticle. We show that PR72, like Naked cuticle, acts as a negative regulator of the classical Wnt signaling cascade, establishing PR72 as a novel regulator of the Wnt signaling pathway. Our data provide evidence that the inhibitory effect of Naked cuticle on Wnt signaling depends on the presence of PR72, both in mammalian cell culture and in Xenopus embryos. Moreover, PR72 is required during early embryonic development to regulate cell morphogenetic movements during body axis formation.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Clonación Molecular , Proteínas del Citoesqueleto/metabolismo , Proteínas Dishevelled , Embrión no Mamífero/metabolismo , Ojo/embriología , Ojo/metabolismo , Gástrula/metabolismo , Humanos , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2 , Transactivadores/metabolismo , Proteínas Wnt , Xenopus , beta Catenina
12.
Genesis ; 32(4): 259-68, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11948913

RESUMEN

The embryonic kidney is a classic developmental model system for studying inductive tissue interactions that govern organogenesis. We report here that Wnt-6 is expressed in the ureter bud, and that cell lines expressing Wnt-6 induce nephrogenesis in vitro. Wnt-6 cells induce tubules with similar kinetics to spinal cord (SPC) and lead to induced expression of Pax2, Pax8, Sfrp2, and E-cadherin genes, early markers of tubulogenesis. Moreover, Wnt-6 signaling rescues tubulogenesis in mesenchyme separated from Wnt-4 mutant embryos and leads to activation of Wnt-4 transcription. Wnt-6 also induces a secondary axis in early Xenopus embryos. We conclude that Wnt-6 is a candidate for the ureter epithelium-derived signal that leads to activation of kidney tubulogenesis via Wnt-4.


Asunto(s)
Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Túbulos Renales/embriología , Proteínas Proto-Oncogénicas/genética , Uréter/embriología , Animales , Línea Celular , Hibridación in Situ , Ratones , Técnicas de Cultivo de Órganos/métodos , Proteínas Wnt
13.
J Biol Chem ; 279(46): 47520-7, 2004 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-15337757

RESUMEN

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.


Asunto(s)
Escherichia coli/metabolismo , Células Eucariotas/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Embrión no Mamífero , Escherichia coli/genética , Genes Reporteros , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Morfogénesis/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Wnt , Proteína Wnt1 , Proteínas de Xenopus , Xenopus laevis/fisiología
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