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1.
Cell ; 169(1): 161-173.e12, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28340341

RESUMO

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.


Assuntos
Embrião de Mamíferos/citologia , Feto/citologia , Desenvolvimento Humano , Imageamento Tridimensional/métodos , Imuno-Histoquímica/métodos , Microscopia/métodos , Desenvolvimento Embrionário , Humanos , Organogênese , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/crescimento & desenvolvimento
2.
Development ; 150(16)2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37497580

RESUMO

Earlier data on liver development demonstrated that morphogenesis of the bile duct, portal mesenchyme and hepatic artery is interdependent, yet how this interdependency is orchestrated remains unknown. Here, using 2D and 3D imaging, we first describe how portal mesenchymal cells become organised to form hepatic arteries. Next, we examined intercellular signalling active during portal area development and found that axon guidance genes are dynamically expressed in developing bile ducts and portal mesenchyme. Using tissue-specific gene inactivation in mice, we show that the repulsive guidance molecule BMP co-receptor A (RGMA)/neogenin (NEO1) receptor/ligand pair is dispensable for portal area development, but that deficient roundabout 2 (ROBO2)/SLIT2 signalling in the portal mesenchyme causes reduced maturation of the vascular smooth muscle cells that form the tunica media of the hepatic artery. This arterial anomaly does not impact liver function in homeostatic conditions, but is associated with significant tissular damage following partial hepatectomy. In conclusion, our work identifies new players in development of the liver vasculature in health and liver regeneration.


Assuntos
Orientação de Axônios , Artéria Hepática , Animais , Camundongos , Ductos Biliares , Morfogênese , Inativação Gênica
3.
Development ; 143(21): 3969-3981, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803058

RESUMO

Fertility in mammals is controlled by hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH). These neurons differentiate in the olfactory placodes during embryogenesis and migrate from the nose to the hypothalamus before birth. Information regarding this process in humans is sparse. Here, we adapted new tissue-clearing and whole-mount immunohistochemical techniques to entire human embryos/fetuses to meticulously study this system during the first trimester of gestation in the largest series of human fetuses examined to date. Combining these cutting-edge techniques with conventional immunohistochemistry, we provide the first chronological and quantitative analysis of GnRH neuron origins, differentiation and migration, as well as a 3D atlas of their distribution in the fetal brain. We reveal not only that the number of GnRH-immunoreactive neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioral and physiological processes.


Assuntos
Encéfalo/embriologia , Diferenciação Celular , Movimento Celular , Fertilidade/fisiologia , Feto/citologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/fisiologia , Anatomia Artística , Atlas como Assunto , Encéfalo/citologia , Encéfalo/metabolismo , Mapeamento Encefálico/métodos , Embrião de Mamíferos , Desenvolvimento Embrionário/fisiologia , Feminino , Feto/embriologia , Feto/metabolismo , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Neurônios/metabolismo
4.
Stem Cells ; 35(5): 1176-1188, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28220575

RESUMO

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73+ photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73+ photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.


Assuntos
Células Alimentadoras/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Preservação Biológica , Epitélio Pigmentado da Retina/citologia , Adesão Celular , Diferenciação Celular , Linhagem Celular , Criopreservação , Humanos , Organoides/ultraestrutura , Células Fotorreceptoras/citologia
5.
Dev Growth Differ ; 58(5): 492-502, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27301906

RESUMO

In the vertebrate retina six types of neurons and one glial cell type are generated from multipotent retinal progenitor cells (RPCs) whose proliferation and differentiation are regulated by intrinsic and extrinsic factors. RPCs proliferate undergoing interkinetic nuclear migration within the neuroblastic layer, with their nuclei moving up and down along the apico-basal axis. Moreover, they only differentiate and therefore exit the cell cycle at the apical side of the neuroblastic layer. Sema6A and its receptors PlexinA4 and PlexinA2 control lamina stratification of the inner plexiform layer in the mouse retina. Nevertheless, their function in earlier developmental stages is still unknown. Here, we analyzed the embryonic retina of PlexinA2 and Sema6A knockout mice. Using time-lapse videomicroscopy we provide evidence that Sema6A/PlexinA2 signaling participates to interkinetic nuclear migration of RPCs around birth. When disrupted, RPCs migration is blocked at the apical side of the neuroblastic layer. This is the first evidence supporting a role for transmembrane molecules in the regulation of interkinetic nuclear migration in the mouse retina.


Assuntos
Movimento Celular/fisiologia , Embrião de Mamíferos/embriologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Retina/embriologia , Semaforinas/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Animais , Embrião de Mamíferos/citologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Retina/citologia , Semaforinas/genética , Células-Tronco/citologia
6.
Vet J ; 308: 106256, 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39419374

RESUMO

Domestic cats use visual cues to communicate with conspecifics and humans. This includes the position and movement of the tail. The tail up signal (i.e. tail held vertically with the tip curled) has been identified as a friendly greeting signal between cats and from cats to humans. We present here a report of two cats unable to display the classic tail up signal due to Spitz-like (curled) tail characteristics, a morphology not previously reported in the scientific literature. The cats, a British Shorthair (Mn, 2 y) and Domestic Longhair (Fn, 8.5 y), belonged to different households and were participants in a study of intraspecific social interactions. Video recordings show the cats displaying normal tail movements (waving, swishing, twitching) and position when the tail was hanging down. However, once the tail was raised to a vertical position, it immediately curled over the back. This Spitz-like tail posture was displayed both when greeting people and other cats. It had reportedly been present since adoption. There was no underlying medical history of injury or pain to indicate this was an acquired state. We suggest this Spitz-like tail is a spontaneously occurring anomaly, that does not seem to noticeably impair the cats' communicative abilities, as normal affiliative behaviours were observed during interactions with the cohoused cat. We discuss the significance of this finding in relation to the formation of social bonds and the signalling of affiliative intent.

7.
Nat Commun ; 14(1): 3809, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37369692

RESUMO

Second messengers, including cAMP, cGMP and Ca2+ are often placed in an integrating position to combine the extracellular cues that orient growing axons in the developing brain. This view suggests that axon repellents share the same set of cellular messenger signals and that axon attractants evoke opposite cAMP, cGMP and Ca2+ changes. Investigating the confinement of these second messengers in cellular nanodomains, we instead demonstrate that two repellent cues, ephrin-A5 and Slit1, induce spatially segregated signals. These guidance molecules activate subcellular-specific second messenger crosstalk, each signaling network controlling distinct axonal morphology changes in vitro and pathfinding decisions in vivo.


Assuntos
Axônios , Sistemas do Segundo Mensageiro , Axônios/fisiologia , GMP Cíclico , Transdução de Sinais
8.
Life Sci Alliance ; 6(6)2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36944420

RESUMO

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.


Assuntos
Folículo Ovariano , Pseudópodes , Feminino , Animais , Camundongos , Folículo Ovariano/metabolismo , Oócitos/metabolismo , Oogênese/fisiologia , Células Germinativas , Miosinas
9.
Commun Biol ; 5(1): 1135, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302949

RESUMO

The ocular vasculature is critically involved in many blinding diseases and is also a popular research model for the exploration of developmental and pathological angiogenesis. The development of ocular vessels is a complex, finely orchestrated sequence of events, involving spatial and temporal coordination of hyaloid, choroidal and retinal networks. Comprehensive studies of the tridimensional dynamics of microvascular remodeling are limited by the fact that preserving the spatial disposition of ocular vascular networks is cumbersome using classical histological procedures. Here, we demonstrate that light-sheet fluorescence microscopy (LFSM) of cleared mouse eyes followed by extensive virtual dissection offers a solution to this problem. To the best of our knowledge, this is the first 3D quantification of the evolution of the hyaloid vasculature and of post-occlusive venous remodeling together with the characterization of spatial distribution of various cell populations in ocular compartments, including the vitreous. These techniques will prove interesting to obtain other insights in scientific questions addressing organ-wide cell interactions.


Assuntos
Corioide , Vasos Retinianos , Camundongos , Animais , Retina , Neovascularização Patológica , Microscopia de Fluorescência
10.
Nat Neurosci ; 25(7): 900-911, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35710984

RESUMO

The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.


Assuntos
Células de Purkinje , Receptores de Dopamina D2 , Animais , Cerebelo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células de Purkinje/fisiologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Comportamento Social
11.
Sci Rep ; 10(1): 8852, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483280

RESUMO

The cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation. Osteoarthritis (OA) is associated with local low-grade inflammation, but the regulatory mechanisms are unclear. Local Ach release could have anti-inflammatory activity since articular cells express Ach receptors involved in inflammatory responses. Using the 3DISCO clearing protocol that allows whole-sample 3-dimensional (3D) analysis, we cleared human OA cartilage-subchondral bone samples to search for cholinergic nerve fibres able to produce Ach locally. We analysed 3 plugs of knee cartilage and subchondral bone from 3 OA patients undergoing arthroplasty. We found no nerves in the superficial and intermediate articular cartilage layers, as evidenced by the lack of Peripherin staining (a peripheral nerves marker). Conversely, peripheral nerves were found in the deepest layer of cartilage and in subchondral bone. Some nerves in the subchondral bone samples were cholinergic because they coexpressed peripherin and choline acetyltransferase (ChAT), a specific marker of cholinergic nerves. However, no cholinergic nerves were found in the cartilage layers. It is therefore feasible to clear human bone to perform 3D immunofluorescence. Human OA subchondral bone is innervated by cholinergic fibres, which may regulate local inflammation through local Ach release.


Assuntos
Imageamento Tridimensional/métodos , Articulação do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia , Nervos Periféricos/patologia , Artroplastia do Joelho , Cartilagem Articular/diagnóstico por imagem , Fibras Colinérgicas/patologia , Humanos , Microscopia de Fluorescência , Osteoartrite do Joelho/terapia
12.
J Comp Neurol ; 527(12): 2009-2029, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-30697732

RESUMO

In Bilaterians, commissural neurons project their axons across the midline of the nervous system to target neurons on the opposite side. In mammals, midline crossing at the level of the hindbrain and spinal cord requires the Robo3 receptor which is transiently expressed by all commissural neurons. Unlike other Robo receptors, mammalian Robo3 receptors do not bind Slit ligands and promote midline crossing. Surprisingly, not much is known about Robo3 distribution and mechanism of action in other vertebrate species. Here, we have used whole-mount immunostaining, tissue clearing and light-sheet fluorescent microscopy to study Robo3 expression pattern in embryonic tissue from diverse representatives of amniotes at distinct stages, including squamate (African house snake), birds (chicken, duck, pigeon, ostrich, emu and zebra finch), early postnatal marsupial mammals (fat-tailed dunnart), and eutherian mammals (mouse and human). The analysis of this rich and unique repertoire of amniote specimens reveals conserved features of Robo3 expression in midbrain, hindbrain and spinal cord commissural circuits, which together with subtle but meaningful modifications could account for species-specific evolution of sensory-motor and cognitive capacities. Our results also highlight important differences of precerebellar nuclei development across amniotes.


Assuntos
Encéfalo/embriologia , Desenvolvimento Embrionário , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Vertebrados/embriologia , Animais , Humanos , Receptores de Superfície Celular/metabolismo
13.
Mol Brain ; 10(1): 33, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28728585

RESUMO

For centuries analyses of tissues have depended on sectioning methods. Recent developments of tissue clearing techniques have now opened a segway from studying tissues in 2 dimensions to 3 dimensions. This particular advantage echoes heavily in the field of neuroscience, where in the last several years there has been an active shift towards understanding the complex orchestration of neural circuits. In the past five years, many tissue-clearing protocols have spawned. This is due to varying strength of each clearing protocol to specific applications. However, two main protocols have shown their applicability to a vast number of applications and thus are exponentially being used by a growing number of laboratories. In this review, we focus specifically on two major tissue-clearing method families, derived from the 3DISCO and the CLARITY clearing protocols. Moreover, we provide a "hands-on" description of each tissue clearing protocol and the steps to look out for when deciding to choose a specific tissue clearing protocol. Lastly, we provide perspectives for the development of tissue clearing protocols into the research community in the fields of embryology and cancer.


Assuntos
Encéfalo/fisiologia , Neurociências , Animais , Fluorescência , Humanos , RNA/metabolismo
14.
Sci Rep ; 7(1): 410, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28341853

RESUMO

DCC, a NETRIN-1 receptor, is considered as a cell-autonomous regulator for midline guidance of many commissural populations in the central nervous system. The corticospinal tract (CST), the principal motor pathway for voluntary movements, crosses the anatomic midline at the pyramidal decussation. CST fails to cross the midline in Kanga mice expressing a truncated DCC protein. Humans with heterozygous DCC mutations have congenital mirror movements (CMM). As CMM has been associated, in some cases, with malformations of the pyramidal decussation, DCC might also be involved in this process in human. Here, we investigated the role of DCC in CST midline crossing both in human and mice. First, we demonstrate by multimodal approaches, that patients with CMM due to DCC mutations have an increased proportion of ipsilateral CST projections. Second, we show that in contrast to Kanga mice, the anatomy of the CST is not altered in mice with a deletion of DCC in the CST. Altogether, these results indicate that DCC controls CST midline crossing in both humans and mice, and that this process is non cell-autonomous in mice. Our data unravel a new level of complexity in the role of DCC in CST guidance at the midline.


Assuntos
Orientação de Axônios , Receptor DCC/fisiologia , Tratos Piramidais/patologia , Tratos Piramidais/fisiopatologia , Adulto , Idoso , Animais , Axônios/metabolismo , Corpo Caloso/metabolismo , Receptor DCC/genética , Potencial Evocado Motor , Feminino , Mãos/inervação , Mãos/fisiopatologia , Humanos , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Córtex Motor/fisiopatologia , Movimento , Neocórtex/metabolismo , Estimulação Magnética Transcraniana
15.
Acta Neuropathol Commun ; 2: 145, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25253021

RESUMO

INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level. RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (µFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied ß-amyloid (Aß) peptides locally to the different cellular compartments. We observed that Aß application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aß deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aß peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aß stress at the network level we developed new µFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aß application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration. CONCLUSION: Thanks to µFD-based reconstructed neuronal networks we evaluated the distant effects of local Aß stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aß production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aß can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Córtex Cerebral/patologia , Rede Nervosa/patologia , Sinapses/patologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Camundongos , Técnicas Analíticas Microfluídicas/métodos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Cultura Primária de Células/métodos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Proteínas tau/metabolismo
16.
Cell Rep ; 9(4): 1191-201, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25456121

RESUMO

Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice.


Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Imageamento Tridimensional/métodos , Coloração e Rotulagem , Animais , Biomarcadores/metabolismo , Receptor DCC , Embrião de Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Knockout , Mutação , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo
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