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3.
Nat Commun ; 12(1): 5056, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417458

RESUMO

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.


Assuntos
Epigênese Genética , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Micrometástase de Neoplasia , Ligação Proteica , Carga Tumoral
4.
Curr Opin Immunol ; 72: 309-317, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34425410

RESUMO

Viral encephalitis is a major neglected medical problem. Host defense mechanisms against viral infection of the central nervous system (CNS) have long remained unclear. The few previous studies of CNS-specific immunity to viruses in mice in vivo and humans in vitro have focused on the contributions of circulating leukocytes, resident microglial cells and astrocytes, with neurons long considered passive victims of viral infection requiring protection from extrinsic antiviral mechanisms. The last decade has witnessed the gradual emergence of the notion that neurons also combat viruses through cell-intrinsic mechanisms. Forward genetic approaches in humans have shown that monogenic inborn errors of TLR3, IFN-α/ß, or snoRNA31 immunity confer susceptibility to herpes simplex virus 1 (HSV-1) infection of the forebrain, whereas inborn errors of DBR1 underlie brainstem infections due to various viruses, including HSV-1. The study of human pluripotent stem cell (hPSC)-derived CNS-resident cells has unraveled known (i.e. TLR3-dependent IFN-α/ß immunity) and new (i.e. snoRNA31-dependent or DBR1-dependent immunity) cell-intrinsic antiviral mechanisms operating in neurons. Reverse genetic approaches in mice have confirmed that some known antiviral mechanisms also operate in mouse neurons (e.g. TLR3 and IFN-α/ß immunity). The search for human inborn errors of immunity (IEIs) underlying various forms of viral encephalitis, coupled with mouse models in vivo, and hPSC-based culture models of CNS and peripheral nervous system cells and organoids in vitro, should shed further light on the cell-specific and tissue-specific mechanisms of host defense against viruses in the human brain.

5.
Nat Commun ; 12(1): 4507, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301951

RESUMO

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.


Assuntos
Processamento Alternativo/genética , Disautonomia Familiar/genética , Mutação , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/metabolismo , Elementos Facilitadores Genéticos/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Camundongos Transgênicos , Estrutura Molecular , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Sítios de Splice de RNA/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Elongação da Transcrição/metabolismo
6.
Cancer Res ; 81(13): 3706-3716, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33941615

RESUMO

Fanconi anemia is an inherited genome instability syndrome characterized by interstrand cross-link hypersensitivity, congenital defects, bone marrow failure, and cancer predisposition. Although DNA repair mediated by Fanconi anemia genes has been extensively studied, how inactivation of these genes leads to specific cellular phenotypic consequences associated with Fanconi anemia is not well understood. Here we report that Fanconi anemia stem cells in the C. elegans germline and in murine embryos display marked nonhomologous end joining (NHEJ)-dependent radiation resistance, leading to survival of progeny cells carrying genetic lesions. In contrast, DNA cross-linking does not induce generational genomic instability in Fanconi anemia stem cells, as widely accepted, but rather drives NHEJ-dependent apoptosis in both species. These findings suggest that Fanconi anemia is a stem cell disease reflecting inappropriate NHEJ, which is mutagenic and carcinogenic as a result of DNA misrepair, while marrow failure represents hematopoietic stem cell apoptosis. SIGNIFICANCE: This study finds that Fanconi anemia stem cells preferentially activate error-prone NHEJ-dependent DNA repair to survive irradiation, thereby conferring generational genomic instability that is instrumental in carcinogenesis.

7.
Cell Stem Cell ; 28(9): 1566-1581.e8, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33951478

RESUMO

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.


Assuntos
Retrovirus Endógenos , Diferenciação Celular , Humanos , Ativação Transcricional
9.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33542154

RESUMO

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.


Assuntos
Endotélio Vascular/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/genética , Animais , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/fisiologia , Endotélio Vascular/fisiologia , Expressão Gênica , Humanos , Camundongos Endogâmicos , Células-Tronco Pluripotentes/fisiologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Análise de Célula Única , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo
10.
Nat Neurosci ; 24(3): 343-354, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33558694

RESUMO

Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APPSWE+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APPSWE+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.


Assuntos
Doença de Alzheimer/metabolismo , Complemento C3/metabolismo , Microglia/metabolismo , Células-Tronco Pluripotentes/patologia , Doença de Alzheimer/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Hematopoese/fisiologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Microglia/patologia , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia
11.
Cell Stem Cell ; 28(2): 217-229.e7, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545080

RESUMO

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.


Assuntos
Dopamina , Células-Tronco Embrionárias Humanas , Animais , Diferenciação Celular , Neurônios Dopaminérgicos , Mesencéfalo , Camundongos , Ratos , Distribuição Tecidual
12.
Cell Stem Cell ; 28(2): 343-355.e5, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545081

RESUMO

Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.


Assuntos
Neurônios Dopaminérgicos , Células-Tronco Embrionárias Humanas , Animais , Diferenciação Celular , Mesencéfalo , Ratos , Via de Sinalização Wnt
13.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393505

RESUMO

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/ß induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-ß protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-ß secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-ß immunity.


Assuntos
Córtex Cerebral/imunologia , Fibroblastos/imunologia , Herpesvirus Humano 1/imunologia , Interferon beta/imunologia , Neurônios/imunologia , Receptor 3 Toll-Like/imunologia , Vesiculovirus/imunologia , Animais , Linhagem Celular , Córtex Cerebral/patologia , Córtex Cerebral/virologia , Fibroblastos/patologia , Fibroblastos/virologia , Humanos , Interferon beta/genética , Camundongos , Camundongos Knockout , Neurônios/patologia , Neurônios/virologia , Receptor 3 Toll-Like/genética
14.
Nat Rev Immunol ; 21(7): 441-453, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33398129

RESUMO

Advancements in human pluripotent stem cell technology offer a unique opportunity for the neuroimmunology field to study host-virus interactions directly in disease-relevant cells of the human central nervous system (CNS). Viral encephalitis is most commonly caused by herpesviruses, arboviruses and enteroviruses targeting distinct CNS cell types and often leading to severe neurological damage with poor clinical outcomes. Furthermore, different neurotropic viruses will affect the CNS at distinct developmental stages, from early prenatal brain development to the aged brain. With the unique flexibility and scalability of human pluripotent stem cell technology, it is now possible to examine the molecular mechanisms underlying acute infection and latency, determine which CNS subpopulations are specifically infected, study temporal aspects of viral susceptibility, perform high-throughput chemical or genetic screens for viral restriction factors and explore complex cell-non-autonomous disease mechanisms. Therefore, human pluripotent stem cell technology has the potential to address key unanswered questions about antiviral immunity in the CNS, including emerging questions on the potential CNS tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).


Assuntos
Sistema Nervoso Central/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Células-Tronco Pluripotentes/imunologia , Tropismo Viral , COVID-19 , Humanos , Microglia , Neuroglia , Neurônios , SARS-CoV-2
15.
Genes Chromosomes Cancer ; 60(4): 272-281, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33336840

RESUMO

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

16.
Front Cell Dev Biol ; 8: 729, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903681

RESUMO

In Parkinson's disease (PD), there are currently no effective therapies to prevent or slow down disease progression. Cell replacement therapy using human pluripotent stem cell (hPSC)-derived dopamine neurons holds considerable promise. It presents a novel, regenerative strategy, building on the extensive history of fetal tissue grafts and capturing the potential of hPSCs to serve as a scalable and standardized cell source. Progress in establishing protocols for the direct differentiation to midbrain dopamine (mDA) neurons from hPSC have catalyzed the development of cell-based therapies for PD. Consequently, several groups have derived clinical-grade mDA neuron precursors under clinical good manufacture practice condition, which are progressing toward clinical testing in PD patients. Here we will review the current status of the field, discuss the remaining key challenges, and highlight future areas for further improvements of hPSC-based technologies in the clinical translation to PD.

17.
Neurobiol Dis ; 144: 105025, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745521

RESUMO

Amyotrophic lateral sclerosis is a disease characterized by progressive paralysis and death. Most ALS-cases are sporadic (sALS) and patient heterogeneity poses challenges for effective therapies. Applying metabolite profiling on 77-sALS patient-derived-fibroblasts and 43-controls, we found ~25% of sALS cases (termed sALS-1) are characterized by transsulfuration pathway upregulation, where methionine-derived-homocysteine is channeled into cysteine for glutathione synthesis. sALS-1 fibroblasts selectively exhibited a growth defect under oxidative conditions, fully-rescued by N-acetylcysteine (NAC). [U13C]-glucose tracing showed transsulfuration pathway activation with accelerated glucose flux into the Krebs cycle. We established a four-metabolite support vector machine model predicting sALS-1 metabotype with 97.5% accuracy. Both sALS-1 metabotype and growth phenotype were validated in an independent cohort of sALS cases. Importantly, plasma metabolite profiling identified a system-wide cysteine metabolism perturbation as a hallmark of sALS-1. Findings reveal that sALS patients can be stratified into distinct metabotypes with differential sensitivity to metabolic stress, providing novel insights for personalized therapy.

18.
Cell Stem Cell ; 27(1): 35-49.e6, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619517

RESUMO

Autism is a clinically heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restricted interests, and repetitive behaviors. Despite significant advances in the genetics of autism, understanding how genetic changes perturb brain development and affect clinical symptoms remains elusive. Here, we present a multiplex human pluripotent stem cell (hPSC) platform, in which 30 isogenic disease lines are pooled in a single dish and differentiated into prefrontal cortex (PFC) lineages to efficiently test early-developmental hypotheses of autism. We define subgroups of autism mutations that perturb PFC neurogenesis and are correlated to abnormal WNT/ßcatenin responses. Class 1 mutations (8 of 27) inhibit while class 2 mutations (5 of 27) enhance PFC neurogenesis. Remarkably, autism patient data reveal that individuals carrying subclass-specific mutations differ clinically in their corresponding language acquisition profiles. Our study provides a framework to disentangle genetic heterogeneity associated with autism and points toward converging molecular and developmental pathways of diverse autism-associated mutations.


Assuntos
Transtorno Autístico , Transtornos do Neurodesenvolvimento , Células-Tronco Pluripotentes , Transtorno Autístico/genética , Diferenciação Celular/genética , Humanos , Neurogênese
19.
Cell Stem Cell ; 27(1): 125-136.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32579880

RESUMO

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Organoides/virologia , Pneumonia Viral/virologia , Tropismo , Enzima de Conversão de Angiotensina 2 , Animais , Autopsia , COVID-19 , Linhagem Celular , Infecções por Coronavirus/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Fígado/patologia , Camundongos , Pâncreas/patologia , Pâncreas/virologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , SARS-CoV-2 , Internalização do Vírus
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