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1.
EClinicalMedicine ; 40: 101099, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34490415

RESUMO

Background: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing urgency to identify pathophysiological characteristics leading to severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors that affect individual immune response to SARS-CoV-2. We sought to evaluate this hypothesis by conducting a multicenter study using HLA sequencing. Methods: We analyzed the association between COVID-19 severity and HLAs in 435 individuals from Germany (n = 135), Spain (n = 133), Switzerland (n = 20) and the United States (n = 147), who had been enrolled from March 2020 to August 2020. This study included patients older than 18 years, diagnosed with COVID-19 and representing the full spectrum of the disease. Finally, we tested our results by meta-analysing data from prior genome-wide association studies (GWAS). Findings: We describe a potential association of HLA-C*04:01 with severe clinical course of COVID-19. Carriers of HLA-C*04:01 had twice the risk of intubation when infected with SARS-CoV-2 (risk ratio 1.5 [95% CI 1.1-2.1], odds ratio 3.5 [95% CI 1.9-6.6], adjusted p-value = 0.0074). These findings are based on data from four countries and corroborated by independent results from GWAS. Our findings are biologically plausible, as HLA-C*04:01 has fewer predicted bindings sites for relevant SARS-CoV-2 peptides compared to other HLA alleles. Interpretation: HLA-C*04:01 carrier state is associated with severe clinical course in SARS-CoV-2. Our findings suggest that HLA class I alleles have a relevant role in immune defense against SARS-CoV-2. Funding: Funded by Roche Sequencing Solutions, Inc.

2.
Cell Stem Cell ; 27(3): 383-395.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32783885

RESUMO

Lineage tracing reveals hematopoietic stem cell (HSC) fates, while single-cell RNA sequencing identifies snapshots of HSC transcriptomes. To obtain information on fate plus transcriptome in the same cell, we developed the PolyloxExpress allele, enabling Cre-recombinase-dependent RNA barcoding in situ. Linking fates to single HSC transcriptomes provided the information required to identify transcriptional signatures of HSC fates, which were not apparent in single-HSC transcriptomes alone. We find that differentiation-inactive, multilineage, and lineage-restricted HSC clones reside in distinct regions of the transcriptional landscape of hematopoiesis. Differentiation-inactive HSC clones are closer to the origin of the transcriptional trajectory, yet they are not characterized by a quiescent gene signature. Fate-specific gene signatures imply coherence of clonal HSC fates, and HSC output toward short-lived lineage progenitors indicates stability of HSC fates over time. These combined analyses of fate and transcriptome under physiological conditions may pave the way toward identifying molecular determinants of HSC fates.


Assuntos
Células-Tronco Hematopoéticas , Transcriptoma , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Clonais , Hematopoese/genética , Transcriptoma/genética
3.
Nat Commun ; 10(1): 5009, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676752

RESUMO

Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hipocampo/metabolismo , Proteínas/genética , RNA/genética , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Ratos Sprague-Dawley
4.
Proc Natl Acad Sci U S A ; 115(34): 8609-8614, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30082403

RESUMO

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded.


Assuntos
Retrovirus Endógenos/genética , Phascolarctidae/genética , Recombinação Genética , Animais , Feminino , Masculino , New South Wales
5.
Nature ; 548(7668): 456-460, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28813413

RESUMO

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.


Assuntos
Sítios de Ligação Microbiológicos/genética , Linhagem da Célula/genética , Rastreamento de Células/métodos , Código de Barras de DNA Taxonômico/métodos , Células-Tronco Hematopoéticas/citologia , Recombinação Genética/genética , Análise de Célula Única/métodos , Animais , Células Clonais/citologia , Células Clonais/metabolismo , Embrião de Mamíferos/citologia , Células Eritroides/citologia , Células Eritroides/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Camundongos , Mosaicismo , Células Mieloides/citologia , Células Mieloides/metabolismo
6.
Mol Syst Biol ; 12(7): 875, 2016 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-27430939

RESUMO

Transcription initiated at alternative sites can produce mRNA isoforms with different 5'UTRs, which are potentially subjected to differential translational regulation. However, the prevalence of such isoform-specific translational control across mammalian genomes is currently unknown. By combining polysome profiling with high-throughput mRNA 5' end sequencing, we directly measured the translational status of mRNA isoforms with distinct start sites. Among 9,951 genes expressed in mouse fibroblasts, we identified 4,153 showed significant initiation at multiple sites, of which 745 genes exhibited significant isoform-divergent translation. Systematic analyses of the isoform-specific translation revealed that isoforms with longer 5'UTRs tended to translate less efficiently. Further investigation of cis-elements within 5'UTRs not only provided novel insights into the regulation by known sequence features, but also led to the discovery of novel regulatory sequence motifs. Quantitative models integrating all these features explained over half of the variance in the observed isoform-divergent translation. Overall, our study demonstrated the extensive translational regulation by usage of alternative transcription start sites and offered comprehensive understanding of translational regulation by diverse sequence features embedded in 5'UTRs.


Assuntos
Polirribossomos/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Sítio de Iniciação de Transcrição , Regiões 5' não Traduzidas , Animais , Regulação da Expressão Gênica , Mamíferos/genética , Camundongos , Células NIH 3T3 , Biossíntese de Proteínas
7.
Nat Neurosci ; 18(4): 603-610, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25714049

RESUMO

Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.


Assuntos
Encéfalo/metabolismo , Dendritos/metabolismo , Plasticidade Neuronal/fisiologia , Neurópilo/metabolismo , RNA/metabolismo , Sinapses/genética , Animais , Encéfalo/crescimento & desenvolvimento , Feminino , Hipocampo/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , RNA Circular , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
8.
EMBO Mol Med ; 5(9): 1431-42, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24000153

RESUMO

RBM10 encodes an RNA binding protein. Mutations in RBM10 are known to cause multiple congenital anomaly syndrome in male humans, the TARP syndrome. However, the molecular function of RBM10 is unknown. Here we used PAR-CLIP to identify thousands of binding sites of RBM10 and observed significant RBM10-RNA interactions in the vicinity of splice sites. Computational analyses of binding sites as well as loss-of-function and gain-of-function experiments provided evidence for the function of RBM10 in regulating exon skipping and suggested an underlying mechanistic model, which could be subsequently validated by minigene experiments. Furthermore, we demonstrated the splicing defects in a patient carrying an RBM10 mutation, which could be explained by disrupted function of RBM10 in splicing regulation. Overall, our study established RBM10 as an important regulator of alternative splicing, presented a mechanistic model for RBM10-mediated splicing regulation and provided a molecular link to understanding a human congenital disorder.


Assuntos
Processamento Alternativo , Pé Torto Equinovaro/genética , Regulação da Expressão Gênica , Cardiopatias Congênitas/genética , Síndrome de Pierre Robin/genética , Proteínas de Ligação a RNA/metabolismo , Humanos
9.
Proc Natl Acad Sci U S A ; 109(19): 7257-62, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22509006

RESUMO

Given worldwide increases in the incidence of obesity and type 2 diabetes, new strategies for preventing and treating metabolic diseases are needed. The nuclear receptor PPARγ (peroxisome proliferator-activated receptor gamma) plays a central role in lipid and glucose metabolism; however, current PPARγ-targeting drugs are characterized by undesirable side effects. Natural products from edible biomaterial provide a structurally diverse resource to alleviate complex disorders via tailored nutritional intervention. We identified a family of natural products, the amorfrutins, from edible parts of two legumes, Glycyrrhiza foetida and Amorpha fruticosa, as structurally new and powerful antidiabetics with unprecedented effects for a dietary molecule. Amorfrutins bind to and activate PPARγ, which results in selective gene expression and physiological profiles markedly different from activation by current synthetic PPARγ drugs. In diet-induced obese and db/db mice, amorfrutin treatment strongly improves insulin resistance and other metabolic and inflammatory parameters without concomitant increase of fat storage or other unwanted side effects such as hepatoxicity. These results show that selective PPARγ-activation by diet-derived ligands may constitute a promising approach to combat metabolic disease.


Assuntos
Produtos Biológicos/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fabaceae/química , Hipoglicemiantes/farmacologia , Salicilatos/farmacologia , Células 3T3-L1 , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Expressão Gênica/efeitos dos fármacos , Glycyrrhiza/química , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/etiologia , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salicilatos/química , Salicilatos/metabolismo
10.
Structure ; 19(9): 1294-306, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21893288

RESUMO

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.


Assuntos
Escherichia coli O157 , Proteínas de Escherichia coli/química , Proteínas do Tecido Nervoso/química , Fragmentos de Peptídeos/química , Receptores de Superfície Celular/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Calorimetria , Chlorocebus aethiops , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Interações Hospedeiro-Patógeno , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imunoprecipitação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/genética , Termodinâmica
11.
Microb Biotechnol ; 4(6): 735-45, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21791029

RESUMO

For possible control of fire blight affecting apple and pear trees, we characterized Erwinia amylovora phages from North America and Germany. The genome size determined by electron microscopy (EM) was confirmed by sequence data and major coat proteins were identified from gel bands by mass spectroscopy. By their morphology from EM data, φEa1h and φEa100 were assigned to the Podoviridae and φEa104 and φEa116 to the Myoviridae. Host ranges were essentially confined to E. amylovora, strains of the species Erwinia pyrifoliae, E. billingiae and even Pantoea stewartii were partially sensitive. The phages φEa1h and φEa100 were dependent on the amylovoran capsule of E. amylovora, φEa104 and φEa116 were not. The Myoviridae efficiently lysed their hosts and protected apple flowers significantly better than the Podoviridae against E. amylovora and should be preferred in biocontrol experiments. We have also isolated and partially characterized E. amylovora phages from apple orchards in Germany. They belong to the Podoviridae or Myoviridae with a host range similar to the phages isolated in North America. In EM measurements, the genome sizes of the Podoviridae were smaller than the genomes of the Myoviridae from North America and from Germany, which differed from each other in corresponding nucleotide sequences.


Assuntos
Bacteriófagos/genética , Bacteriófagos/fisiologia , Erwinia amylovora/virologia , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/isolamento & purificação , DNA Viral/química , DNA Viral/genética , Eletroforese , Erwinia amylovora/isolamento & purificação , Genoma Viral , Alemanha , Especificidade de Hospedeiro , Malus , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Myoviridae/genética , Myoviridae/crescimento & desenvolvimento , Myoviridae/isolamento & purificação , Myoviridae/fisiologia , América do Norte , Pantoea/virologia , Doenças das Plantas/microbiologia , Podoviridae/genética , Podoviridae/crescimento & desenvolvimento , Podoviridae/isolamento & purificação , Podoviridae/fisiologia , Pyrus , Análise de Sequência de DNA , Proteínas Virais/análise
12.
J Struct Biol ; 175(2): 159-70, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21382497

RESUMO

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Vetores Genéticos/normas , Complexos Multiproteicos/biossíntese , Proteínas Recombinantes/biossíntese , Academias e Institutos , Fator de Ligação a CCAAT/biossíntese , Fator de Ligação a CCAAT/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Europa (Continente) , Geminina , Cooperação Internacional , Israel , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição TFII/biossíntese , Fatores de Transcrição TFII/genética
13.
FEBS J ; 275(18): 4627-40, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18699778

RESUMO

Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca2+. Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2+. Furthermore, no binding of Ca2+ up to 0.1 mM was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação a DNA/química , Proteínas dos Microfilamentos/química , Actinas/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Dimerização , Células HeLa , Humanos , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Shigella/patogenicidade
14.
Genome Biol ; 5(9): R71, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15345055

RESUMO

We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.


Assuntos
Clonagem Molecular/métodos , DNA Complementar/biossíntese , Biblioteca Gênica , Genômica/métodos , Catálogos como Assunto , Cristalografia por Raios X/métodos , Bases de Dados Genéticas , Expressão Gênica/genética , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Valor Preditivo dos Testes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência de DNA/métodos , Solubilidade
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