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1.
Mol Cell ; 77(5): 1066-1079.e9, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-31902667

RESUMEN

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1 wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S phase-dependent manner to assist in the eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among the potential Ddi1 targets, we found the core component of Pol II and show that its genotoxin-induced degradation is impaired in ddi1. We propose that the Ddi1 protease contributes to DPC proteolysis.


Asunto(s)
Daño del ADN , Reparación del ADN , ADN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Animales , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Proteolisis , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9 , Spodoptera , Transcripción Genética
2.
EMBO J ; 41(7): e109998, 2022 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-35188676

RESUMEN

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non-vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1-an uncharacterized protein harboring a Chorein-N lipid transport motif-for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.


Asunto(s)
Lípidos de la Membrana , Orgánulos , Transporte Biológico , Homeostasis , Metabolismo de los Lípidos/genética , Lípidos de la Membrana/genética , Lípidos de la Membrana/metabolismo , Orgánulos/metabolismo , Fosfolípidos/genética , Fosfolípidos/metabolismo
3.
PLoS Genet ; 17(3): e1009414, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33690632

RESUMEN

Indole-3-acetic acid (IAA) is the most common, naturally occurring phytohormone that regulates cell division, differentiation, and senescence in plants. The capacity to synthesize IAA is also widespread among plant-associated bacterial and fungal species, which may use IAA as an effector molecule to define their relationships with plants or to coordinate their physiological behavior through cell-cell communication. Fungi, including many species that do not entertain a plant-associated life style, are also able to synthesize IAA, but the physiological role of IAA in these fungi has largely remained enigmatic. Interestingly, in this context, growth of the budding yeast Saccharomyces cerevisiae is sensitive to extracellular IAA. Here, we use a combination of various genetic approaches including chemical-genetic profiling, SAturated Transposon Analysis in Yeast (SATAY), and genetic epistasis analyses to identify the mode-of-action by which IAA inhibits growth in yeast. Surprisingly, these analyses pinpointed the target of rapamycin complex 1 (TORC1), a central regulator of eukaryotic cell growth, as the major growth-limiting target of IAA. Our biochemical analyses further demonstrate that IAA inhibits TORC1 both in vivo and in vitro. Intriguingly, we also show that yeast cells are able to synthesize IAA and specifically accumulate IAA upon entry into stationary phase. Our data therefore suggest that IAA contributes to proper entry of yeast cells into a quiescent state by acting as a metabolic inhibitor of TORC1.


Asunto(s)
Hongos/efectos de los fármacos , Hongos/enzimología , Ácidos Indolacéticos/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Elementos Transponibles de ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática , Hongos/genética , Ácidos Indolacéticos/química , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Inhibidores de Proteínas Quinasas/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacos
4.
Genes Dev ; 23(5): 602-18, 2009 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-19270160

RESUMEN

In many species, a dosage compensation complex (DCC) is targeted to X chromosomes of one sex to equalize levels of X-gene products between males (1X) and females (2X). Here we identify cis-acting regulatory elements that target the Caenorhabditis elegans X chromosome for repression by the DCC. The DCC binds to discrete, dispersed sites on X of two types. rex sites (recruitment elements on X) recruit the DCC in an autonomous, DNA sequence-dependent manner using a 12-base-pair (bp) consensus motif that is enriched on X. This motif is critical for DCC binding, is clustered in rex sites, and confers much of X-chromosome specificity. Motif variants enriched on X by 3.8-fold or more are highly predictive (95%) for rex sites. In contrast, dox sites (dependent on X) lack the X-enriched variants and cannot bind the DCC when detached from X. dox sites are more prevalent than rex sites and, unlike rex sites, reside preferentially in promoters of some expressed genes. These findings fulfill predictions for a targeting model in which the DCC binds to recruitment sites on X and disperses to discrete sites lacking autonomous recruitment ability. To relate DCC binding to function, we identified dosage-compensated and noncompensated genes on X. Unexpectedly, many genes of both types have bound DCC, but many do not, suggesting the DCC acts over long distances to repress X-gene expression. Remarkably, the DCC binds to autosomes, but at far fewer sites and rarely at consensus motifs. DCC disruption causes opposite effects on expression of X and autosomal genes. The DCC thus acts at a distance to impact expression throughout the genome.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/metabolismo , Compensación de Dosificación (Genética)/fisiología , Regulación del Desarrollo de la Expresión Génica , Genoma de los Helmintos/fisiología , Complejos Multiproteicos/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia de Consenso/genética , Femenino , Genoma de los Helmintos/genética , Masculino , Unión Proteica , Elementos Reguladores de la Transcripción , Cromosoma X/genética , Cromosoma X/metabolismo
5.
Biochim Biophys Acta ; 1833(11): 2526-41, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23380708

RESUMEN

Membrane-bound organelles are a wonderful evolutionary acquisition of the eukaryotic cell, allowing the segregation of sometimes incompatible biochemical reactions into specific compartments with tailored microenvironments. On the flip side, these isolating membranes that crowd the interior of the cell, constitute a hindrance to the diffusion of metabolites and information to all corners of the cell. To ensure coordination of cellular activities, cells use a network of contact sites between the membranes of different organelles. These membrane contact sites (MCSs) are domains where two membranes come to close proximity, typically less than 30nm. Such contacts create microdomains that favor exchange between two organelles. MCSs are established and maintained in durable or transient states by tethering structures, which keep the two membranes in proximity, but fusion between the membranes does not take place. Since the endoplasmic reticulum (ER) is the most extensive cellular membrane network, it is thus not surprising to find the ER involved in most MCSs within the cell. The ER contacts diverse compartments such as mitochondria, lysosomes, lipid droplets, the Golgi apparatus, endosomes and the plasma membrane. In this review, we will focus on the common organizing principles underlying the many MCSs found between the ER and virtually all compartments of the cell, and on how the ER establishes a network of MCSs for the trafficking of vital metabolites and information. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.


Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Orgánulos/metabolismo , Animales , Humanos , Transporte de Proteínas
6.
Biochem Soc Trans ; 40(2): 445-50, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22435828

RESUMEN

Cellular organelles need to communicate in order to co-ordinate homoeostasis of the compartmentalized eukaryotic cell. Such communication involves the formation of membrane contact sites between adjacent organelles, allowing privileged exchange of metabolites and information. Using a synthetic protein designed to artificially tether the ER (endoplasmic reticulum) to mitochondria, we have discovered a yeast protein complex naturally involved in establishing and maintaining contact sites between these two organelles. This protein complex is physiologically involved in a plethora of mitochondrial processes, suggesting that ER-mitochondria connections play a central co-ordinating role in the regulation of mitochondrial biology. Recent biochemical characterization of this protein complex led to the discovery that GTPases of the Miro family are part of ER-mitochondria connections. The yeast Miro GTPase Gem1 localizes to ER-mitochondria interface and influences the size and distribution of mitochondria. Thus Miro GTPases may serve as regulators of the ER-mitochondria connection.


Asunto(s)
Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , GTP Fosfohidrolasas/metabolismo
7.
Methods Mol Biol ; 2477: 349-379, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35524127

RESUMEN

Genome-wide transposon mutagenesis followed by deep sequencing allows the genome-wide mapping of growth-affecting loci in a straightforward and time-efficient way.SAturated Transposon Analysis in Yeast (SATAY) takes advantage of a modified maize transposon that is highly mobilizable in S. cerevisiae. SATAY allows not only the genome-wide mapping of genes required for growth in select conditions (such as genetic interactions or drug sensitivity/resistance), but also of protein sub-domains, as well as the creation of gain- and separation-of-function alleles. From strain preparation to the mapping of sequencing reads, we detail all the steps for the making and analysis of SATAY libraries in any S. cerevisiae lab, requiring only ordinary equipment and access to a Next-Gen sequencing platform.


Asunto(s)
Elementos Transponibles de ADN , Saccharomyces cerevisiae , Alelos , Mapeo Cromosómico , Elementos Transponibles de ADN/genética , Mutagénesis Insercional , Saccharomyces cerevisiae/genética
8.
Nat Commun ; 13(1): 1490, 2022 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-35314699

RESUMEN

Due to epistasis, the same mutation can have drastically different phenotypic consequences in different individuals. This phenomenon is pertinent to precision medicine as well as antimicrobial drug development, but its general characteristics are largely unknown. We approach this question by genome-wide assessment of gene essentiality polymorphism in 16 Saccharomyces cerevisiae strains using transposon insertional mutagenesis. Essentiality polymorphism is observed for 9.8% of genes, most of which have had repeated essentiality switches in evolution. Genes exhibiting essentiality polymorphism lean toward having intermediate numbers of genetic and protein interactions. Gene essentiality changes tend to occur concordantly among components of the same protein complex or metabolic pathway and among a group of over 100 mitochondrial proteins, revealing molecular machines or functional modules as units of gene essentiality variation. Most essential genes tolerate transposon insertions consistently among strains in one or more coding segments, delineating nonessential regions within essential genes.


Asunto(s)
Elementos Transponibles de ADN , Saccharomyces cerevisiae , Elementos Transponibles de ADN/genética , Genes Esenciales/genética , Humanos , Redes y Vías Metabólicas , Mutagénesis/genética , Mutagénesis Insercional/genética , Saccharomyces cerevisiae/genética
9.
Cell Rep ; 37(8): 110034, 2021 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-34818558

RESUMEN

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.


Asunto(s)
Reparación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Cisteína Endopeptidasas/metabolismo , ADN/metabolismo , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/genética , Sumoilación/fisiología , Ubiquitina-Proteína Ligasas/metabolismo
10.
Mol Biol Cell ; 31(12): 1302-1313, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32267208

RESUMEN

Eukaryotic cells are compartmentalized into organelles by intracellular membranes. While the organelles are distinct, many of them make intimate contact with one another. These contacts were first observed in the 1950s, but only recently have the functions of these contact sites begun to be understood. In yeast, the endoplasmic reticulum (ER) makes extensive intermembrane contacts with the plasma membrane (PM), covering ∼40% of the PM. Many functions of ER-PM contacts have been proposed, including nonvesicular lipid trafficking, ion transfer, and as signaling hubs. Surprisingly, cells that lack ER-PM contacts grow well, indicating that alternative pathways may be compensating for the loss of ER-PM contact. To better understand the function of ER-PM contact sites we used saturating transposon mutagenesis to identify synthetic lethal mutants in a yeast strain lacking ER-PM contact sites. The strongest hits were components of the ESCRT complexes. The synthetic lethal mutants have low levels of some lipid species but accumulate free fatty acids and lipid droplets. We found that only ESCRT-III components are synthetic lethal, indicating that Vps4 and other ESCRT complexes do not function in this pathway. These data suggest that ESCRT-III proteins and ER-PM contact sites act in independent pathways to maintain lipid homeostasis.


Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Lípidos/genética , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Transporte de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Mol Biol Cell ; 30(22): 2814-2826, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31509475

RESUMEN

Hereditary sensory and autonomic neuropathy (HSAN) types IA and IC (IA/C) are caused by elevated levels of an atypical class of lipid named 1-deoxysphingolipid (DoxSL). How elevated levels of DoxSL perturb the physiology of the cell and how the perturbations lead to HSAN IA/C are largely unknown. In this study, we show that C26-1-deoxydihydroceramide (C26-DoxDHCer) is highly toxic to the cell, while C16- and C18-DoxDHCer are less toxic. Genome-wide genetic screens and lipidomics revealed the dynamics of DoxSL accumulation and DoxSL species responsible for the toxicity over the course of DoxSL accumulation. Moreover, we show that disruption of F-actin organization, alteration of mitochondrial shape, and accumulation of hydrophobic bodies by DoxSL are not sufficient to cause complete cellular failure. We found that cell death coincides with collapsed ER membrane, although we cannot rule out other possible causes of cell death. Thus, we have unraveled key principles of DoxSL cytotoxicity that may help to explain the clinical features of HSAN IA/C.


Asunto(s)
Neuropatías Hereditarias Sensoriales y Autónomas/metabolismo , Esfingolípidos/metabolismo , Actinas/metabolismo , Ceramidas/toxicidad , Neuropatías Hereditarias Sensoriales y Autónomas/fisiopatología , Metabolismo de los Lípidos , Lipidómica , Lípidos , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolípidos/genética
12.
J Cell Biol ; 217(3): 959-974, 2018 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-29279306

RESUMEN

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes.


Asunto(s)
Retículo Endoplásmico/metabolismo , Kluyveromyces/metabolismo , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Complejos Multiproteicos/metabolismo , Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Activo/fisiología , Retículo Endoplásmico/genética , Kluyveromyces/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Complejos Multiproteicos/genética , Fosfolípidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-Actividad
13.
Elife ; 62017 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-28481201

RESUMEN

Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput.


Asunto(s)
Genes Fúngicos , Genética Microbiana/métodos , Anotación de Secuencia Molecular/métodos , Mutagénesis Insercional/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Elementos Transponibles de ADN , Genoma Fúngico , Análisis de Secuencia de ADN
14.
Genes Dev ; 19(10): 1199-210, 2005 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15905408

RESUMEN

We show that in budding yeast large rDNA deletions arise frequently and cause an increase in telomeric and mating-type gene silencing proportional to repeat loss. Paradoxically, this increase in silencing is correlated with a highly specific down-regulation of SIR2, which encodes a deacetylase enzyme required for silencing. These apparently conflicting observations suggest that a large nucleolar pool of Sir2 is released upon rDNA loss and made available for telomeric and HM silencing, as well as down-regulation of SIR2 itself. Indeed, we present evidence for a reduction in the fraction of Sir2 colocalizing with the nucleolar marker Nop1, and for SIR2 autoregulation. Despite a decrease in the fraction of nucleolar Sir2, and in overall Sir2 protein levels, short rDNA strains display normal rDNA silencing and a lifespan indistinguishable from wild type. These observations reveal an unexpectedly large clonal variation in rDNA cluster size and point to the existence of a novel regulatory circuit, sensitive to rDNA copy number, that balances nucleolar and nonnucleolar pools of Sir2 protein.


Asunto(s)
ADN de Hongos/metabolismo , ADN Ribosómico/metabolismo , Regulación hacia Abajo/fisiología , Epigénesis Genética , Dosificación de Gen , Silenciador del Gen/fisiología , Histona Desacetilasas/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuinas/fisiología , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/fisiología , Histona Desacetilasas/genética , Homeostasis/genética , Homeostasis/fisiología , Mutación , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2 , Sirtuinas/genética , Especificidad de la Especie , Telómero/fisiología
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