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1.
Heliyon ; 10(7): e28055, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38560224

RESUMO

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

2.
J Biol Chem ; 299(11): 105282, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37742923

RESUMO

The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ-binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ-binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ-binding motif is critical for proper endoplasmic reticulum (ER) export of Cx36. Removing the PDZ-binding motif of Cx36 results in ER retention and the formation of multimembrane vesicles containing gap junction-like connexin aggregates. Using a combination of site-directed mutagenesis and electron micrographs, we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.


Assuntos
Conexinas , Retículo Endoplasmático , Junções Comunicantes , Humanos , Conexinas/genética , Conexinas/metabolismo , Retículo Endoplasmático/metabolismo , Junções Comunicantes/metabolismo , Células HEK293 , Domínios Proteicos , Motivos de Aminoácidos , Sinapses Elétricas/fisiologia , Mutação , Transporte Proteico/genética , Vesículas Sinápticas/patologia , Vesículas Sinápticas/ultraestrutura , Microscopia Eletrônica de Varredura , Proteína delta-2 de Junções Comunicantes
3.
Nat Commun ; 14(1): 1240, 2023 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-36870997

RESUMO

The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.


Assuntos
Salmonella enterica , Imagem Individual de Molécula , Salmonella , Proteínas de Membrana , Transporte Biológico
4.
PLoS Pathog ; 17(6): e1009319, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34143852

RESUMO

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Human-restricted typhoidal S. enterica serovars Typhi (STY) or Paratyphi A (SPA) cause severe typhoid or paratyphoid fever, while many S. enterica serovar Typhimurium (STM) strains have a broad host range and in human hosts usually lead to a self-limiting gastroenteritis. Due to restriction of STY and SPA to primate hosts, experimental systems for studying the pathogenesis of typhoid and paratyphoid fever are limited. Therefore, STM infection of susceptible mice is commonly considered as model system for studying these diseases. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2-T3SS) is a key factor for intracellular survival of Salmonella. Inside host cells, the pathogen resides within the Salmonella-containing vacuole (SCV) and induces tubular structures extending from the SCV, termed Salmonella-induced filaments (SIF). This study applies single cell analyses approaches, which are flow cytometry of Salmonella harboring dual fluorescent protein reporters, effector translocation, and correlative light and electron microscopy to investigate the fate and activities of intracellular STY and SPA. The SPI2-T3SS of STY and SPA is functional in translocation of effector proteins, SCV and SIF formation. However, only a low proportion of intracellular STY and SPA are actively deploying SPI2-T3SS and STY and SPA exhibited a rapid decline of protein biosynthesis upon experimental induction. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for survival or proliferation in phagocytic host cells. Our results indicate that reduced intracellular activities are factors of the stealth strategy of STY and SPA and facilitate systemic spread and persistence of the typhoidal Salmonella.


Assuntos
Salmonella paratyphi A/patogenicidade , Salmonella typhi/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Proliferação de Células , Células HeLa , Humanos , Camundongos , Células RAW 264.7 , Salmonella paratyphi A/metabolismo , Salmonella typhi/metabolismo , Análise de Célula Única , Células U937 , Fatores de Virulência/metabolismo
5.
PLoS Pathog ; 16(7): e1008220, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658937

RESUMO

The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation of SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, which is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, providing a potential link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed us to characterize completely new host factor interactions, and to strengthen observations from several previous studies.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Endossomos/metabolismo , Endossomos/microbiologia , Células HeLa , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , RNA Interferente Pequeno
6.
Mol Cell Proteomics ; 19(5): 900-912, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32102972

RESUMO

Systemic infection and proliferation of intracellular pathogens require the biogenesis of a growth-stimulating compartment. The gastrointestinal pathogen Salmonella enterica commonly forms highly dynamic and extensive tubular membrane compartments built from Salmonella-modified membranes (SMMs) in diverse host cells. Although the general mechanism involved in the formation of replication-permissive compartments of S. enterica is well researched, much less is known regarding specific adaptations to different host cell types. Using an affinity-based proteome approach, we explored the composition of SMMs in murine macrophages. The systematic characterization provides a broader landscape of host players to the maturation of Salmonella-containing compartments and reveals core host elements targeted by Salmonella in macrophages as well as epithelial cells. However, we also identified subtle host specific adaptations. Some of these observations, such as the differential involvement of the COPII system, Rab GTPases 2A, 8B, 11 and ER transport proteins Sec61 and Sec22B may explain cell line-dependent variations in the pathophysiology of Salmonella infections. In summary, our system-wide approach demonstrates a hitherto underappreciated impact of the host cell type in the formation of intracellular compartments by Salmonella.


Assuntos
Adaptação Fisiológica , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Proteômica , Salmonella enterica/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Membranas , Camundongos , Proteoma/metabolismo , Células RAW 264.7
7.
mSphere ; 4(6)2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31826974

RESUMO

The tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (ΔfumABC) elicited a unique metabolic profile. Alongside fumarate, S Typhimurium ΔfumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S Typhimurium and cellular functions and virulence.IMPORTANCE We performed perturbation analyses of the tricarboxylic acid cycle of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium. The defect of fumarase activity led to accumulation of fumarate but also resulted in a global alteration of carbon fluxes, leading to increased storage of glycogen. Gross alterations were observed in proteome and metabolome compositions of fumarase-deficient Salmonella In turn, these changes were linked to aberrant motility patterns of the mutant strain and resulted in highly increased phagocytic uptake by macrophages. Our findings indicate that basic cellular functions and specific virulence functions in Salmonella critically depend on the proper function of the primary metabolism.


Assuntos
Carbono/metabolismo , Ciclo do Ácido Cítrico , Fumaratos/metabolismo , Interações Hospedeiro-Patógeno , Locomoção , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Fumarato Hidratase/deficiência , Glicólise , Macrófagos/imunologia , Macrófagos/microbiologia , Análise do Fluxo Metabólico , Erros Inatos do Metabolismo , Metaboloma , Hipotonia Muscular , Via de Pentose Fosfato , Fagocitose , Proteoma , Transtornos Psicomotores , Salmonella typhimurium/enzimologia , Salmonella typhimurium/imunologia , Virulência
8.
mBio ; 10(3)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239375

RESUMO

Type III secretion systems (T3SS) are molecular machines in Gram-negative pathogens that translocate effector proteins with central roles in virulence. The analyses of the translocation, subcellular localization, and mode of action of T3SS effector proteins are of central importance for the understanding of host-pathogen interaction and pathogenesis of bacterial infections. The analysis of translocation requires dedicated techniques to address the temporal and spatial dynamics of translocation. Here we describe a novel approach to deploy self-labeling enzymes (SLE) as universal tags for localization and tracking of translocated effector proteins. Effector-SLE fusion proteins allow live-cell imaging of translocation by T3SS, superresolution microscopy, and single-molecule tracking of effector motility in living host cells. We describe the application of the approach to T3SS effector proteins for invasion and intracellular lifestyle of Salmonella enterica serovar Typhimurium and to a T3SS effector of Yersinia enterocolitica The novel approach enables analyses of the role of T3SS in host-pathogen interaction at the highest temporal and spatial resolution, toward understanding the molecular mechanisms of their effector proteins.IMPORTANCE Type III secretion systems mediate translocation of effector proteins into mammalian cells. These proteins interfere with host cell functions, being main virulence factors of Gram-negative pathogens. Analyses of the process of translocation, the subcellular distribution, and the dynamics of effector proteins in host cells have been hampered by the lack of suitable tags and detection systems. Here we describe the use of self-labeling enzyme tags for generation of fusions with effector proteins that are translocated and functional in host cell manipulation. Self-labeling reactions with cell-permeable ligand dyes are possible prior to or after translocation. We applied the new approach to superresolution microscopy for effector protein translocation. For the first time, we show the dynamic properties of effector proteins in living host cells after translocation by intracellular bacteria. The new approach of self-labeling enzyme tags fusions will enable analyses of type III secretion system effector proteins with new dimensions of temporal and spatial resolution.


Assuntos
Transporte Proteico , Salmonella typhimurium/enzimologia , Sistemas de Secreção Tipo III/análise , Yersinia enterocolitica/enzimologia , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Salmonella typhimurium/genética , Coloração e Rotulagem , Fatores de Virulência , Yersinia enterocolitica/genética
9.
Sci Rep ; 7(1): 10326, 2017 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-28871183

RESUMO

Adhesins are crucial virulence factors of pathogenic bacteria involved in colonization, transmission and pathogenesis. Many bacterial genomes contain the information for a surprisingly large number of diverse adhesive structures. One prominent example is the invasive and facultative intracellular pathogen Salmonella enterica with an adhesiome of up to 20 adhesins. Such large repertoire of adhesins contributes to colonization of a broad range of host species and may allow adaptation to various environments within the host, as well as in non-host environments. For S. enterica, only few members of the adhesiome are functionally expressed under laboratory conditions, and accordingly the structural and functional understanding of the majority of adhesins is sparse. We have devised a simple and versatile approach to functionally express all adhesins of S. enterica serotype Typhimurium, either within Salmonella or within heterologous hosts such as Escherichia coli. We demonstrate the surface expression of various so far cryptic adhesins and show ultrastructural features using atomic force microscopy and transmission electron microscopy. In summary, we report for the first time the expression of the entire adhesiome of S. enterica serotype Typhimurium.


Assuntos
Adesinas Bacterianas/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/fisiologia , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Ordem dos Genes , Vetores Genéticos , Microscopia de Força Atômica , Óperon , Salmonella typhimurium/ultraestrutura , Fatores de Virulência/genética
10.
Cell Host Microbe ; 21(3): 390-402, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28238623

RESUMO

Salmonella enterica is a facultative intracellular pathogen that survives and proliferates in the Salmonella-containing vacuole (SCV), yet how these vacuolar bacteria acquire nutrition remains to be determined. Intracellular Salmonella convert the host endosomal system into an extensive network of interconnected tubular vesicles, of which Salmonella-induced filaments (SIFs) are the most prominent. We found that membranes and lumen of SIFs and SCVs form a continuum, giving vacuolar Salmonella access to various types of endocytosed material. Membrane proteins and luminal content rapidly diffuse between SIFs and SCVs. Salmonella in SCVs without connection to SIFs have reduced access to endocytosed components. On a single-cell level, Salmonella within the SCV-SIF continuum were found to exhibit higher metabolic activity than vacuolar bacteria lacking SIFs. Our data demonstrate that formation of the SCV-SIF continuum allows Salmonella to bypass nutritional restriction in the intracellular environment by acquiring nutrients from the host cell endosomal system.


Assuntos
Endossomos/microbiologia , Interações Hospedeiro-Patógeno , Biogênese de Organelas , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Vacúolos/microbiologia , Células HeLa , Humanos , Análise de Célula Única
11.
PLoS One ; 11(2): e0149187, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26882435

RESUMO

Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Nanopartículas/química , Proteínas Quinases/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/química , Histidina Quinase , Bicamadas Lipídicas/metabolismo , Nanopartículas/ultraestrutura , Ligação Proteica , Proteínas Quinases/química
12.
Sci Rep ; 5: 17740, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26643905

RESUMO

Research in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches.


Assuntos
Enzimas , Corantes Fluorescentes , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Animais , Enzimas/química , Humanos , Ligantes , Rodaminas
13.
Cell Microbiol ; 17(5): 639-47, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25802001

RESUMO

Salmonella enterica is a facultative intracellular pathogen residing in a unique host cell-derived membrane compartment, termed Salmonella-containing vacuole or SCV. By the activity of effector proteins translocated by the SPI2-endoced type III secretion system (T3SS), the biogenesis of the SCV is manipulated to generate a habitat permissive for intracellular proliferation. By taking control of the host cell vesicle fusion machinery, intracellular Salmonella creates an extensive interconnected system of tubular membranes arising from vesicles of various origins, collectively termed Salmonella-induced tubules (SIT). Recent work investigated the dynamic properties of these manipulations. New host cell targets of SPI2-T3SS effector proteins were identified. By applying combinations of live cell imaging and ultrastructural analyses, the detailed organization of membrane compartments inhabited and modified by intracellular Salmonella is now available. These studies provided unexpected new details on the intracellular environments of Salmonella. For example, one kind of SIT, the LAMP1-positive Salmonella-induced filaments (SIF), are composed of double-membrane tubules, with an inner lumen containing host cell cytosol and cytoskeletal filaments, and an outer lumen containing endocytosed cargo. The novel findings call for new models for the biogenesis of SCV and SIT and give raise to many open questions we discuss in this review.


Assuntos
Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Salmonella enterica/fisiologia , Vacúolos/microbiologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Ilhas Genômicas , Fatores de Virulência/metabolismo
14.
PLoS One ; 9(12): e115423, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25522146

RESUMO

Intracellular Salmonella enterica induce a massive remodeling of the endosomal system in infected host cells. One dramatic consequence of this interference is the induction of various extensive tubular aggregations of membrane vesicles, and tubules positive for late endosomal/lysosomal markers are referred to as Salmonella-induced filaments or SIF. SIF are highly dynamic in nature with extension and collapse velocities of 0.4-0.5 µm x sec-1. The induction of SIF depends on the function of the Salmonella Pathogenicity Island 2 (SPI2) encoded type III secretion system (T3SS) and a subset of effector proteins. In this study, we applied live cell imaging and electron microscopy to analyze the role of individual effector proteins in SIF morphology and dynamic properties of SIF. SIF in cells infected with sifB, sseJ, sseK1, sseK2, sseI, sseL, sspH1, sspH2, slrP, steC, gogB or pipB mutant strains showed a morphology and dynamics comparable to SIF induced by WT Salmonella. SIF were absent in cells infected with the sifA-deficient strain and live cell analyses allowed tracking of the loss of the SCV membrane of intracellular sifA Salmonella. In contrast to analyses in fixed cells, in living host cells SIF induced by sseF- or sseG-deficient strains were not discontinuous, but rather continuous and thinner in diameter. A very dramatic phenotype was observed for the pipB2-deficient strain that induced very bulky, non-dynamic aggregations of membrane vesicles. Our study underlines the requirement of the study of Salmonella-host interaction in living systems and reveals new phenotypes due to the intracellular activities of Salmonella.


Assuntos
Proteínas de Bactérias/metabolismo , Endossomos/microbiologia , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Salmonella enterica/patogenicidade , Proteínas de Bactérias/genética , Endossomos/metabolismo , Glicoproteínas/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Salmonella enterica/metabolismo
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